Klebsiella pneumoniae outer membrane vesicles induce strong IL-8 expression via NF-κB activation in normal pulmonary bronchial cells.

BACKGROUND: Outer membrane vesicles (OMVs) derived from bacteria are known to play a crucial role in the interactions between bacteria and their environment, as well as bacteria-bacteria and bacteria-host interactions.Specifically, OMVs derived from Klebsiella pneumoniae have been implicated in contributing to the pathogenesis of this bacterium.Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a global pathogen of great concern due to its heightened virulence compared to classical K. pneumoniae (cKp), and its ability to cause community-acquired infections, even in healthy individuals.The objective of this study was to investigate potential differences between hvKp-derived OMVs and cKp-derived OMVs in their interactions with microorganisms and host cells. METHODS: Four strains of K. pneumoniae were used to produce OMVs: hvKp strain NTUH-K2044 (K1, ST23), hvKp clinical strain AP8555, and two cKP clinical strains C19 and C250. To examine the morphology and size of the bacterial OMVs, transmission electron microscopy (TEM) was utilized. Additionally, dynamic light scattering (DLS) was used to analyze the size characterization of the OMVs.The normal pulmonary bronchial cell line HBE was exposed to OMVs derived from hvKp and cKP. Interleukin 8 (IL-8) messenger RNA (mRNA) expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR), while IL-8 secretion was analyzed using enzyme-linked immunosorbent assay (ELISA).Furthermore, the activation of nuclear factor kappa B (NF-κB) was evaluated using both Western blotting and confocal microscopy. RESULTS: After purification, OMVs appeared as electron-dense particles with a uniform spherical morphology when observed through TEM.DLS analysis indicated that hvKp-derived OMVs from K2044 and AP8555 measured an average size of 116.87 ± 4.95 nm and 96.23 ± 2.16 nm, respectively, while cKP-derived OMVs from C19 and C250 measured an average size of 297.67 ± 26.3 nm and 325 ± 6.06 nm, respectively. The average diameter of hvKp-derived OMVs was smaller than that of cKP-derived OMVs.A total vesicular protein amount of 47.35 mg, 41.90 mg, 16.44 mg, and 12.65 mg was generated by hvKp-K2044, hvKp-AP8555, cKP-C19, and cKP-C250, respectively, obtained from 750 mL of culture supernatant. Both hvKp-derived OMVs and cKP-derived OMVs induced similar expression levels of IL-8 mRNA and protein. However, IL-8 expression was reduced when cells were exposed to BAY11-7028, an inhibitor of the NF-κB pathway.Western blotting and confocal microscopy revealed increased phosphorylation of p65 in cells exposed to OMVs. CONCLUSIONS: Klebsiella pneumoniae produces outer membrane vesicles (OMVs) that play a key role in microorganism-host interactions. HvKp, a hypervirulent strain of K. pneumoniae, generates more OMVs than cKP.The average size of OMVs derived from hvKp is smaller than that of cKP-derived OMVs.Despite these differences, both hvKp-derived and cKP-derived OMVs induce a similar level of expression of IL-8 mRNA and protein.OMVs secreted by K. pneumoniae stimulate the secretion of interleukin 8 by activating the nuclear factor NF-κB.

High Prevalence of Klebsiella pneumoniae in European Food Products: a Multicentric Study Comparing Culture and Molecular Detection Methods.

The Klebsiella pneumoniae species complex (KpSC) is a leading cause of multidrug-resistant human infections. To better understand the potential contribution of food as a vehicle of KpSC, we conducted a multicentric study to define an optimal culture method for its recovery from food matrices and to characterize food isolates phenotypically and genotypically. Chicken meat (n = 160) and salad (n = 145) samples were collected in five European countries and screened for the presence of KpSC using culture-based and zur-khe intergenic region (ZKIR) quantitative PCR (qPCR) methods. Enrichment using buffered peptone water followed by streaking on Simmons citrate agar with inositol (44°C for 48 h) was defined as the most suitable selective culture method for KpSC recovery. A high prevalence of KpSC was found in chicken meat (60% and 52% by ZKIR qPCR and the culture approach, respectively) and salad (30% and 21%, respectively) samples. Genomic analyses revealed high genetic diversity with the dominance of phylogroups Kp1 (91%) and Kp3 (6%). A total of 82% of isolates presented a natural antimicrobial susceptibility phenotype and genotype, with only four CTX-M-15-producing isolates detected. Notably, identical genotypes were found across samples-same food type and same country (15 cases), different food types and same country (1), and same food type and two countries (1)-suggesting high rates of transmission of KpSC within the food sector. Our study provides a novel isolation strategy for KpSC from food matrices and reinforces the view of food as a potential source of KpSC colonization in humans. IMPORTANCE Bacteria of the Klebsiella pneumoniae species complex (KpSC) are ubiquitous, and K. pneumoniae is a leading cause of antibiotic-resistant infections in humans. Despite the urgent public health threat represented by K. pneumoniae, there is a lack of knowledge of the contribution of food sources to colonization and subsequent infection in humans. This is partly due to the absence of standardized methods for characterizing the presence of KpSC in food matrices. Our multicentric study provides and implements a novel isolation strategy for KpSC from food matrices and shows that KpSC members are highly prevalent in salads and chicken meat, reinforcing the view of food as a potential source of KpSC colonization in humans. Despite the large genetic diversity and the low levels of resistance detected, the occurrence of identical genotypes across samples suggests high rates of transmission of KpSC within the food sector, which need to be further explored to define possible control strategies.

Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147.

The rapid emergence of multidrug-resistant Klebsiella pneumoniae is being driven largely by the spread of specific clonal groups (CGs). Of these, CG147 includes 7-gene multilocus sequence typing (MLST) sequence types (STs) ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Antimicrobial-susceptibility testing following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and genome sequencing (Illumina and Oxford Nanopore Technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes [National Center for Biotechnology Information (NCBI), May 2019]. CG147 evolution was inferred within a temporal phylogenetic framework (beast) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, phaster, ICEfinder and CRISPRCasFinder). Strain DJ had a pandrug-resistance phenotype. Its genome comprised the chromosome, seven plasmids and one linear phage-plasmid. Four carbapenemase genes were detected: blaNDM-5 and two copies of blaOXA-181 in the chromosome, and a second copy of blaNDM-5 on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63 % carried a carbapenemase gene and 83 % harboured blaCTX-M. All CG147 genomes presented GyrA and ParC mutations and a common subtype I-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74 %, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20 %, emerged in 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Furthermore, subclades within ST147-KL64 differed at the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system. K. pneumoniae CG147 comprises pandrug-resistant or extensively resistant isolates, and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal blaNDM-5. Its emergence is being driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo-spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.

regentrans: a framework and R package for using genomics to study regional pathogen transmission.

Increasing evidence of regional pathogen transmission networks highlights the importance of investigating the dissemination of multidrug-resistant organisms (MDROs) across a region to identify where transmission is occurring and how pathogens move across regions. We developed a framework for investigating MDRO regional transmission dynamics using whole-genome sequencing data and created regentrans, an easy-to-use, open source R package that implements these methods (https://github.com/Snitkin-Lab-Umich/regentrans). Using a dataset of over 400 carbapenem-resistant isolates of Klebsiella pneumoniae collected from patients in 21 long-term acute care hospitals over a one-year period, we demonstrate how to use our framework to gain insights into differences in inter- and intra-facility transmission across different facilities and over time. This framework and corresponding R package will allow investigators to better understand the origins and transmission patterns of MDROs, which is the first step in understanding how to stop transmission at the regional level.

Context-aware genomic surveillance reveals hidden transmission of a carbapenemase-producing Klebsiella pneumoniae.

Genomic surveillance can inform effective public health responses to pathogen outbreaks. However, integration of non-local data is rarely done. We investigate two large hospital outbreaks of a carbapenemase-carrying Klebsiella pneumoniae strain in Germany and show the value of contextual data. By screening about 10 000 genomes, over 400 000 metagenomes and two culture collections using in silico and in vitro methods, we identify a total of 415 closely related genomes reported in 28 studies. We identify the relationship between the two outbreaks through time-dated phylogeny, including their respective origin. One of the outbreaks presents extensive hidden transmission, with descendant isolates only identified in other studies. We then leverage the genome collection from this meta-analysis to identify genes under positive selection. We thereby identify an inner membrane transporter (ynjC) with a putative role in colistin resistance. Contextual data from other sources can thus enhance local genomic surveillance at multiple levels and should be integrated by default when available.

Comparison of Four Carbapenemase Detection Methods for blaKPC-2 Variants.

Recently, various blaKPC-2 variants resistant to ceftazidime-avibactam have begun to emerge in clinical settings, but it is unclear which testing method is most appropriate for detecting these variants. Strains were subjected to antimicrobial susceptibility testing using the broth microdilution method. Four carbapenemase detection methods, modified carbapenem inactivation method (mCIM) and EDTA carbapenem inactivation method (eCIM), APB/EDTA (carbapenemase inhibitor APB [3-aminophenylboronic acid] and EDTA enhancement method), NG-test Carba 5, and GeneXpert Carba-R were used to try to detect KPC-2 variants in 19 Klebsiella pneumoniae isolates. Among those blaKPC-2 variants, blaKPC-33-, blaKPC-35-, blaKPC-71-, blaKPC-76-, blaKPC-78-, and blaKPC-79-positive isolates accounted for 26.3% (5/19), 15.8% (3/19), 5.3% (1/19), % 42.1% (8/19), 5.3% (1/19), and 5.3% (1/19), respectively. All 19 K. pneumoniae carrying blaKPC-2 variants showed resistance to ceftazidime-avibactam (MICs:16 to >64 mg/L), and 14 strains were susceptible to imipenem (MICs: 0.25 to 1 mg/L). None of the blaKPC-2 variants could be detected using either the mCIM or the APB/EDTA method, while five strains carrying blaKPC-2 variants (blaKPC-35, blaKPC-78, and blaKPC-79) tested KPC positive when using NG-test Carba 5. However, GeneXpert Carba-R was able to detect blaKPC-2 variants (harboring blaKPC-33, blaKPC-35, blaKPC-71, blaKPC-76, blaKPC-78, and blaKPC-79) carried by all 19 K. pneumoniae. The emergence of new KPC variants poses an increased challenge for carbapenemase detection methods, and laboratories should use the appropriate assays to accurately detect these variants. IMPORTANCE Carbapenemase detection is essential for the appropriate treatment of CRE infections. Several clinical laboratories have begun using relevant carbapenemase assays such as mCIM and eCIM, the APB/EDTA method, NG-test Carba 5, and GeneXpert Carba-R to detect carbapenemases. Nevertheless, some of these methods may have limitations for detecting blaKPC-2 variants. Additionally, there has been little relevant research on evaluate the differences between these standard methods for detecting blaKPC-2 variants. Therefore, we investigated the reliability of these classic methods for assessing 19 K. pneumoniae with blaKPC-2 variants. Our results showed that none of the blaKPC-2 variants could be detected using either the mCIM or APB/EDTA method, while five strains (harboring blaKPC-35, blaKPC-78,and blaKPC-79) tested KPC positive when using NG-test Carba 5. GeneXpert Carba-R could detect six blaKPC-2 variants carried by all 19 K. pneumoniae. This study may be valuable for clinical laboratories in their efforts to test for various blaKPC-2 variants.

Novel strains of Klebsiella africana and Klebsiella pneumoniae in Australian fruit bats (Pteropus poliocephalus).

Over the past decade human associated multidrug resistant (MDR) and hypervirulent Klebsiella pneumoniae lineages have been increasingly detected in wildlife. This study investigated the occurrence of K. pneumoniae species complex (KpSC) in grey-headed flying foxes (GHFF), an Australian fruit bat. Thirty-nine KpSC isolates were cultured from 275 GHFF faecal samples (14.2%), comprising K. pneumoniae (n = 30), Klebsiella africana (n = 8) and Klebsiella variicola subsp. variicola (n = 1). The majority (79.5%) of isolates belonged to novel sequence types (ST), including two novel K. africana STs. This is the first report of K. africana outside of Africa and in a non-human host. A minority (15.4%) of GHFF KpSC isolates shared STs with human clinical K. pneumoniae strains, of which, none belonged to MDR clonal lineages that cause frequent nosocomial outbreaks, and no isolates were characterised as hypervirulent. The occurrence of KpSC isolates carrying acquired antimicrobial resistance genes in GHFF was low (1.1%), with three K. pneumoniae isolates harbouring both fluoroquinolone and trimethoprim resistance genes. This study indicates that GHFF are not reservoirs for MDR and hypervirulent KpSC strains, but they do carry novel K. africana lineages. Health risks associated with KpSC carriage by GHFF are deemed low for the public and GHFF.

A LAMP-based system for rapid detection of eight common pathogens causing lower respiratory tract infections.

Lower respiratory tract infections (LRTIs) are a leading cause of morbidity and mortality worldwide and lack a rapid diagnostic method. To improve the diagnosis of LRTIs, we established an available loop-mediated isothermal amplification (LAMP) assay for the detection of eight common lower respiratory pathogens, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. The whole process can be achieved within 1 h (sample to results read out). We established an extraction free isothermal system. 528 sputum samples collected from patients suspected to have LRTIs were analyzed by the system (8 tests in each sample, a total of 4224 tests) and compared with the standard culture method (SCM). The samples with inconsistent results were further verified by Sanger sequencing and High-throughput sequencing (NGS). The detection limits of the LAMP assay for the 8 pathogens ranged from 103 to 104 CFU/mL. Upon testing 528 samples, the Kappa coefficients of all pathogens ranged between 0.5 and 0.7 indicated a moderate agreement between the LAMP assay and the SCM. All inconsistent samples were further verified by Sanger sequencing, we found that the developed LAMP assay had a higher consistency level with Sanger sequencing than the SCM for all pathogens. Additionally, when the NGS was set to a diagnostic gold standard, the specificity and sensitivity of the LAMP assay for LRTIs were 94.49% and 75.00%. The present study demonstrated that the developed LAMP has high consistency with the sequencing methods. Meanwhile, the LAMP assay has a higher detection rate compared to the SCM. It may be a powerful tool for rapid and reliable clinical diagnosis of LRTIs in primary hospitals.

Detection of a NDM-5-producing Klebsiella pneumoniae sequence type 340 (CG258) high-risk clone in swine.

The emergence and rapid increase of carbapenem-resistant Enterobacteriaceae among food-producing animals poses a serious threat to public health. The aim of this study was to investigate the presence and dissemination of blaNDM-5 in porcine Klebsiella pneumoniae isolates. Of 19 meropenem-resistant K. pneumoniae isolates, 18 were blaNDM-5-positive and one carried blaNDM-1. Susceptibility testing indicated that all blaNDM-carrying K. pneumoniae showed a multiple drug resistance (MDR) profile. The blaNDM-5 gene was located on a conjugative IncX3 plasmid of ∼46-kb in all 18 blaNDM-5-carrying isolates. MLST analysis revealed that ST340 (n = 8), a member of the worldwide existing high-risk epidemic clonal group 258 (CG258), was predominant. Furthermore, whole genome sequence (WGS) analysis for one representative ST340 K. pneumoniae Kp19110124 showed a MDR profile for a wide range of antimicrobial agents, including meropenem, various cephalosporins, azteonam, gentamicin, ciprofloxacin, and florfenicol. Phylogenetic analysis exhibited that K. pneumoniae ST340 strains were clustered into one branch, which had spread across host species and across continents. Among them, K. pneumoniae Kp19110124 displayed a very close relationship with a clinical isolate collected from a patient in Canada. In conclusion, these results reveal the presence of a blaNDM-5-carrying conjugative IncX3 type plasmid into K. pneumoniae ST340 clone, which then may accelerate the dissemination of the blaNDM-5 gene in porcine K. pneumoniae isolates. The detection of high-risk blaNDM-5-carrying K. pneumoniae ST340 in food-producing animal poses a serious threat to public health.

A novel hypermucoviscous Klebsiella pneumoniae ST3994-K2 clone belonging to Clonal Group 86.

Emergent hypervirulent Klebsiella pneumoniae has been responsible for severe diseases, representing a serious threat to public health. We report the whole-genome sequencing of a novel ST3994-K2 clone, a single locus variant of ST86 K2, which is considered a worrying hypervirulent clone that emerged in several parts of the world. The strain K. pneumonia Kpi144 was isolated in 2013 from a blood culture of a 69-year-old male patient admitted to a tertiary hospital in Teresina, state of Piauí, northeastern Brazil. The strain was susceptible to 41 antibiotics tested, presented hypermucoviscous phenotype and a virulent behavior was observed in the Galleria mellonella infection model. Moreover, the virulome showed several virulence genes. To the best of our knowledge, this is the first worldwide report of a novel ST3994-K2 K. pneumoniae clone, an SLV of ST86 K2, which is considered a worrying virulent clone that has emerged in several parts of the world, including South America and Brazil.

Outer Membrane Vesicles Derived from Klebsiella pneumoniae Are a Driving Force for Horizontal Gene Transfer.

Gram-negative bacteria release Outer Membrane Vesicles (OMVs) into the extracellular environment. Recent studies recognized these vesicles as vectors to horizontal gene transfer; however, the parameters that mediate OMVs transfer within bacterial communities remain unclear. The present study highlights for the first time the transfer of plasmids containing resistance genes via OMVs derived from Klebsiella pneumoniae (K. pneumoniae). This mechanism confers DNA protection, it is plasmid copy number dependent with a ratio of 3.6 times among high copy number plasmid (pGR) versus low copy number plasmid (PRM), and the transformation efficiency was 3.6 times greater. Therefore, the DNA amount in the vesicular lumen and the efficacy of horizontal gene transfer was strictly dependent on the identity of the plasmid. Moreover, the role of K. pneumoniae-OMVs in interspecies transfer was described. The transfer ability was not related to the phylogenetic characteristics between the donor and the recipient species. K. pneumoniae-OMVs transferred plasmid to Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Burkholderia cepacia. These findings address the pivotal role of K. pneumoniae-OMVs as vectors for antimicrobial resistance genes spread, contributing to the development of antibiotic resistance in the microbial communities.

Harmonization of whole-genome sequencing for outbreak surveillance of Enterobacteriaceae and Enterococci.

Whole-genome sequencing (WGS) is becoming the de facto standard for bacterial typing and outbreak surveillance of resistant bacterial pathogens. However, interoperability for WGS of bacterial outbreaks is poorly understood. We hypothesized that harmonization of WGS for outbreak surveillance is achievable through the use of identical protocols for both data generation and data analysis. A set of 30 bacterial isolates, comprising of various species belonging to the Enterobacteriaceae family and Enterococcus genera, were selected and sequenced using the same protocol on the Illumina MiSeq platform in each individual centre. All generated sequencing data were analysed by one centre using BioNumerics (6.7.3) for (i) genotyping origin of replications and antimicrobial resistance genes, (ii) core-genome multi-locus sequence typing (cgMLST) for Escherichia coli and Klebsiella pneumoniae and whole-genome multi-locus sequencing typing (wgMLST) for all species. Additionally, a split k-mer analysis was performed to determine the number of SNPs between samples. A precision of 99.0% and an accuracy of 99.2% was achieved for genotyping. Based on cgMLST, a discrepant allele was called only in 2/27 and 3/15 comparisons between two genomes, for E. coli and K. pneumoniae, respectively. Based on wgMLST, the number of discrepant alleles ranged from 0 to 7 (average 1.6). For SNPs, this ranged from 0 to 11 SNPs (average 3.4). Furthermore, we demonstrate that using different de novo assemblers to analyse the same dataset introduces up to 150 SNPs, which surpasses most thresholds for bacterial outbreaks. This shows the importance of harmonization of data-processing surveillance of bacterial outbreaks. In summary, multi-centre WGS for bacterial surveillance is achievable, but only if protocols are harmonized.

Resistance evolution of hypervirulent carbapenem-resistant Klebsiella pneumoniae ST11 during treatment with tigecycline and polymyxin.

Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has recently aroused increasing attention, especially ST11, the predominant CRKP clone in China. Here, we report a case of hv-CRKP-associated infection and reveal the in-host evolution of its mechanism of resistance to tigecycline and polymyxin under clinical therapy. A total of 11 K. pneumoniae carbapenemase (KPC)-producing CRKP strains were consecutively isolated from a male patient who suffered from continuous and multisite infections. String and antimicrobial susceptibility tests identified seven hypermucoviscous strains and three tigecycline-resistant and four colistin-resistant strains. Galleria mellonella larvae infection model confirmed the hypervirulence. Pulsed-field gel electrophoresis (PFGE) separated three PFGE clusters among all strains, and further Southern blotting detected that blaKPC-2 was located on the same-sized plasmid. Whole-genome sequencing showed that all strains belonged to the hv-CRKP ST11-KL64 clone. Diverse hypervirulence factors and resistance genes were identified. Further sequencing with the Nanopore platform was performed on the CRKP-Urine1 strain, which contained one virulence plasmid (pVi-CRKP-Urine1) and two resistance plasmids (pKPC-CRKP-Urine1 and pqnrS1-CRKP-Urine1). The gene mutations responsible for tigecycline or colistin resistance were then amplified with PCR followed by sequencing, which indicated that mutations of ramR and lon were the potential loci for tigecycline resistance and that the pmrB, phoQ and mgrB genes for colistin resistance. A novel frameshift mutation of lon was identified in the high-level tigecycline-resistant strain (MIC, 128 mg/L). The results indicate that the hypervirulent ST11-KL64 clone is a potential threat to antiinfection treatment and is capable of rapid and diverse evolution of resistance during tigecycline and polymyxin treatment.

Gastrointestinal carriage of Klebsiella pneumoniae in a general adult population: a cross-sectional study of risk factors and bacterial genomic diversity.

Antibiotic resistant Klebsiella pneumoniae is a leading public health threat and gastrointestinal carriage is an established risk factor for subsequent infections during hospitalization. Our study contributes new knowledge of risk factors for gastrointestinal carriage and the genomic population structure of K. pneumoniae colonizing humans in a representative sample of a general population in a community setting. Altogether, 2,975 participants (54% women) >40 y in the population-based Tromsø Study: Tromsø7, Norway (2015-2016) were included. Fecal samples were screened for K. pneumoniae, which were characterized using whole-genome sequencing. Risk factors for carriage were analyzed using multivariable logistic regression on data from questionnaires and the Norwegian Prescription Database. Prevalence of K. pneumoniae gastrointestinal carriage was 16.3% (95% CI 15.0-17.7, no gender difference). Risk factors associated with carriage included age ≥60 y, travel to Greece or Asia past 12 months (adjusted odds ratio 1.49, 95% CI 1.11-2.00), Crohn's disease/ulcerative colitis (2.26, 1.20-4.27), use of proton pump inhibitors (1.62, 1.18-2.22) and non-steroidal anti-inflammatory drugs past 6 months (1.38, 1.04-1.84), and antibiotic use the last month (1.73, 1.05-2.86). Prevalence was higher among those having used combinations of drug classes and decreased over time with respect to preceding antibiotic use. The K. pneumoniae population was diverse with 300 sequence types among 484 isolates distributed across four phylogroups. Only 5.2% of isolates harbored acquired resistance and 11.6% had virulence factors. Identification of risk factors for gastrointestinal carriage allows for identification of individuals that may have higher risk of extraintestinal infection during hospitalization. The findings that specific diseases and drugs used were associated with carriage show an impact of these possibly through modulating the human gut microbiota promoting colonization. The diverse population structure of carriage isolates reflects the ecologically adaptive capacity of the bacterium and challenges for vaccine prospects and the identification of reservoirs as a potential source for human colonization.

Proposal for reunification of the genus Raoultella with the genus Klebsiella and reclassification of Raoultella electrica as Klebsiella electrica comb. nov.

The order Enterobacterales was divided into seven families including the family Enterobacteriaceae in 2016. The genus Klebsiella within the family Enterobacteriaceae was divided into two genera Klebsiella and Raoultella in 2001. Here, our phylogenomic analysis shows that the genus Raoultella is nested within the genus Klebsiella. Klebsiella and Raoultella together are monophyletic and share average amino acid identities (AAIs) of 86.9-89.6% above the AAI threshold (86%) for genus delimitation within the family Enterobacteriaceae. Klebsiella and Raoultella share AAIs of 79.9%-85.0% with the other genera within the subfamily "Klebsiella clade", which are in the range of inter-genus AAIs (74-85%) within the family Enterobacteriaceae. Klebsiella and Raoultella also share six known conserved signature indels. Therefore, we propose to reunify Klebsiella and Raoultella to the single genus Klebsiella and reclassify Raoultella electrica as Klebsiella electrica comb. nov. Our genome-based taxonomic analyses also identified seven potential novel species within the unified genus Klebsiella.

Characterization of multidrug-resistant and virulent Klebsiella pneumoniae strains belonging to the high-risk clonal group 258 (CG258) isolated from inpatients in northeastern Brazil.

Multidrug-resistant (MDR) and hypervirulent Klebsiella pneumoniae (hvKp) clones have become a major threat to global public health. The clonal group 258 (CG258) is considered a high-risk CG and the K. pneumoniae strains belonging to it are often multi-resistant and to spread mainly in the hospital environment. This study aimed to characterize the antimicrobial resistance profile, virulence factors, and the clonal relationships among 13 K. pneumoniae strains belonging to CG258 from patients admitted to a tertiary hospital in Teresina, in the state of Piauí, northeastern Brazil. Ten strains were classified as MDR and three as extensively drug-resistant (XDR). Three different ß-lactamase-encoding genes (blaKPC, blaOXA-1-like, and blaCTX-M-Gp1) and six virulence genes (fimH, ycfM, mrkD, entB, ybtS, and kfu) were detected. Moreover, two hypermucoviscous K. pneumoniae strains and one capsular K-type 2 were found. Multilocus sequence typing analysis revealed ten different sequence types (STs) (ST14, ST17, ST20, ST29, ST45, ST101, ST268, ST1800, ST3995, and ST3996) belonging to CG258, being two (ST3995 and ST3996) described for the first time in this study.

Determination of incompatibility group plasmids and copy number of the bla NDM-1 gene in carbapenem-resistant Klebsiella pneumoniae strains recovered from different hospitals in Kerman, Iran.

Introduction. New Delhi metallo-ß-lactamase (NDM)-producing Klebsiella pneumoniae has become a serious global health concern.Hypothesis/Gap Statement. Due to the high genetic diversity among NDM-positive K. pneumoniae, we need further surveillance and studies to better understand the relationships between them. In addition, the coexistence of several plasmid replicon types in NDM-positive K. pneumoniae may affect the copy number of bla NDM, the MIC level to antibiotics, as well as increasing the chance of horizontal gene transfer.Aim. The aim of this study was to determine incompatible plasmid groups and copy numbers of bla NDM, and to investigate the genetic relationship of 37 NDM-positive K. pneumoniae in Kerman, Iran.Methodology. The bla NDM-1 gene was detected and confirmed by PCR-sequencing. The plasmid replicon types were determined by PCR-based replicon typing (PBRT) and the copy number of bla NDM-1 was determined by quantitaive real time-PCR (qPCR). Random amplified polymorphic DNA (RAPD)-PCR typing was used to detect genetic relationships between the strains.Results. In this study, 10 different replicon types, including Frep [n=25 (67.5 %)], FIIAs [n=11 (29.7 %)], FIA [n=5 (13.5 %)], FIB [n=3 (8.1 %)], I1-Iγ [n=2 (5.4 %)], L/M [n=7 (18.9 %)], A/C [n=7 (18.9 %)], Y [n=3 (8.1 %)], P [n=1 (2.7 %)] and FIC [n=1 (2.7 %)] were reported. The copy numbers of the bla NDM-1 gene varied from 30.00 to 5.0×106 and no statistically significant correlation was observed between a rise of the MIC to imipenem and the copy numbers of bla NDM-1 (P>0.05). According to RAPD typing results, 35 strains were divided into five clusters, while two strains were non-typeable.Conclusion. The spread of NDM-1-producing K. pneumoniae strains that carry several plasmid replicon types increases the chance of horizontal transfer of antibiotic resistance genes in hospital settings. In this study, 10 different replicon types were identified. We could not find any relationship between the increase of MIC levels to imipenem and the copy numbers of bla NDM-1. Therefore, due to the identification of different replicon types in this study, the type and genetic characteristics of bla NDM-1-carrying plasmids, and other factors such as antibiotic selective pressure, probably affect the copy number of bla NDM-1 and change the MIC level to imipenem.

Clonal Dissemination of KPC-2, VIM-1, OXA-48-Producing Klebsiella pneumoniae ST147 in Katowice, Poland.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important bacterium of nosocomial infections. In this study, CRKP strains, which were mainly isolated from fecal samples of 14 patients in three wards of the hospital in the Silesia Voivodship, rapidly increased from February to August 2018. Therefore, we conducted microbiological and molecular studies of the CRKP isolates analyzed. Colonized patients had critical underlying diseases and comorbidities; one developed bloodstream infection, and five died (33.3%). Antibiotic susceptibilities were determined by the E-test method. A disc synergy test confirmed carbapenemase production. CTX-Mplex PCR evaluated the presence of resistance genes bla CTX-M-type, bla CTX-M-1, bla CTX-M-9, and the genes bla SHV, bla TEM, bla KPC-2, bla NDM-1, bla OXA-48, bla IMP, and bla VIM-1 was detected with the PCR method. Clonality was evaluated by Multi Locus Sequence Typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE). Six (40%) strains were of XDR (Extensively Drug-Resistant) phenotype, and nine (60%) of the isolates exhibited MDR (Multidrug-Resistant) phenotype. The range of carbapenem minimal inhibitory concentrations (MICs, µg/mL) was as follows doripenem (16 to >32), ertapenem (> 32), imipenem (4 to > 32), and meropenem (> 32). PCR and sequencing confirmed the bla CTX-M-15, bla KPC-2, bla OXA-48, and bla VIM-1 genes in all strains. The isolates formed one large PFGE cluster (clone A). MLST assigned them to the emerging high-risk clone of ST147 (CC147) pandemic lineage harboring the bla OXA-48 gene. This study showed that the K. pneumoniae isolates detected in the multi-profile medical centre in Katowice represented a single strain of the microorganism spreading in the hospital environment.

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.