Transcriptional regulation of the yersiniabactin receptor fyuA gene by the ferric uptake regulator in Klebsiella pneumoniae NTUH-K2044.

The ferric uptake regulator (Fur) is a global regulator that influences the expression of virulence genes in Klebsiella pneumoniae. Bioinformatics analysis suggests Fur may involve in iron acquisition via the identified regulatory box upstream of the yersiniabactin receptor gene fyuA. To observe the impact of the gene fyuA on the virulence of K. pneumoniae, the gene fyuA knockout strain and complementation strain were constructed and then conducted a series of phenotypic experiments including chrome azurol S (CAS) detection, crystal violet staining, and wax moth virulence experiment. To examine the regulatory relationship between Fur and the gene fyuA, green fluorescent protein (GFP) reporter gene fusion assay, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), gel migration assay (EMSA), and DNase I footprinting assay were used to clarify the regulatory mechanism of Fur on fyuA. CAS detection revealed that the gene fyuA could affect the generation of iron carriers in K. pneumoniae. Crystal violet staining experiment showed that fyuA could positively influence biofilm formation. Wax moth virulence experiment indicated that the deletion of the fyuA could weaken bacterial virulence. GFP reporter gene fusion experiment and RT-qPCR analysis revealed that Fur negatively regulated the expression of fyuA in iron-sufficient environment. EMSA experiment demonstrated that Fur could directly bind to the promoter region of fyuA, and DNase I footprinting assay further identified the specific binding site sequences. The study showed that Fur negatively regulated the transcriptional expression of fyuA by binding to upstream of the gene promoter region, and then affected the virulence of K. pneumoniae.

Domination of an emerging erythromycin-resistant ptxP3 Bordetella pertussis clone in Shanghai, China.

The number of cases of pertussis has been increasing since 2014 in China, with high prevalence of macrolide resistance in ptxP1 isolates and low prevalence in ptxP3 isolates. This study aimed to investigate the dynamic changes in the B. pertussis population from paediatric patients and household contacts in Shanghai between 2018 and 2022. Clinical data of laboratory-confirmed cases of pertussis were analysed, while isolates recovered from hospitalized children and household contacts were characterized by antimicrobial susceptibility testing, whole-genome sequencing, vaccine antigen gene typing and phylogenetical analysis. Among 640 laboratory-confirmed cases, 340 (53.1%) were fully vaccinated with DTaP and 114 (17.8%) were hospitalized for treatment. The frequency of erythromycin resistance in the 103 B. pertussis isolates from inpatients (n=73) and household contacts (n=30) was 78.6% (81/103), increasing from 65% (13/20) in 2018 to 100% (26/26) in 2022. The proportion of ptxP3 isolates increased from 35% (7/20) in 2018 to 100% (26/26) in 2022. Based on genomic analysis, a novel ptxP3 clone (MT28-Shanghai) belonging to sublineage IVd was discovered and dominated in 2021-2022, which was characterized with ptxP3, erythromycin resistance and prn150. Twelve (11.7%) predicted pertactin-deficient isolates were found; of these, nine were ptxP3 isolates and three were ptxP1 isolates. A complete shift from ptxP1 to ptxP3 in Shanghai, China, which may have been accelerated by the domination of a novel erythromycin-resistant MT28 clone, challenges the pertussis vaccines used at present in China.

Penicillin and Cefotaxime Resistance of Quinolone-Resistant Neisseria meningitidis Clonal Complex 4821, Shanghai, China, 1965-2020.

Clonal complex 4821 (CC4821) Neisseria meningitidis, usually resistant to quinolones but susceptible to penicillin and third-generation cephalosporins, is increasing worldwide. To characterize the penicillin-nonsusceptible (PenNS) meningococci, we analyzed 491 meningococci and 724 commensal Neisseria isolates in Shanghai, China, during 1965-2020. The PenNS proportion increased from 0.3% in 1965-1985 to 7.0% in 2005-2014 and to 33.3% in 2015-2020. Of the 26 PenNS meningococci, 11 (42.3%) belonged to the CC4821 cluster; all possessed mutations in penicillin-binding protein 2, mostly from commensal Neisseria. Genetic analyses and transformation identified potential donors of 6 penA alleles. Three PenNS meningococci were resistant to cefotaxime, 2 within the CC4821 cluster. With 96% of the PenNS meningococci beyond the coverage of scheduled vaccination and the cefotaxime-resistant isolates all from toddlers, quinolone-resistant CC4821 has acquired penicillin and cefotaxime resistance closely related to the internationally disseminated ceftriaxone-resistant gonococcal FC428 clone, posing a greater threat especially to young children.

Serogroup Y Clonal Complex 23 Meningococcus in China Acquiring Penicillin Resistance from Commensal Neisseria lactamica Species.

Invasive meningococcal disease (IMD) due to serogroup Y Neisseria meningitidis (NmY) is rare in China; recently, an invasive NmY isolate, Nm512, was discovered in Shanghai with decreased susceptibility to penicillin (PenNS). Here, we investigated the epidemiology of NmY isolates in Shanghai and explored the potential commensal Neisseria lactamica donor of the PenNS NmY isolate. A total of 491 N. meningitidis and 724 commensal Neisseria spp. isolates were collected. Eleven NmY isolates were discovered from IMD (n = 1) and carriers (n = 10), including two PenNS isolates with five-key-mutation-harboring (F504L-A510V-I515V-H541N-I566V) penA genes. Five of the eight ST-175 complex (CC175) isolates had a genotype [Y:P1.5-1,2-2:F5-8:ST-175(CC175)] identical to that of the predominant invasive clone found in South Africa. Only one invasive NmY CC23 isolate (Nm512) was discovered; this isolate carried a novel PenNSpenA832 allele, which was identified in commensal N. lactamica isolates locally. Recombination analysis and transformation of the penA allele highlighted that N. meningitidis Nm512 may acquire resistance from its commensal donor; this was supported by the similar distribution of transformation-required DNA uptake sequence variants and the highly cognate receptor ComP between N. meningitidis and N. lactamica. In 2,309 NmY CC23 genomes from the PubMLST database, isolates with key-mutation-harboring penA genes comprised 12% and have been increasing since the 1990s, accompanied by recruitment of the blaROB-1 and/or quinolone resistance allele. Moreover, penA22 was predominant among genomes without key mutations in penA. These results strongly suggest that Nm512 is a descendant of the penA22-harboring CC23 isolate from Europe and acquired its penicillin resistance locally from commensal N. lactamica species by natural transformation.

Regulation of ECP fimbriae-related genes by the transcriptional regulator RcsAB in Klebsiella pneumoniae NTUH-K2044.

Klebsiella pneumoniae is one of the major pathogens causing nosocomial infections. The regulator of capsule synthesis (Rcs) system is a complex signal transduction pathway that is involved in the regulation of virulence factors of K. pneumoniae as an important transcriptional regulator. The RcsAB box-like sequence was found to be present in the promoter-proximal regions of ykgK, one of the ECP fimbriae-related genes, which suggested the expression of ECP fimbriae may be regulated by RcsAB. The ykgK gene in K. pneumoniae has 86% similarity to the ecpR gene in Escherichia coli. Nucleotide sequence alignment revealed a similar ECP fimbriae gene cluster including six genes in K. pneumoniae, which was proved to be on the same operon in this study. The electrophoretic mobility shift assay and DNase I assay, relative fluorescence expression, ß-galactosidase activity, and relative gene expression of ykgK in the wild-type and mutant strains were performed to determine the transcriptional regulation mechanism of RcsAB on ECP fimbriae. The mutant ΔykgK and complementary strain ΔykgK/cΔykgK were constructed to complete the Galleria mellonella larvae infection experiment and biofilm formation assay. This study showed that RcsAB binds directly to the promoter region of the ykgK gene to positively regulate ECP fimbriae-related gene clusters, and then positively affect the biofilm formation.

Genome sequencing and comparative genome analysis of 6 hypervirulent Klebsiella pneumoniae strains isolated in China.

Hypervirulent Klebsiella pneumoniae (hvKP) has been increasingly reported over the past three decades and causes severe infections. To increase our understanding of hvKP at the genome level, genome sequencing and comparative genome analysis were performed on 6 hvKPs. The whole genome DNA from 6 hvKPs with different capsular serotypes isolated in China was extracted. The genome sequencing and assembly results showed the genome size of the six hvKPs and GC content. Comparative analyses of the genomes revealed the gene homology and genome rearrangement in the 6 hvKPs compared with Klebsiella pneumonia NTUH-K2044. The phylogenetic tree based on full-genome SNPs of the 7 hvKPs showed that NTUH-K2044 formed a single clade, showing distant evolutionary distances with the other six strains, and the non-K1 hvKP strains had a relatively closer phylogenetic relationship. BLAST comparison analysis found that some selected virulence genes had different degrees of deletion in the non-K1 hvKPs. SNP-based virulence gene mutation analysis showed that some virulence genes had different degrees of SNP mutations. The whole-genome sequencing and comparative genome analysis of six hvKP strains with NTUH-K2044 provide us with a basic understanding of the genome composition, genetic polymorphism, evolution and virulence genes of hvKP and a basis for further research on these genes and the pathogenesis of hvKP.

Risk factor for lung infection in recipients after liver transplantation: A meta-analysis.

Lung infection (LI) often occurs in patients with liver transplantation (LT). This meta-analysis was conducted to determine the risk factors associated with LI after LT. We retrieved relevant research published as of February 2020 from eight electronic databases. The studies were reviewed against the inclusion and exclusion criteria. The Z test was used to determine the combined odds ratio (OR) or the standardized mean difference (SMD) of the risk factors. We used the OR and its corresponding 95% confidence interval (CI) or the SMD and its corresponding 95% CI to identify significant differences in risk factors. A total of nine studies were included, comprising a total of 1624 recipients. Six risk factors associated with LI were identified after LT: Model for end-stage liver disease score (MELD score) (SMD = 0.40), Child-Pugh class C (OR = 3.00), intensive care unit (ICU) hospital stay (SMD = 1.35), mechanical ventilation (SMD = 1.03), bilirubin (SMD = 0.39), and atelectasis (OR = 7.28). Although certain risk factors have been identified as important factors for LI after LT, which may provide a basis for clinical prevention, a well-designed prospective study should be conducted to validate the findings of this study.

[Pathogenic role leukotriene B4 in lung injury induced by lung-protective mechanical ventilation in rabbits].

OBJECTIVE: To elucidate the pathogenic role of leukotriene B4 (LTB4) in pulmonary hyper-permeability and inflammation induced by lung-protective mechanical ventilation (LPMV) in rabbits. METHODS: Thirty-two healthy Japanese white rabbits were randomized into 4 groups for treatment with vehicle or bestatin (a leukotriene A4 hydrolase inhibitor that inhibits LTB4 production) administered intragastrically at the daily dose of 8 mg/kg for 5 days, followed by sham operation (group S and group BS, respectively, in which the rabbits were anesthetized only) or LPMV (group PM and group BPM, respectively, in which the rabbits received ventilation with 50% oxygen at a tidal volume of 8 mL/kg for 5 h). The concentrations of LTB4 and cyclic adenosine monophosphate (cAMP) in the lung tissues were analyzed by ELISA. cAMP content, protein kinase A (PKA) protein expression and the Rap1-GTP protein to total Rap1 protein ratio were determined to assess the activities of cAMP/PKA and Rap1 signaling pathways. The lung injury was evaluated by assessing lung permeability index, lung wet/dry weight ratio, polymorphonuclear leukocyte (PMN) count in bronchoalveolar lavage fluid (BALF), pulmonary myeloperoxidase (MPO) activity and lung histological scores. RESULTS: None of the examined parameters differed significantly between group S and group BS. All the parameters with the exception of lung histological score increased significantly in group PM and group BPM as compared to those in group S (P < 0.05). Compared with those in PM group, the rabbits in group BPM showed significantly reduced LTB4 production in the lungs (P < 0.05), up-regulated cAMP/ PKA and Rap1 signaling pathway activities (P < 0.05), and alleviated lung hyper-permeability and inflammation (P < 0.05). CONCLUSIONS: LPMV can induce LTB4 overproduction to down-regulate cAMP/PKA and Rap1 signaling pathways in the lungs of rabbits, which results in lung hyper-permeability and inflammation. Bestatin can inhibit LTB4 production in the lungs to protect against LPMV-induced lung hyper-permeability and inflammation.

RcsAB and Fur Coregulate the Iron-Acquisition System via entC in Klebsiella pneumoniae NTUH-K2044 in Response to Iron Availability.

The iron acquisition system is an essential virulence factor for human infection and is under tight regulatory control in a variety of pathogens. Ferric-uptake regulator (Fur) is one of Fe2+-responsive transcription factor that maintains iron homeostasis, and the regulator of capsule synthesis (Rcs) is known to regulate exopolysaccharide biosynthesis. We speculate the Rcs may involve in iron-acquisition given the identified regulator box in the upstream of entC that participated in the biosynthesis of enterobactin. To study the coregulation by RcsAB and Fur of entC, we measured the ß-galactosidase activity and relative mRNA expression of entC in WT and mutant strains. The RcsAB- and Fur-protected regions were identified by an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay. A regulatory cascade was identified with which Fur repressed rcsA expression and reduced RcsAB and entC expression. Our study demonstrated that entC was coregulated by two different transcriptional regulators, namely, RcsAB and Fur, in response to iron availability in Klebsiella pneumoniae.

Target-inspired Pb2+-dependent DNAzyme for ultrasensitive electrochemical sensor based on MoS2-AuPt nanocomposites and hemin/G-quadruplex DNAzyme as signal amplifier.

In this work, a novel Pb2+ electrochemical DNAzyme sensor was developed for ultrasensitive detection of lead ions (Pb2+) in water environment by coupling with the MoS2-AuPt nanomaterials and hemin/G-quadruplex DNAzyme, which acting as the electrocatalytic signal tag. Streptavidin (SA) modified tin dioxide-functionalized reduced graphene oxide (rGO-SnO2) /gold nanoparticles (AuNPs) served as a sensor platform for enhancing conductivity and immobilizing more Pb2+-specific DNAzyme. In the presence of Pb2+, the Pb2+-dependent DNAzyme specifically reacted with Pb2+, cleaving the substrate strand (SS) into two free fragment and releasing the biotin-modified enzyme strand (Bio-ES) on the electrode. Connecting MoS2-AuPt nanocomposites labeled with G-rich DNA (G-DNA) strand and exposure of Bio-ES through the Helper DNA, as well as adding hemin to form a hemin/G-quadruplex, the biosensor achieved signal amplification. Chronoamperometry was used to record the current signal, which was primarily derived from the cocatalysis reduction of H2O2 by MoS2-AuPt nanocomposites and the hemin/G-quadruplex. Under optimal conditions, the designed biosensor exhibited sensitive detection of Pb2+ from 0.1 pg mL-1 to 1000 ng mL-1, with a lower detection limit of 38 fg mL-1 (based on 3σ). This proposed biosensor is ultrasensitive and specific, representing a potential application for the detection of Pb2+ in a water environment.