Klebsiella pneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniae-induced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation.
The increasing prevalence of antibiotic resistant pathogens poses a serious threat to global health. However, less emphasis has been placed to co-relate the gene expression and metabolism of antibiotic resistant pathogens. This study aims to elucidate gene expression and variations in metabolism of multidrug resistant Klebsiella pneumoniae after exposure to antibiotics. Phenotypic responses of three genotypically distinct carbapenem resistant Klebsiella pneumoniae (CRKP) strains untreated and treated with sub-lethal concentrations of imipenem were investigated via phenotype microarrays (PM). The gene expression and metabolism of the strain harboring blaNDM-1 before and after exposure to sub-lethal concentration of imipenem were further investigated by RNA-sequencing (RNA-Seq) and 1H NMR spectroscopy respectively. Most genes related to cell division, central carbon metabolism and nucleotide metabolism were downregulated after imipenem treatment. Similarly, 1H NMR spectra obtained from treated CRKP showed decrease in levels of bacterial end products (acetate, pyruvate, succinate, formate) and metabolites involved in nucleotide metabolism (uracil, xanthine, hypoxanthine) but elevated levels of glycerophosphocholine. The presence of anserine was also observed for the treated CRKP while FAPγ-adenine and methyladenine were only present in untreated bacterial cells. As a conclusion, the studied CRKP strain exhibited decrease in central carbon metabolism, cell division and nucleotide metabolism after exposure to sub-lethal concentrations of imipenem. The understanding of the complex biological system of this multidrug resistant bacterium may help in the development of novel strategies and potential targets for the management of the infections.
Klebsiella pneumoniae (KP) remains the most prevalent nosocomial pathogen and carries the carbapenemase (KPC) gene which confers resistance towards carbapenem. Thus, it is necessary to discover novel antimicrobials to address the issue of antimicrobial resistance in such pathogens. Natural products such as essential oils are a promising source due to their complex composition. Essential oils have been shown to be effective against pathogens, but the overall mechanisms have yet to be fully explained. Understanding the molecular mechanisms of essential oil towards KPC-KP cells would provide a deeper understanding of their potential use in clinical settings. Therefore, we aimed to investigate the mode of action of essential oil against KPC-KP cells from a proteomic perspective by comparing the overall proteome profile of KPC-KP cells treated with cinnamon bark (Cinnamomum verum J. Presl) essential oil (CBO) at their sub-inhibitory concentration of 0.08% (v/v). A total of 384 proteins were successfully identified from the non-treated cells, whereas only 242 proteins were identified from the CBO-treated cells. Proteins were then categorized based on their biological processes, cellular components and molecular function prior to pathway analysis. Pathway analysis showed that CBO induced oxidative stress in the KPC-KP cells as indicated by the abundance of oxidative stress regulator proteins such as glycyl radical cofactor, catalase peroxidase and DNA mismatch repair protein. Oxidative stress is likely to oxidize and disrupt the bacterial membrane as shown by the loss of major membrane proteins. Several genes selected for qRT-PCR analysis validated the proteomic profile and were congruent with the proteomic abundance profiles. In conclusion, KPC-KP cells exposed to CBO undergo oxidative stress that eventually disrupts the bacterial membrane possibly via interaction with the phospholipid bilayer. Interestingly, several pathways involved in the bacterial membrane repair system were also affected by oxidative stress, contributing to the loss of cells viability.
Aims: The high prevalence of multidrug resistance (MDR) and extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae associated with nosocomial infections has caused serious therapeutic challenges. The objectives of this study were to determine the genotypic and phenotypic characteristics of K. pneumoniae strains isolated from Malaysian swine farms and the transferability of ESBL genes by plasmids. Results: A total of 50 K. pneumoniae strains were isolated from 389 samples, which were collected from healthy and unhealthy pigs (swine rectum and oral cavities), healthy farmers (human rectum, urine, and nasal cavities), farm's environment, and animal feeds from seven Malaysian swine farms. Antimicrobial susceptibility analysis of these 50 K. pneumoniae strains showed that the majority (86%) were resistant to tetracycline, while 44% and 36% of these strains were MDR and ESBL producers, respectively. PCR and DNA sequencing of the amplicons showed the occurrence of blaTEM (15/18), blaSHV (15/18), blaCTX-M-1 group (7/18), and blaCTX-M-2 group (2/18), while only class 1 integron-encoded integrase was detected. Conjugation experiments and plasmid analysis indicated that the majority of the ESBL genes were plasmid encoded and the plasmids in 11 strains were conjugative. Genotyping by pulsed-field gel electrophoresis and repetitive extragenic palindrome-polymerase chain reaction (REP-PCR) showed that these 50 strains were genetically diverse with 44 pulsotypes and 43 REP-PCR subtypes. Conclusions: ESBL-producing K. pneumoniae strains showed high resistance to tetracycline as this antibiotic is used for prophylaxis and therapeutic purposes at the swine farms. The findings in this study have drawn attention to the issue of increasing MDR in animal husbandry and it should be taken seriously to prevent the spread and treatment failure due to antimicrobial resistance.
In addition to beta-lactamase production, loss of porins confers resistance to extended-spectrum beta-lactams in Klebsiella pneumoniae and Escherichia coli infection. This study describes the detection of SHV-12 extended-spectrum beta-lactamase (ESBL) subtype and the loss of OmpK35 porin in 4 strains of K. pneumoniae and E. coli.
Background: Klebsiella pneumoniae is a major opportunistic pathogen frequently associated with nosocomial infections, and often poses a major threat to immunocompromised patients. In our previous study, two K. pneumoniae (K36 and B13), which displayed resistance to almost all major antibiotics, including colistin, were isolated. Both isolates were not associated with infection and isolated from the stools of two preterm neonates admitted to the neonatal intensive care unit (NICU) during their first week of life. Materials and Methods: In this study, whole genome sequencing was performed on these two clinical multidrug resistant K. pneumoniae. We aimed to determine the genetic factors that underline the antibiotic-resistance phenotypes of these isolates. Results: The strains harbored blaSHV-27, blaSHV-71, and oqxAB genes conferring resistance to cephalosporins, carbapenems, and fluoroquinolones, respectively, but not harboring any known plasmid-borne colistin resistance determinants such as mcr-1. However, genome analysis discovered interruption of mgrB gene by insertion sequences gaining insight into the development of colistin resistance. Conclusion: The observed finding that points to a scenario of potential gut-associated resistance genes to Gram negative (K. pneumoniae) host in the NICU environment warrants attention and further investigation.
Multidrug-resistant (MDR) and extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae associated with nosocomial infections have caused serious problems in antibiotic management with limited therapeutic choices. This study aimed to determine the genotypic and phenotypic characteristics of K. pneumoniae strains isolated from a tertiary hospital in Malaysia. Ninety-seven clinical K. pneumoniae strains were analyzed for antimicrobial susceptibility, all of which were sensitive to amikacin and colistin (except one strain), while 31.9 % and 27.8 % were MDR and ESBL producers, respectively. PCR and DNA sequencing of the amplicons indicated that the majority of MDR strains (26/27) were positive for blaTEM, followed by blaSHV (24/27), blaCTX-M-1 group (23/27), blaCTX-M-9 group (2/27), and mcr-1 (1/27). Thirty-seven strains were hypervirulent and PCR detection of virulence genes showed 38.1 %, 22.7 %, and 16.5 % of the strains were positive for K1, wabG, and uge genes, respectively. Genotyping by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) showed that these strains were genetically diverse and heterogeneous. Sequence types, ST23, ST22, and ST412 were the predominant genotypes. This is the first report of colistin-resistant K. pneumoniae among clinical strains associated with mcr-1 plasmid in Malaysia. The findings in this study have contributed to the effort in combating the increase in antimicrobial resistance by providing better understanding of genotypic characteristics and resistance mechanisms of the organisms.