sRNA expression profile of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae: Functional role of sRNA51.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has significant challenges to human health and clinical treatment, with KPC-2-producing CRKP being the predominant epidemic strain. Therefore, there is an urgent need to identify new therapeutic targets and strategies. Non-coding small RNA (sRNA) is a post-transcriptional regulator of genes involved in important biological processes in bacteria and represents an emerging therapeutic strategy for antibiotic-resistant bacteria. In this study, we analyzed the transcription profile of KPC-2-producing CRKP using RNA-seq. Of the 4693 known genes detected, the expression of 307 genes was significantly different from that of carbapenem-sensitive Klebsiella pneumoniae (CSKP), including 133 up-regulated and 174 down-regulated genes. Both the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly related to metabolism. In addition, we identified the sRNA expression profile of KPC-2-producing CRKP for the first time and detected 115 sRNAs, including 112 newly discovered sRNAs. Compared to CSKP, 43 sRNAs were differentially expressed in KPC-2-producing CRKP, including 39 up-regulated and 4 down-regulated sRNAs. We chose sRNA51, the most significantly differentially expressed sRNA in KPC-2-producing CRKP, as our research subject. By constructing sRNA51-overexpressing KPC-2-producing CRKP strains, we found that sRNA51 overexpression down-regulated the expression of acrA and alleviated resistance to meropenem and ertapenem in KPC-2-producing CRKP, while overexpression of acrA in sRNA51-overexpressing strains restored the reduction of resistance. Therefore, we speculated that sRNA51 could affect the resistance of KPC-2-producing CRKP by inhibiting acrA expression and affecting the formation of efflux pumps. This provides a new approach for developing antibiotic adjuvants to restore the sensitivity of CRKP.

A curated collection of Klebsiella metabolic models reveals variable substrate usage and gene essentiality.

The Klebsiella pneumoniae species complex (KpSC) is a set of seven Klebsiella taxa that are found in a variety of niches and are an important cause of opportunistic health care-associated infections in humans. Because of increasing rates of multi-drug resistance within the KpSC, there is a growing interest in better understanding the biology and metabolism of these organisms to inform novel control strategies. We collated 37 sequenced KpSC isolates isolated from a variety of niches, representing all seven taxa. We generated strain-specific genome-scale metabolic models (GEMs) for all 37 isolates and simulated growth phenotypes on 511 distinct carbon, nitrogen, sulfur, and phosphorus substrates. Models were curated and their accuracy was assessed using matched phenotypic growth data for 94 substrates (median accuracy of 96%). We explored species-specific growth capabilities and examined the impact of all possible single gene deletions using growth simulations in 145 core carbon substrates. These analyses revealed multiple strain-specific differences, within and between species, and highlight the importance of selecting a diverse range of strains when exploring KpSC metabolism. This diverse set of highly accurate GEMs could be used to inform novel drug design, enhance genomic analyses, and identify novel virulence and resistance determinants. We envisage that these 37 curated strain-specific GEMs, covering all seven taxa of the KpSC, provide a valuable resource to the Klebsiella research community.

Klebsiella pneumoniae causes bacteremia using factors that mediate tissue-specific fitness and resistance to oxidative stress.

Gram-negative bacteremia is a major cause of global morbidity involving three phases of pathogenesis: initial site infection, dissemination, and survival in the blood and filtering organs. Klebsiella pneumoniae is a leading cause of bacteremia and pneumonia is often the initial infection. In the lung, K. pneumoniae relies on many factors like capsular polysaccharide and branched chain amino acid biosynthesis for virulence and fitness. However, mechanisms directly enabling bloodstream fitness are unclear. Here, we performed transposon insertion sequencing (TnSeq) in a tail-vein injection model of bacteremia and identified 58 K. pneumoniae bloodstream fitness genes. These factors are diverse and represent a variety of cellular processes. In vivo validation revealed tissue-specific mechanisms by which distinct factors support bacteremia. ArnD, involved in Lipid A modification, was required across blood filtering organs and supported resistance to soluble splenic factors. The purine biosynthesis enzyme PurD supported liver fitness in vivo and was required for replication in serum. PdxA, a member of the endogenous vitamin B6 biosynthesis pathway, optimized replication in serum and lung fitness. The stringent response regulator SspA was required for splenic fitness yet was dispensable in the liver. In a bacteremic pneumonia model that incorporates initial site infection and dissemination, splenic fitness defects were enhanced. ArnD, PurD, DsbA, SspA, and PdxA increased fitness across bacteremia phases and each demonstrated unique fitness dynamics within compartments in this model. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA, but not PdxA, specifically resists oxidative stress produced by NADPH oxidase Nox2 in the lung, spleen, and liver, as it was a fitness factor in wild-type but not Nox2-deficient (Cybb-/-) mice. These results identify site-specific fitness factors that act during the progression of Gram-negative bacteremia. Defining K. pneumoniae fitness strategies across bacteremia phases could illuminate therapeutic targets that prevent infection and sepsis.

Understanding blaNDM-1 gene regulation in CRKP infections: toward novel antimicrobial strategies for hospital-acquired pneumonia.

BACKGROUND: The escalating challenge of Carbapenem-resistant Klebsiella pneumoniae (CRKP) in hospital-acquired pneumonia (HAP) is closely linked to the blaNDM-1 gene. This study explores the regulatory mechanisms of blaNDM-1 expression and aims to enhance antibacterial tactics to counteract the spread and infection of resistant bacteria. METHODS: KP and CRKP strains were isolated from HAP patients' blood samples. Transcriptomic sequencing (RNA-seq) identified significant upregulation of blaNDM-1 gene expression in CRKP strains. Bioinformatics analysis revealed blaNDM-1 gene involvement in beta-lactam resistance pathways. CRISPR-Cas9 was used to delete the blaNDM-1 gene, restoring sensitivity. In vitro and in vivo experiments demonstrated enhanced efficacy with Imipenem and Thanatin or Subatan combination therapy. RESULTS: KP and CRKP strains were isolated with significant upregulation of blaNDM-1 in CRKP strains identified by RNA-seq. The Beta-lactam resistance pathway was implicated in bioinformatics analysis. Knockout of blaNDM-1 reinstated sensitivity in CRKP strains. Further, co-treatment with Imipenem, Thanatin, or Subactam markedly improved antimicrobial effectiveness. CONCLUSION: Silencing blaNDM-1 in CRKP strains from HAP patients weakens their Carbapenem resistance and optimizes antibacterial strategies. These results provide new theoretical insights and practical methods for treating resistant bacterial infections.

Characterization of blaOXA-232 carrying carbapenem-resistant Klebsiella pneumoniae (CRKP) & their expression profiles under selective carbapenem pressure: An in-depth study from India.

Background & objectives OXA-232 is a five amino acid substitution variant of OXA-48 and is reported in carbapenem-resistant Klebsiella pneumoniae (CRKP), which is associated with nosocomial infections among immunocompromised patients in the intensive care unit. This study aimed to characterise blaOXA-232 in CRKP of clinical origin and investigate its transcriptional response against sub-inhibitory levels of carbapenems. Methods CRKP was isolated from blood (pathogens) and stool cultures (colonisers) of neonates and was characterized for blaOXA-232. Co-existing resistance determinants were investigated in blaOXA-232 positive isolates, followed by horizontal gene transferability assay and PCR-based replicon typing (PBRT). Cloning of blaOXA-232 was performed, and expression of blaOXA-232 in the isolates and their clones under sub-inhibitory concentrations of carbapenems was checked via RT-PCR. Mobile genetic elements associated with blaOXA-232 were investigated, followed by DNA fingerprinting through enterobacterial repetitive intergenic consensus (ERIC) PCR. Results blaOXA-232 with co-carriage of extended-spectrum beta-lactamases (ESBLs), sulphonamides and quinolones were identified in seven CRPK isolates recovered from blood samples of neonates. Transformation and cloning of blaOXA-232 was successful. The sub-inhibitory concentration of carbapenems induces elevated expression of this resistant determinant. ISEcp1 was associated with blaOXA-232 in the upstream region within two haplotypes of CRKP isolates of clinical origin. Interpretation & conclusions Selective carbapenem pressure resulted in higher expression of this gene, which could account for treatment failure. With frequent reports of occurrence among clinical isolates, monitoring and further investigation of this novel variant are necessary to understand its transmission dynamics and to thwart its further dissemination.

The Response of Murine Gut Microbiome in the Presence of Altered rpoS Gene of Klebsiella pneumoniae.

The murine model is invaluable for studying intricate interactions among gut microbes; hosts; and diseases. However; the impact of genetic variations in the murine microbiome; especially in disease contexts such as Klebsiella pneumoniae (Kp) infection; still needs to be explored. Kp; an opportunistic global pathogen; is becoming increasingly prevalent in regions like Asia; especially China. This study explored the role of the gut microbiota during Kp infection using mouse model; including wild-type and rpoS mutants of Kp138; KpC4; and KpE4 from human; maize; and ditch water; respectively. Under stress conditions; RpoS reconfigures global gene expression in bacteria; shifting the cells from active growth to survival mode. Our study examined notable differences in microbiome composition; finding that Lactobacillus and Klebsiella (particularly in WKp138) were the most abundant genera in mice guts at the genus level in all wild-type treated mice. In contrast; Firmicutes were predominant in the healthy control mice. Furthermore; Clostridium was the dominant genus in all mutants; mainly in ∆KpC4; and was absent in wild-type treated mice. Differential abundance analysis identified that these candidate taxa potentially influence disease progression and pathogen virulence. Functional prediction analysis showed that most bacterial groups were functionally involved in biosynthesis; precursor metabolites; degradation; energy generation; and metabolic cluster formation. These findings challenge the conventional understanding and highlight the need for nuanced interpretations in murine studies. Additionally; this study sheds light on microbiome-immune interactions in K. pneumoniae infection and proposes new potential therapeutic strategies.

Genomic Insights into CRISPR-Harboring Plasmids in the Klebsiella Genus: Distribution, Backbone Structures, Antibiotic Resistance, and Virulence Determinant Profiles.

CRISPR systems are often encoded by many prokaryotes as adaptive defense against mobile genetic elements (MGEs), but several MGEs also recruit CRISPR components to perform additional biological functions. Type IV-A systems are identified in Klebsiella plasmids, yet the distribution, characterization, and role of these plasmids carrying CRISPR systems in the whole Klebsiella genus remain unclear. Here, we performed large-scale comparative analysis of these plasmids using publicly available plasmid genomes. CRISPR-harboring plasmids were mainly distributed in Klebsiella pneumoniae (9.09%), covering 19.23% of sequence types, but sparse in Klebsiella species outside Klebsiella pneumoniae (3.92%). Plasmid genome comparison reiterated that these plasmids often carried the cointegrates of IncFIB and IncHI1B replicons, occasionally linked to other replicons, such as IncFIA, IncFII, IncR, IncQ, and IncU. Comparative genome analysis showed that CRISPR-carrying Klebsiella plasmids shared a conserved pNDM-MAR-like conjugation module as their backbones and served as an important vector for the accretion of antibiotic resistance genes (ARGs) and even virulence genes (VGs). Moreover, compared with CRISPR-negative IncFIB/IncHIB plasmids, CRISPR-positive IncFIB/IncHIB plasmids displayed high divergences in terms of ARGs, VGs, GC content, plasmid length, and backbone structures, suggesting their divergent evolutionary paths. The network analysis revealed that CRISPR-positive plasmids yielded fierce competitions with other plasmid types, especially conjugative plasmids, thereby affecting the dynamics of plasmid transmission. Overall, our study provides valuable insights into the role of CRISPR-positive plasmids in the spread of ARGs and VGs in Klebsiella genus.

Molecular Characteristics of an NDM-4 and OXA-181 Co-Producing K51-ST16 Carbapenem-Resistant Klebsiella pneumoniae: Study of Its Potential Dissemination Mediated by Conjugative Plasmids and Insertion Sequences.

The carbapenem-resistant Klebsiella pneumoniae (CRKP) strain GX34 was recovered from the respiratory tract of an elderly male with severe pneumonia, and only susceptible to amikacin, tigecycline, and colistin. Complete genome suggested that it belonged to K51-ST16 and harbored plasmid-encoded NDM-4 and OXA-181, located on IncFIB plasmid GX34p1_NDM-4 and ColKP3/IncX3 plasmid GX34p4_OXA-181, respectively. A series of transconjugants generated in the plasmid conjugation assays, including Escherichia coli J53-N1 (harboring a self-transmissible and blaNDM-1-producing plasmid Eco-N-1-p), J53-N2 (harboring a blaNDM-4-producing plasmid and a helper plasmid GX34p5), and J53-O (harboring a blaOXA-181-producing plasmid), could be stably inherited after 10 days of serial passage and no significant biological fitness costs were detected. Furthermore, we first reported the blaNDM-1 gene, derived from blaNDM-4 mutation (460C>A) under meropenem pressure, could be in vitro transferred into a self-conjugative, recombined plasmid Eco-N-1-p of J53-N1. Eco-N-1-p was mainly recombined by GX34p4_OXA-181 (40,449 bp, 75.16%) and GX34p1_NDM-4 (8,553 bp, 15.89%), in which IS26 and IS5-like probably played a major role. Eco-N-1-p could be transferred into the conjugation recipient K. pneumoniae KP54 and make the latter sacrifice fitness. The retention rates of blaNDM-1 remained high stability (>80% after 200 generations). The comparative genomic analysis of GX34 and those carrying blaNDM-4 or blaOXA-181 genes retrieved from the NCBI RefSeq database showed all blaNDM-4 (26/26, 100.00%) and blaOXA-181 (13/13, 100.00%) were surrounded by IS26. The immediate environment of blaNDM-4 and blaOXA-181 in GX34 and some retrieved strains shared identical features, hinting at their possible dissemination. Effective measures should be taken to monitor the spread of this clone.

TAM mediates adaptation of carbapenem-resistant Klebsiella pneumoniae to antimicrobial stress during host colonization and infection.

Gram-negative pathogens, such as Klebsiella pneumoniae, remodel their outer membrane (OM) in response to stress to maintain its integrity as an effective barrier and thus to promote their survival in the host. The emergence of carbapenem-resistant K. pneumoniae (CR-Kp) strains that are resistant to virtually all antibiotics is an increasing clinical problem and OM impermeability has limited development of antimicrobial agents because higher molecular weight antibiotics cannot access sites of activity. Here, we demonstrate that TAM (translocation and assembly module) deletion increases CR-Kp OM permeability under stress conditions and enhances sensitivity to high-molecular weight antimicrobials. SILAC-based proteomic analyses revealed mis-localization of membrane proteins in the TAM deficient strain. Stress-induced sensitization enhances clearance of TAM-deficient CR-Kp from the gut lumen following fecal microbiota transplantation and from infection sites following pulmonary or systemic infection. Our study suggests that TAM, as a regulator of OM permeability, represents a potential target for development of agents that enhance the effectiveness of existing antibiotics.

Reduced virulence in tigecycline-resistant Klebsiella pneumoniae caused by overexpression of ompR and down-regulation of ompK35.

BACKGROUND: The development of tigecycline resistance in hypervirulent Klebsiella pneumoniae strains has resulted in decreased virulence that is associated with reduced production of capsular polysaccharides (CPS). In this study, we investigated the mechanisms that link tigecycline susceptibility to decreased virulence. METHODS: We compared transcriptomes from tigecycline-susceptible wild-type strains and tigecycline-resistant mutants using mRNA sequencing. ompR-overexpressed and ompR-deleted mutants were constructed from wild-type strains and tigecycline-resistant mutants, respectively. Antibiotic susceptibility tests were performed, and string tests and precipitation assays were conducted to identify phenotypic changes related to tigecycline susceptibility and ompR expression. Bacterial virulence was assessed by serum resistance and Galleria mellonella infection assays. RESULTS: Transcriptomic analyses demonstrated a significant decrease in the expression of ompK35 in the tigecycline-resistant mutants. We observed that tigecycline-resistant mutants overexpressed ompR, and that the expression of ompK35 was regulated negatively by ompR. While tigecycline-resistant mutants and ompR-overexpressed mutants exhibited reduced hypermucoviscosity and virulence, deletion of ompR from tigecycline-resistant mutants restored their hypermucoviscosity and virulence. CONCLUSIONS: In hypervirulent K. pneumoniae strains, ompR expression, which is regulated by exposure to tigecycline, may affect the production of CPS, leading to bacterial virulence.

Magnitude and Molecular Characterization of Extended-Spectrum ß-Lactamase Genes among Klebsiella pneumoniae Isolates in a Large Tertiary Hospital in Ethiopia.

Background Extended-spectrum ß-lactamases (ESBLs)-producing Klebsiella pneumoniae is reported worldwide increasingly. However, studies on ESBLs are still scarce in Ethiopia. Therefore, the current study aimed to determine the magnitude and resistance patterns of ESBL-producing K. pneumoniae as well as the frequency of ESBL-encoding genes.Methods A cross-sectional study was conducted from September 2018 to February 2019 at Tikur Anbessa Specialized Hospital, Addis Ababa, Ethiopia among a total of 132 non-duplicate K. pneumoniae isolates. Phenotypic detection of ESBL production was done using Combined Disc Test. ESBL-encoding genes of blaCTX-M, blaTEM, and blaSHV were detected through multiplex PCR.Results The magnitude of ESBL production was 102/132 (77.3%). ESBL positive isolates were 100% resistant to ceftriaxone, cefotaxime, and cefuroxime. Co-resistance of ESBL-positive isolates to other non ß-lactam antimicrobials was high to trimethoprim-sulfamethoxazole (96.1%) followed by tetracycline (75.5%) and gentamicin (73.5%). However, these isolates showed high susceptibility to amikacin (96.1%) and meropenem (89.2%). From the total ESBL-positive isolates, 82.6%, 73.5%, and 75% carried blaCTX-M, blaTEM, and blaSHV genes, respectively. The majority 78/102 (76.5%) of ESBL-positive isolates harbored all three types of ESBL genes simultaneously.Conclusions The magnitude of ESBL-producing K. pneumoniae isolates was very alarming in the study area. The co-occurrence of blaCTX-M, blaTEM, and blaSHV genes is high, demanding large-scale studies to evaluate the presence of antimicrobial resistance super-clones. ESBL-producing isolates showed high resistance to most of the antimicrobials, needing phenotypic detection of ESBL regularly for better management of patients.

Integrated chromosomal and plasmid sequence analyses reveal diverse modes of carbapenemase gene spread among Klebsiella pneumoniae.

Molecular and genomic surveillance systems for bacterial pathogens currently rely on tracking clonally evolving lineages. By contrast, plasmids are usually excluded or analyzed with low-resolution techniques, despite being the primary vectors of antibiotic resistance genes across many key pathogens. Here, we used a combination of long- and short-read sequence data of Klebsiella pneumoniae isolates (n = 1,717) from a European survey to perform an integrated, continent-wide study of chromosomal and plasmid diversity. This revealed three contrasting modes of dissemination used by carbapenemase genes, which confer resistance to last-line carbapenems. First, blaOXA-48-like genes have spread primarily via the single epidemic pOXA-48-like plasmid, which emerged recently in clinical settings and spread rapidly to numerous lineages. Second, blaVIM and blaNDM genes have spread via transient associations of many diverse plasmids with numerous lineages. Third, blaKPC genes have transmitted predominantly by stable association with one successful clonal lineage (ST258/512) yet have been mobilized among diverse plasmids within this lineage. We show that these plasmids, which include pKpQIL-like and IncX3 plasmids, have a long association (and are coevolving) with the lineage, although frequent recombination and rearrangement events between them have led to a complex array of mosaic plasmids carrying blaKPC Taken altogether, these results reveal the diverse trajectories of antibiotic resistance genes in clinical settings, summarized as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage. Our study provides a framework for the much needed incorporation of plasmid data into genomic surveillance systems, an essential step toward a more comprehensive understanding of resistance spread.

Klebsiella oxytoca enterotoxins tilimycin and tilivalline have distinct host DNA-damaging and microtubule-stabilizing activities.

Establishing causal links between bacterial metabolites and human intestinal disease is a significant challenge. This study reveals the molecular basis of antibiotic-associated hemorrhagic colitis (AAHC) caused by intestinal resident Klebsiella oxytoca Colitogenic strains produce the nonribosomal peptides tilivalline and tilimycin. Here, we verify that these enterotoxins are present in the human intestine during active colitis and determine their concentrations in a murine disease model. Although both toxins share a pyrrolobenzodiazepine structure, they have distinct molecular targets. Tilimycin acts as a genotoxin. Its interaction with DNA activates damage repair mechanisms in cultured cells and causes DNA strand breakage and an increased lesion burden in cecal enterocytes of colonized mice. In contrast, tilivalline binds tubulin and stabilizes microtubules leading to mitotic arrest. To our knowledge, this activity is unique for microbiota-derived metabolites of the human intestine. The capacity of both toxins to induce apoptosis in intestinal epithelial cells-a hallmark feature of AAHC-by independent modes of action, strengthens our proposal that these metabolites act collectively in the pathogenicity of colitis.

Molecular genetic characteristics of resistome and virulome of carbapenem-resistant Klebsiella pneumoniae clinical strains.

The characteristics of resistome and virulome structure of four carbapenem-resistant Klebsiella pneumoniae clinical strains are present in the work. Two strains belonged to the sequence-type ST395, one strain - ST2262, one strain - to the new sequence-type 5816. The genes of fimbriae, enterobactin, beta-lactamase SHV type, resistance to fosfomycin fosA and transport of fluoroquinolones oqxAB in all Klebsiella strains chromosome structure were identified. The determinants of yersineobactin and aerobactin are enriched the virulome of ST395 NNKP315 and NNKP343 strains. The aerobactin genes are located on IncHI1B plasmids (IncHI1B/FIB) which highly homologous to the virulence pLVPK and pK2044 plasmids. IncR, IncL, IncQ plasmids carrying blaOXA-48, blaCTX-M-15, blaOXA-1, blaTEM-1, qnrS1, tetA, sul1, dfrA1, aac(6 ')-Ib-cr, catA1, catB3 etc. were identified in these strains. As a result of in silico analysis, an assumption about the localization of the blaOXA-48 in the structure of the IncHI1B plasmid of NNKP315 strain was made. This plasmid also contains the aminoglycosidases genes inserted into a class 1 integron In822. The mutations were found in the porin proteins OmpK35, OmpK36 and OmpK37 genes, which increases the carbapenem resistance. The virulome of NNKP16 (ST2262) strain additionally includes of the iron utilization system kfuABC chromosomal genes, and the virulome of NNKP15 (ST5816) strain contains of the capsular polysaccharide kvgAS and microcin E492 genes. Additional determinants of resistance were not identified in the resistome structure of K. pneumoniae NNKP16 and only the blaCTX-M-15 gene was found in the NNKP15 strain. The absence of acquired resistance genes seems to be due to the presence of the type I-E CRISPR-Cas system. Multiple drug resistance of the studied strains is associated with mutations identified in the gene structure of porin proteins OmpK36 and OmpK37, as well as the activity of efflux systems. It was showed the stop codon formation in the nucleotide sequence of the regulatory gene ramR to both strains, which can potentially provide overexpression of AcrAB efflux proteins.

Host adaptation and convergent evolution increases antibiotic resistance without loss of virulence in a major human pathogen.

As human population density and antibiotic exposure increase, specialised bacterial subtypes have begun to emerge. Arising among species that are common commensals and infrequent pathogens, antibiotic-resistant 'high-risk clones' have evolved to better survive in the modern human. Here, we show that the major matrix porin (OmpK35) of Klebsiella pneumoniae is not required in the mammalian host for colonisation, pathogenesis, nor for antibiotic resistance, and that it is commonly absent in pathogenic isolates. This is found in association with, but apparently independent of, a highly specific change in the co-regulated partner porin, the osmoporin (OmpK36), which provides enhanced antibiotic resistance without significant loss of fitness in the mammalian host. These features are common in well-described 'high-risk clones' of K. pneumoniae, as well as in unrelated members of this species and similar adaptations are found in other members of the Enterobacteriaceae that share this lifestyle. Available sequence data indicate evolutionary convergence, with implications for the spread of lethal antibiotic-resistant pathogens in humans.

Prophylaxis and Treatment against Klebsiella pneumoniae: Current Insights on This Emerging Anti-Microbial Resistant Global Threat.

Klebsiella pneumoniae (Kp) is an opportunistic pathogen and the leading cause of healthcare-associated infections, mostly affecting subjects with compromised immune systems or suffering from concurrent bacterial infections. However, the dramatic increase in hypervirulent strains and the emergence of new multidrug-resistant clones resulted in Kp occurrence among previously healthy people and in increased morbidity and mortality, including neonatal sepsis and death across low- and middle-income countries. As a consequence, carbapenem-resistant and extended spectrum ß-lactamase-producing Kp have been prioritized as a critical anti-microbial resistance threat by the World Health Organization and this has renewed the interest of the scientific community in developing a vaccine as well as treatments alternative to the now ineffective antibiotics. Capsule polysaccharide is the most important virulence factor of Kp and plays major roles in the pathogenesis but its high variability (more than 100 different types have been reported) makes the identification of a universal treatment or prevention strategy very challenging. However, less variable virulence factors such as the O-Antigen, outer membrane proteins as fimbriae and siderophores might also be key players in the fight against Kp infections. Here, we review elements of the current status of the epidemiology and the molecular pathogenesis of Kp and explore specific bacterial antigens as potential targets for both prophylactic and therapeutic solutions.

Regulation of Pulmonary Bacterial Immunity by Follistatin-Like Protein 1.

Klebsiella pneumoniae is a common cause of antibiotic-resistant pneumonia. Follistatin-like protein 1 (FSTL-1) is highly expressed in the lung and is critical for lung homeostasis. The role of FSTL-1 in immunity to bacterial pneumonia is unknown. Wild-type (WT) and FSTL-1 hypomorphic (Hypo) mice were infected with Klebsiella pneumoniae to determine infectious burden, immune cell abundance, and cytokine production. FSTL-1 Hypo/TCRδ-/- and FSTL-1 Hypo/IL17ra-/- were also generated to assess the role of γδT17 cells in this model. FSTL-1 Hypo mice had reduced K. pneumoniae lung burden compared with that of WT controls. FSTL-1 Hypo mice had increased Il17a/interleukin-17A (IL-17A) and IL-17-dependent cytokine expression. FSTL-1 Hypo lungs also had increased IL-17A+ and TCRγδ+ cells. FSTL-1 Hypo/TCRδ-/- displayed a lung burden similar to that of FSTL-1 Hypo and reduced lung burden compared with the TCRδ-/- controls. However, FSTL-1 Hypo/TCRδ-/- mice had greater bacterial dissemination than FSTL-1 Hypo mice, suggesting that gamma delta T (γδT) cells are dispensable for FSTL-1 Hypo control of pulmonary infection but are required for dissemination control. Confusing these observations, FSTL-1 Hypo/TCRδ-/- lungs had an increased percentage of IL-17A-producing cells compared with that of TCRδ-/- mice. Removal of IL-17A signaling in the FSTL-1 Hypo mouse resulted in an increased lung burden. These findings identify a novel role for FSTL-1 in innate lung immunity to bacterial infection, suggesting that FSTL-1 influences type-17 pulmonary bacterial immunity.

Emergence of an NDM-5-Producing Hypervirulent Klebsiella pneumoniae Sequence Type 35 Strain with Chromosomal Integration of an Integrative and Conjugative Element, ICEKp1.

Here, we report an NDM-5-producing sequence type 35 (ST35) hypervirulent Klebsiella pneumoniae strain, isolated from the blood of a male patient. It showed a remarkable resistance to serum killing and neutrophil phagocytosis and high virulence in a mouse peritonitis infection model. Instead of carrying a pLVPK-like virulence plasmid, chromosomal integration of ICEKp1 (∼76 kb) was identified in a specific asparagine-tRNA gene, harboring the iron acquisition system salmochelin genes (iroBCDN), a yersiniabactin gene, and a variant of the rmpA gene.

Acyloxyacyl hydrolase promotes the resolution of lipopolysaccharide-induced acute lung injury.

Pulmonary infection is the most common risk factor for acute lung injury (ALI). Innate immune responses induced by Microbe-Associated Molecular Pattern (MAMP) molecules are essential for lung defense but can lead to tissue injury. Little is known about how MAMP molecules are degraded in the lung or how MAMP degradation/inactivation helps prevent or ameliorate the harmful inflammation that produces ALI. Acyloxyacyl hydrolase (AOAH) is a host lipase that inactivates Gram-negative bacterial endotoxin (lipopolysaccharide, or LPS). We report here that alveolar macrophages increase AOAH expression upon exposure to LPS and that Aoah+/+ mice recover more rapidly than do Aoah-/- mice from ALI induced by nasally instilled LPS or Klebsiella pneumoniae. Aoah-/- mouse lungs had more prolonged leukocyte infiltration, greater pro- and anti-inflammatory cytokine expression, and longer-lasting alveolar barrier damage. We also describe evidence that the persistently bioactive LPS in Aoah-/- alveoli can stimulate alveolar macrophages directly and epithelial cells indirectly to produce chemoattractants that recruit neutrophils to the lung and may prevent their clearance. Distinct from the prolonged tolerance observed in LPS-exposed Aoah-/- peritoneal macrophages, alveolar macrophages that lacked AOAH maintained or increased their responses to bioactive LPS and sustained inflammation. Inactivation of LPS by AOAH is a previously unappreciated mechanism for promoting resolution of pulmonary inflammation/injury induced by Gram-negative bacterial infection.

Nbeal2 Deficiency Increases Organ Damage but Does Not Affect Host Defense During Gram-Negative Pneumonia-Derived Sepsis.

Objective- Nbeal2-/- mice, a model of human gray platelet syndrome, have reduced neutrophil granularity and impaired host defense against systemic Staphylococcus aureus infection. We here aimed to study the role of Nbeal2 deficiency in both leukocytes and platelets during gram-negative pneumonia and sepsis. Approach and Results- We studied the role of Nbeal2 in platelets and leukocytes during murine pneumonia and sepsis by Klebsiella pneumoniae. Apart from platelet α-granule deficiency and reduced neutrophil granularity, also monocyte granularity was reduced in Nbeal2-/- mice, whereas plasma levels of MPO (myeloperoxidase), elastase, NGAL (neutrophil gelatinase-associated lipocalin), and MMP-9 (matrix metalloproteinase 9), and leukocyte CD11b expression were increased. Nbeal2-/- leukocytes showed unaltered in vitro antibacterial response and phagocytosis capacity against Klebsiella, and unchanged reactive nitrogen species and cytokine production. Also during Klebsiella pneumonia and sepsis, Nbeal2-/- mice had similar bacterial growth in lung and distant body sites, with enhanced leukocyte migration to the bronchoalveolar space. Despite similar infection-induced inflammation, organ damage was increased in Nbeal2-/- mice, which was also seen during endotoxemia. Platelet-specific Nbeal2 deficiency did not influence leukocyte functions, indicating that Nbeal2 directly modifies leukocytes. Transfusion of Nbeal2-/- but not of Nbeal2+/+ platelets into thrombocytopenic mice was associated with bleeding in the lung but similar host defense, pointing at a role for platelet α-granules in maintaining vascular integrity but not host defense during Klebsiella pneumosepsis. Conclusions- These data show that Nbeal2 deficiency-resulting in gray platelet syndrome-affects platelets, neutrophils, and monocytes, with intact host defense but increased organ damage during gram-negative pneumosepsis.