Dataset Information

The role of EII complex in the bacterial responses to the glucose-survey in clinical Klebsiella pneumoniae isolates.

ABSTRACT: Type 3 fimbriae in Klebsiella pneumoniae are important for bacterial colonization on abiotic and biotic surfaces. The major subunit of type 3 fimbriae (MrkA) is increased by overexpression of EtcABC, an EII complex of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs), through cAMP-cAMP receptor protein (cAMP-CRP) in K. pneumoniae STU1. Here, we further characterized the relations between the amount of etcABC mRNA and MrkA in 78 clinical K. pneumoniae isolates incubated in high levels of glucose. By Western blotting, we observed that MrkA of 29 isolates were not decreased much by high levels of glucose (Group A) but MrkA of other 49 isolates were significantly reduced (Group B) in the same condition. The bacterial biofilms on abiotic surfaces and colonization in the Caenorhabditis elegans of representative isolates in the Group A were not affected by high levels of glucose. However, the biofilm and colonization in the worm of clinical isolates in the Group B were much reduced by high levels of glucose. After quantification by real time RT-PCR, 76% of Group A but just 10% of Group B showed high amount of etcA mRNA. In summary, our results suggested that for most of K. pneumoniae clinical isolates, the amount of etcABC mRNA was positively related to their type 3 fimbriae production in a high level of glucose, thereby to their biofilm formation and colonization in the worm.

SUBMITTER: Horng YT

PROVIDER: S-EPMC10406186 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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The role of EII complex in the bacterial responses to the glucose-survey in clinical Klebsiella pneumoniae isolates.

Horng Yu-Tze YT Panjaitan Novaria Sari Dewi NSD Tsai Yi-Jhen YJ Su Pin-Wei PW Yang Hung-Chi HC Soo Po-Chi PC

PloS one 20230807 8

Type 3 fimbriae in Klebsiella pneumoniae are important for bacterial colonization on abiotic and biotic surfaces. The major subunit of type 3 fimbriae (MrkA) is increased by overexpression of EtcABC, an EII complex of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs), through cAMP-cAMP receptor protein (cAMP-CRP) in K. pneumoniae STU1. Here, we further characterized the relations between the amount of etcABC mRNA and MrkA in 78 clinical K. pneumoniae isolates incubated in high l ...[more]

PMID: 37549172

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Project description:Biofilm formation by Klebsiella pneumoniae on indwelling medical devices increases the risk of infection. Both type 1 and type 3 fimbriae are important factors in biofilm formation by K. pneumoniae. We found that a putative enzyme II (EII) complex of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), etcA (EIIA)-etcB (EIIB)-etcC (EIIC), regulated biofilm and type 3 fimbriae formation by K. pneumoniae STU1. In this study, the regulatory mechanism of etcABC in K. pneumoniae type 3 fimbriae formation was investigated. We found via quantitative RT-PCR that overexpression of etcABC enhanced the transcription level of the mrk operon, which is involved in type 3 fimbriae synthesis, and reduced the transcription level of the fim operon, which is involved in type 1 fimbriae synthesis. To gain further insight into the role of etcABC in type 3 fimbriae synthesis, we analyzed the region upstream of the mrk operon and found the potential cyclic 3'5'-adenosine monophosphate (cAMP) receptor protein (CRP) binding site. After crp was deleted in K. pneumoniae STU1 and two clinical isolates, these three crp mutant strains could not express MrkA, the major subunit of the fimbrial shaft, indicating that CRP positively regulated type 3 fimbriae synthesis. Moreover, a crp mutant overexpressing etcABC could not express MrkA, indicating that the regulation of type 3 fimbriae by etcABC was dependent on CRP. In addition, deletion of cyaA, which encodes the adenylyl cyclase that synthesizes cAMP, and deletion of crr, which encodes the glucose-specific EIIA, led to a reduction in lac operon regulation and therefore bacterial lactose uptake in K. pneumoniae. Exogenous cAMP but not etcABC overexpression compensated for the role of cyaA in bacterial lactose uptake. However, either etcABC overexpression or exogenous cAMP compensated for the role of crr in bacterial lac operon regulation that would eventually restore lactose uptake. We also found via ELISA and the luxCDABE reporter system that overexpression of etcABC increased intracellular cAMP levels and the transcription level of crp, respectively, in K. pneumoniae. In conclusion, overexpression of etcABC positively regulated cAMP production and cAMP-CRP activity to activate the mrk operon, resulting in increased type 3 fimbriae synthesis in K. pneumoniae.

Project description:Here, we conducted a comprehensive analysis of 356 Klebsiella pneumoniae species complex (KpSC) isolates that were classified as classical (cl), presumptive hypervirulent (p-hv) and hypermucoviscous-like (hmv-like). Overall, K. pneumoniae (82.3%), K. variicola (2.5%) and K. quasipneumoniae (2.5%) were identified. These isolates comprised 321 cl-KpSC, 7 p-hv-KpSC and 18 hmv-like-KpSC. A large proportion of cl-KpSC isolates were extended-spectrum-β-lactamases (ESBLs)-producers (64.4%) and 3.4% of isolates were colistin-resistant carrying carbapenemase and ESBL genes. All p-hv-KpSC showed an antibiotic susceptible phenotype and hmv-like isolates were found to be ESBL-producers (8/18). Assays for capsule production and capsule-dependent virulence phenotypes and whole-genome sequencing (WGS) were performed in a subset of isolates. Capsule amount differed in all p-hv strains and hmv-like produced higher capsule amounts than cl strains; these variations had important implications in phagocytosis and virulence. Murine sepsis model showed that most cl strains were nonlethal and the hmv-like caused 100% mortality with 3 × 108 CFUs. Unexpectedly, 3/7 (42.9%) of p-hv strains required 108 CFUs to cause 100% mortality (atypical hypervirulent), and 4/7 (57.1%) strains were considered truly hypervirulent (hv). Genomic analyses confirmed the diverse population, including isolates belonging to hv clonal groups (CG) CG23, CG86, CG380 and CG25 (this corresponded to the ST3999 a novel hv clone) and MDR clones such as CG258 and CG147 (ST392) among others. We noted that the hmv-like and hv-ST3999 isolates showed a close phylogenetic relationship with cl-MDR K. pneumoniae. The information collected here is important to understand the evolution of clinically important phenotypes such as hypervirulent and ESBL-producing-hypermucoviscous-like amongst the KpSC in Mexican healthcare settings. Likewise, this study shows that mgrB inactivation is the main mechanism of colistin resistance in K. pneumoniae isolates from Mexico.

Project description:Klebsiella pneumoniae is a major threat to public health, causing significant morbidity and mortality worldwide. The emergence of highly drug-resistant strains is particularly concerning. There has been a recognition and division of Klebsiella pneumoniae into three distinct phylogenetic groups: Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae. K. variicola and K. quasipneumoniae have often been described as opportunistic pathogens that have less virulence in humans than K. pneumoniae does. We recently sequenced the genomes of 1,777 extended-spectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates recovered from human infections and discovered that 28 strains were phylogenetically related to K. variicola and K. quasipneumoniae. Whole-genome sequencing of 95 additional non-ESBL-producing K. pneumoniae isolates recovered from patients found 12 K. quasipneumoniae strains. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis initially identified all patient isolates as K. pneumoniae, suggesting a potential pitfall in conventional clinical microbiology laboratory identification methods. Whole-genome sequence analysis revealed extensive sharing of core gene content and plasmid replicons among the Klebsiella species. For the first time, strains of both K. variicola and K. quasipneumoniae were found to carry the Klebsiella pneumoniae carbapenemase (KPC) gene, while another K. variicola strain was found to carry the New Delhi metallo-beta-lactamase 1 (NDM-1) gene. K. variicola and K. quasipneumoniae infections were not less virulent than K. pneumoniae infections, as assessed by in-hospital mortality and infection type. We also discovered evidence of homologous recombination in one K. variicola strain, as well as one strain from a novel Klebsiella species, which challenge the current understanding of interrelationships between clades of Klebsiella. IMPORTANCEKlebsiella pneumoniae is a serious human pathogen associated with resistance to multiple antibiotics and high mortality. K. variicola and K. quasipneumoniae are closely related organisms that are generally considered to be less-virulent opportunistic pathogens. We used a large, comprehensive, population-based strain collection and whole-genome sequencing to investigate infections caused by these organisms in our hospital system. We discovered that K. variicola and K. quasipneumoniae isolates are often misidentified as K. pneumoniae by routine clinical microbiology diagnostics and frequently cause severe life-threatening infections similar to K. pneumoniae. The presence of KPC in K. variicola and K. quasipneumoniae strains as well as NDM-1 metallo-beta-lactamase in one K. variicola strain is particularly concerning because these genes confer resistance to many different beta-lactam antibiotics. The sharing of plasmids, as well as evidence of homologous recombination, between these three species of Klebsiella is cause for additional concern.

Project description:Phagocytosis is a key process of the immune system. The human pathogen Klebsiella pneumoniae is a well known example of a pathogen highly resistant to phagocytosis. A wealth of evidence demonstrates that the capsule polysaccharide (CPS) plays a crucial role in resistance to phagocytosis. The amoeba Dictyostelium discoideum shares with mammalian macrophages the ability to phagocytose and kill bacteria. The fact that K. pneumoniae is ubiquitous in nature and, therefore, should avoid predation by amoebae, poses the question whether K. pneumoniae employs similar means to counteract amoebae and mammalian phagocytes. Here we developed an assay to evaluate K. pneumoniae-D. discoideum interaction. The richness of the growth medium affected the threshold at which the cps mutant was permissive for Dictyostelium and only at lower nutrient concentrations the cps mutant was susceptible to predation by amoebae. Given the critical role of bacterial surface elements on host-pathogen interactions, we explored the possible contribution of the lipopolysaccharide (LPS) and outer membrane proteins (OMPs) to combat phagoyctosis by D. discoideum. We uncover that, in addition to the CPS, the LPS O-polysaccharide and the first core sugar participate in Klebsiella resistance to predation by D. discoideum. K. pneumoniae LPS lipid A decorations are also necessary to avoid predation by amoebae although PagP-dependent palmitoylation plays a more important role than the lipid A modification with aminoarabinose. Mutants lacking OMPs OmpA or OmpK36 were also permissive for D. discoideium growth. Except the LPS O-polysaccharide mutants, all mutants were more susceptible to phagocytosis by mouse alveolar macrophages. Finally, we found a correlation between virulence, using the pneumonia mouse model, and resistance to phagocytosis. Altogether, this work reveals novel K. pneumoniae determinants involved in resistance to phagocytosis and supports the notion that Dictyostelium amoebae might be useful as host model to measure K. pneumoniae virulence and not only phagocytosis.

Project description:Klebsiella pneumoniae is among the most common causes of hospital-acquired infections and has emerged as an urgent threat to public health due to carbapenem antimicrobial resistance. K. pneumoniae commonly colonizes hospitalized patients and causes extraintestinal infections such as urinary tract infection, bloodstream infection (septicemia), and pneumonia. If colonization is an intermediate step before infection, then detection and characterization of colonizing isolates could enable strategies to prevent or empirically treat K. pneumoniae infections in hospitalized patients. However, the strength of the association between colonization and infection is unclear. To test the hypothesis that hospitalized patients become infected with their colonizing strain, 1,765 patients were screened for rectal colonization with K. pneumoniae, and extraintestinal isolates from these same patients were collected over a 3-month period in a cohort study design. The overall colonization prevalence was 23.0%. After adjustment for other patient factors, colonization was significantly associated with subsequent infection: 21 of 406 (5.2%) colonized patients later had extraintestinal infection, compared to 18 of 1,359 (1.3%) noncolonized patients (adjusted odds ratio [OR], 4.01; 95% confidence interval, 2.08 to 7.73; P < 0.001). Despite a high diversity of colonizing isolates, 7/7 respiratory, 4/4 urinary, and 2/5 bloodstream isolates from colonized patients matched the patient corresponding rectal swab isolates, based on wzi capsular typing, multilocus sequence typing (MLST), and whole-genome sequence analysis. These results suggest that K. pneumoniae colonization is directly associated with progression to extraintestinal infection. IMPORTANCE K. pneumoniae commonly infects hospitalized patients, and these infections are increasingly resistant to carbapenems, the antibiotics of last resort for life-threatening bacterial infections. To prevent and treat these infections, we must better understand how K. pneumoniae causes disease and discover new ways to predict and detect infections. This study demonstrates that colonization with K. pneumoniae in the intestinal tract is strongly linked to subsequent infection. This finding helps to identify a potential time frame and possible approach for intervention: the colonizing strain from a patient could be isolated as part of a risk assessment, and antibiotic susceptibility testing could guide empirical therapy if the patient becomes acutely ill.

Dataset Information

The role of EII complex in the bacterial responses to the glucose-survey in clinical Klebsiella pneumoniae isolates.

Publications

The role of EII complex in the bacterial responses to the glucose-survey in clinical Klebsiella pneumoniae isolates.

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