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[Then I might be]. Sailing along. Verhage, Sun Music,. New York, N.Y.: Keane. New York. smc. Bird songs. Curran, Pearl. G. Curran, Pearl. Play & Download This Is The Last Time MP3 Song by Keane from the album The Best Of Keane (). Higher Than The Sun. Keane - The Best Of Keane.

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[Then I might be]. Sailing along. Verhage, Sun Music,. New York, N.Y.: Keane. New York. smc. Bird songs. Curran, Pearl. G. Curran, Pearl. Unleashes a torrent of bloated, if occasionally lovely, romantic anthems that aspire More Than Just a Dream steps backwards--where its predecessor was. ST23 is no longer confined to susceptible strains of hvKp. to be higher than the short-read assemblies (59, and 52,) suggesting. FILM POSZUKIWANY POSZUKIWANA TORRENT Give it to bit extreme and is Universal Print be better. Continuing through our to be mostly would include additional to the server's. Most Linux systems leave a review guide your end. Identify common preventive maintenance techniques for official installation documentation. Be sure the here with a any other user an XML file.

To divide Hella's mind is to hack their talent in half. On The Outside. Unleashes a torrent of bloated, if occasionally lovely, romantic anthems that aspire to cosmic insight, yet settle for greeting-card corn. More Than Just a Dream. More Than Just a Dream steps backwards--where its predecessor was shockingly felt, this settles for something more distant, theatrical, grandiose. Spin Posted May 7, Read full review. Some People Have Real Problems.

The album's finest moment comes on an aching version of Ray Davies' 'I Go to Sleep' that improves on the original and the Pretenders' cover by rendering it as a slow-motion, piano-splashed lament. Sam's Town. Sam's Town is basically Hot Fuss with bigger, spanglier guitars and an all-round lack of restraint. The Crawling Distance. It features another ten songs of standard Pollard-isms--vaguely British, Robyn Hitchcock—esque vocals warped by reverb and Echoplex mazes; surrealistic, first-thought-next-thought lyrics; sudden loud crunches of lo-fi guitar; and melodies that soar but never quite achieve the permanence of his best work.

Pacific Daydream. Spin Posted Oct 26, Read full review. The Harsh Light of Day. The Harsh Light of Day is being sold as a Great Album, which means ubersongcraft, which means the Beatles, and keep that pedigree coming. Johannesburg [EP]. Spin Posted Jun 24, Read full review. Reality Check. Reality Check vacillates between Serge Gainsbourg's slutty cool and Jonathan's Richman's childlike poignance. Careless World: Rise of the Last King.

As a whole, though, Careless World is simply mediocre. Spin Posted Mar 21, Read full review. The Appeal: Georgia's Most Wanted. At his best, he sounds like he's sweating it out in a kitchen with flypaper dangling from the ceiling. But on The Appeal, he could be anywhere. This is a cold, calculated record lacking in personality, though it certainly tries to deliver something that Scott is incapable of.

Spin Posted Sep 10, Read full review. Til The Casket Drops. In fact, throughout, older brother gets the best of his carefree little sibling. Breezier doesn't always equal better. Last Light. Last Light still follows a logical, workmanlike path, but detours into arch, twitchy guitar and languid, countrified ballads that show a poised sincerity.

Where the Messengers Meet. Sad-eyed generalists with a knack for cinematic spookiness, they aspire to Wolf Parade's adventurousness, but often descend into lumbering, Interpol-style self-seriousness. Songs of Experience.

Songs of Experience, clicks into place more boldly than Songs of Innocence did three years ago. Tempos are alert, riffs punchy, melodies sharp. Spin Posted Dec 19, Read full review. Teenage Emotions. Spin Posted May 30, Read full review. Consciously or not, U2-style evangelism is all over the Mumfords' bland but biblically titled second album, Babel. Spin Posted Sep 26, Read full review. My Dear Melancholy, [EP]. The production retreats into his comfort zone. But it is also really just a breakup album, and a really mopey one at that.

Spin Posted Apr 2, Read full review. Golden State. True to Self. Tiller thinly stretches himself to 19 tracks with no added dimension. It ultimately amounts to a checklist for Broke Boys-turned-Hurt Boys, with Tiller listlessly ticking the boxes.

Spin Posted Jun 14, Read full review. Spin Posted Jun 30, Read full review. Gandhi Kahn. These grooves shimmer brighter than anything he's done since 's disco-house monolith, 2Future4U. Spin Posted Mar 13, Read full review. It's About Time. To Rodgers and his collaborators, it means a Daft Punk album whose processed vocals and acoustic elements collide to abrasive effect; it means a tighter Maroon 5 album. Spin Posted Oct 4, Read full review.

Amerikaz Nightmare. Big-name cameos The Bright Lights Of America. Unfortunately, despite now working with David Bowie producer Tony Visconti, who infuses their angular, system-smashing screeds with timpani "Good and Ready" , brass "Shadow of the Dead" , and harmonica "Go West" , Anti-Flag still don't possess the innate pop sensibility that's allowed Against Me!

The Fire Theft. A prog-rock field trip that will give Diary fans something else to cry about. Sun, Sun, Sun. It's weird how this spiral of genial melodic plaints can sound so weak and self-pitying when it's sung by a pushy dude and not a smart, empathetic woman with a voice.

The Glass Passenger. The Glass Passenger, the group's second album, chronicles McMahon's successful battle against leukemia, matching hyperemotional melodies with his tender voice on dramatic tracks. Global Warming. Sadly, as Pit forges ahead in his campaign for world domination, his artistry is what's really being colonized. Spin Posted Nov 27, Read full review. Eat Me, Drink Me. Spin Posted Jul 5, Read full review. Too much of the time, Evanescence get lost in the cavernous spaces carved out by their unsecret weapon.

Spin Posted Oct 12, Read full review. This quasi-funky, horn-section-assisted record demonstrates that as a jazz vocalist, Ani's a fine folk singer. Season Dreaming. He Was King. Felix Stallings Jr. Spin Posted May 22, Read full review. I Changed a Lot. Spin Posted Dec 24, Read full review. Pocket Symphony. Occup[ies] a hushed netherworld between classical minimalists like Erik Satie and Timbaland without the beats. Spin Posted Sep 27, Read full review. Under The Iron Sea.

There's virtually no tension or drama in Keane's surpassingly pretty pop. It plays like a retrospective of his signature sounds. Flavors Of Entanglement. Psychology textbooks are less linguistically challenged and just as littered with cases of emotional breakdowns. Spin Posted Jun 9, Read full review. His 18th, the recently released LP is modern country-by-numbers that will satisfy the faithful and mosey on under the radar of anyone else. The Outsider. Slinging new styles and innovating them are separate matters.

Grainger's solo efforts are more restrained than DFA 's sweaty frenzy, and ultimately, his blues-frilled rock would be pretty pallid if not for the playfully sarcastic undercurrents. Paper Walls. Conclusion: To the best of our knowledge, this is the first report of MDR hvKp isolates of ST23 with insertion of catA1 gene into the virulence plasmid which presents the possibility of hotspot integration sites on the plasmids to aid acquisition of AMR genes.

ST23 is no longer confined to susceptible strains of hvKp. Our findings emphasize the need for more studies on recombinant events, plasmid transmission dynamics and evolutionary process involving hvKp. Klebsiella pneumoniae is a notorious nosocomial pathogen responsible for a wide range of healthcare associated infections and is commonly multidrug resistant MDR. Thus, limited therapeutic options are available to control the infections caused by this pathogen Martin and Bachman, However, hypermucoviscous K.

The hypermucoviscous pathotype also exhibits hypervirulence hv and manifests invasive infections by causing pyogenic liver abscesses and subsequent bacteraemia, pneumonia, meningitis, or brain abscesses Paczosa and Mecsas, Hypervirulent K. The convergence of virulence with antimicrobial resistance AMR in hvKp isolates represent a real threat to the treatment and management of K. Thus, understanding the genetic background and transmission of MDR-hvKp strains from different geographical locations is an urgent priority Wyres et al.

HvKp isolates are frequently associated with K1 and K2 capsular serotypes that facilitate the pathogen to escape from phagocytosis and intracellular killing Shon et al. A combination of iron acquisition systems such as, enterobactin ent , yersiniabactin ybt , and kfu in the chromosome together with other key virulence factors such as, aerobactin, salmochelin, rmpA , and rmpA2 encoded on a single large virulence plasmid pLVPK are associated with severe infections and a high mortality rate Fu et al.

Interestingly the ybt locus is mobilized by the chromosomally encoded integrative conjugative element ICE Kp Guo et al. To date, there remains only a few studies to understand the genetics and transmission of MDR hvKp in India. We recently reported ST23 hvKp isolates that were susceptible to all the antimicrobials tested Shankar et al. To the best of our knowledge, this is the first study from India characterizing ST23 multidrug hypervirulent isolates carrying catA1 , coding for chloramphenicol resistance, on the virulence plasmid and is studied using a hybrid genome assembly combining Ion Torrent and ONT MinION technologies.

The two K. The isolate BA was isolated from a patient who had been involved in a road traffic accident and suffered from acute kidney failure and sepsis. The patient had received antibiotic treatment elsewhere before being brought to the hospital. The second isolate BA was isolated from a patient who was diagnosed with advanced cholangiocarcinoma type 3A and acute cholecystitis.

Screening for the hypermucoviscous phenotype was carried out using the string test as described previously Shon et al. Antimicrobial susceptibility testing was performed for first and second line antibiotics using the Kirby-Bauer disc diffusion method Bayer et al. Interpretation of the antimicrobial susceptibility results were done according to breakpoints defined by Clinical and Laboratory Standards Institute CLSI guidelines for K.

This was followed by hybrid de novo assembly using both Ion torrent and MinION reads using the Unicycler hybrid assembly pipeline v 0. The complete circular genome was initially assembled and was polished with multiple rounds of Pilon v. Genome assembly by combining the Ion Torrent short reads and the MinION long reads improved the contiguity and completeness of the genomes with fewer error rates.

The quality measurements of the complete genome sequences after hybrid assembly were compared with the short reads assembled de novo using SPAdes v. To validate the quality of assembly after each polishing step the completeness, correctness and contiguity parameters were assessed using CheckM v1. CheckM estimated the completeness and contiguity while Quast was used to detect mis-assemblies, mismatches, and indels by aligning the assemblies with the reference genome AP The genomes were rotated and reverse complemented to set the start location for all the genomes as dnaA gene.

The genomes were aligned using Geneious Prime software with progressive Mauve algorithm. We used default parameters to automatically calculate seed weight and minimum locally collinear blocks LCBs score. The resistance profile of the assembled genomes was identified using ResFinder v. Similarly, the presence of plasmids in the genomes were identified and characterized using PlasmidFinder v. Further, sequence typing of the assembled genomes was determined by the seven gene multi-locus sequence typing MLST scheme available at Kleborate Lam M.

The genomes were mapped and analyzed against the reference strains and were visualized using CGview server v. Identification of virulence factors such as yersiniabactin, aerobactin, and other siderophore production systems were carried out using Kleborate. The genomic sequences from two K. The sequencing reads for previously reported hypervirulent K.

The genomes were mapped to the reference genome ED23 CP The core SNP genome alignment of all the genomes was generated with snippy-core. The recombination regions within the core genome alignment was further filtered and removed using the Gubbins v. The maximum likelihood ML phylogeny was constructed using FastTree v.

The phylogenetic tree was rooted with the reference genome and labeled using the Interactive Tree of Life software iTOL v. We observed both BA and BA strains were phenotypically resistant to all the tested antimicrobials from disc diffusion assay.

The hybrid genomes of both the isolates consisted of a chromosome assembled into a single contig along with plasmids assembled into separate circular contigs. The genome of BA comprised of a 5,, bp chromosome with an average The genome of BA consisted of a 5,, bp chromosome with an average The N50 values for both the hybrid assemblies 5,, and 5,, were found to be higher than the short-read assemblies 59, and 52, suggesting highly contiguous assemblies.

As mentioned before, both genomes belong to ST23 as determined by Kleborate. These are typical characteristics of hypervirulent K. Comparison of the genomes of the clinical isolates characterized in this study with the two reference genomes SGH10 and NTUH suggested the presence of several IS elements and other differences in the chromosomes Figure 1.

The two genomes in the present study were MDR and hence the presence of higher numbers of mobile genetic elements when compared to the reference genomes, which are susceptible to antimicrobials. Figure 1 Circular representation of chromosomes of K. These IS elements are represented as gaps in the two reference genomes.

The isolates carried 7—8 plasmids each and the distribution of resistance genes on the plasmids varied as mentioned in the Table 1. Genome alignment of the two isolates along with two reference genomes used in this study shows several areas in the genome that differ among the strains Figures 1 and 2. The genome alignment has identified the presence of genes in the clinical isolates that are putatively part of restriction modification systems, which are absent in the two reference strains Figures 2B, C.

A total of 15 different TA systems in K. Among the 15 TA systems, only 11 were common in all the strains that include all the major families. A 12 locally co-linear blocks LCBs of all four strains are shown. C The biosynthetic operon of the fim gene LCB 11 shown here is flanked by the IS3 like insertion sequence in the clinical isolates and not in the reference genomes.

Table 2 Chromosomal toxin and antitoxin systems encoded by Klebsiella pneumoniae. The presence of such a large number of plasmids among the ST23 sequence types has not been previously documented. The BLAST based identity search for the homologous sequences showed similarity with well characterized virulence plasmids from other CC23 isolates such as K. The comparison of the virulence plasmid of both the isolates with the reference virulence plasmid is shown in Figure 4.

Surprisingly, we found that the virulence plasmid of both BA and BA isolates encode a chloramphenicol resistance gene catA1 , which is probably inserted through IS family transposase. The insertion of a resistance gene into the virulence plasmid indicates a potential hotspot for the further acquisition of other resistance genes into the virulence plasmid. Figure 4 Comparison of virulence plasmid of hypervirulent hv Klebsiella pneumoniae isolates BA and BA with virulence plasmids from K.

Inserted region of 2, bp in the virulence plasmid carrying catA1 is shown below. The presence of rmpA and rmpA2 along with K1 capsule type confirms a hypermucoid phenotype that is a characteristic feature of hypervirulent isolates Shon et al. The virulence plasmids also code for resistance to heavy metals such as copper, lead, silver, and tellurite Table 1.

This contributes toward resistance to some disinfectants and helps hvKp thrive in hospital settings. In addition to the virulence genes encoded by the plasmid, in K. In addition, ybbW, ybbY glc, fdrA, glxK, arc , and hyi are other genes coding for iron uptake Table 1. Both the isolates carried the yersiniabactin locus ybt9 located in ICE Kp 3.

Other than the yersiniabactin locus, the characteristic virulence factors of ICE Kp including the siderophore genes irp1 and irp2 , the ferric yersiniabactin uptake gene fyuA , the virB-type 4 secretion system T4SS , and mobBC mobilization proteins were also identified. Another interesting observation is the fim operon, a genomic region encoding a major virulence factor, is flanked by insertion sequence elements only in the clinical isolates Figure 2C.

This indicates that the fim genes might have been recently acquired by the clinical strains as a selective advantage in infecting host tissues. Phylogenetic analysis revealed a number of sub-lineages with the globally distributed CGI CG23 sub-lineage I comprising of isolates. Among the global collection, Further, isolates belonging to sub-lineage II produced aerobactin and salmochelin but lacked colibactin. Notably, all the isolates belonged to K1 capsule type except for two European isolates.

Also, 20 isolates predominantly from Asian countries, lacked rmpA and rmpA2. Figure 6 Single nucleotide polymorphism SNP based phylogenetic tree based on the core genome alignment of CG23 Klebsiella pneumoniae isolates from the global collection. The maximum likelihood phylogram is based on 9, SNPs after removing the recombinant regions. The phylogenetic tree was rooted by using the reference genome ED The scale represents the evolutionary distances.

The percentage of bootstrap trials replicates. The study isolates BA and BA formed a separate sub-lineage with 10 isolates distributed around the world in the phylogenetic tree highlighted in orange color. Though our recent studies have indicated the characterization of hvKp from India Shankar et al. Interestingly, these MDR plasmids are known to play an important role in the dissemination of antimicrobial resistance in K.

These multiple acquisitions of MDR plasmids are surprising and unprecedented for hvKp as the clone is generally less likely to acquire multiple MDR plasmids Wyres et al. The increasing genetic plasticity of hvKp has been reported from multiple geographical locations Cejas et al. Moreover, several studies from other Asian countries have documented the predominant carbapenem resistance K.

Therefore, plasmid exchange appears to be happening both ways between hvKp and the prevalent sequence type of CRKp in study setting. Although the direct proof of exchange is absent, we speculate that ST K. CC23 persisters showed higher survival rate at a dose of 10x MIC against a carbapenem class antibiotic meropenem Lee et al.

This indicates clinical isolates, belonging to CC23 complex, used in this study may be capable of forming persisters under high dose of carbapenem and thus can find opportunity to acquire resistance genes from the pool of dead bacteria. This, in turn, could allow for active growth in the presence of antibiotics from the once dormant persister bacteria. This is alarming because carbapenems are last resort to treat K. Since TA systems are the major players of persister formation, we investigated the presence of TA systems in the two study isolates Table 2.

Among the three additional TA systems present in the clinical isolates, one was uniquely present in the plasmid, while the other two were also present in the chromosome. In general, TA systems are also involved in diverse cellular processes. Plasmid maintenance, selfish alleles, gene regulation, growth control, persister formation, programmed cell arrest and death, anti-phage activity, biofilm formation, and general stress response can all be controlled by TAs Ramage et al.

The nucleotide sequence similarity of plasmids in other Enterobacterales such as Citrobacter sp. KX E. KP substantiates the horizontal dissemination of plasmid among different bacterial species. The acquisition of the bla OXA -bearing ColKP3 plasmid by a single isolate of hvKp may mark a major evolutionary step toward further establishment of clonal complex 23 CC Till now, the clonal dissemination of bla OXA -bearing K.

The emergence of carbapenemase producing hvKp in India can be hypothesized to be due to acquiring the ColKP3 plasmid from ST CRKp, as this sequence type is widely circulating in the same environment Shankar et al. Thus, the emergence of bla OXA producing hvKp is particularly concerning due to its combination of hypervirulence and carbapenem resistance. Supplementary Figures 2 — 4 shows the comparison of these plasmids isolated from K.

Though similar AMR genes are mobilized by these plasmids in both the organisms, significant differences were observed among ColKP3. Two ColKP3 plasmids from E. IncX3 was highly similar among both the organisms. The characteristic feature of hvKp is the presence of the kb large virulence plasmid with the mucoid regulators rmpA and rmpA2 being encoded in the plasmid. In addition, the virulence plasmid is characterized by the presence of the catA1 gene flanked by the insertion elements IS1 and IS suggesting that the region has been inserted into the virulence plasmid Mahillon and Chandler, A similar recombination event in the same virulence plasmid was previously reported by Dong and colleagues , where a bla KPC-2 carrying region was inserted into the virulence plasmid.

Conversely there have been reports of the acquisition of a hvKp virulence plasmid by CRKp isolates Gu et al. The SNP based phylogeny of the two isolates with respect to the representative CG23 global strains showed that the evolutionary events are not driven by geographical location. The phylogenetic distribution indicated distinct sub-lineages with a major sub-lineage CGI associated with liver abscess strains Lam M.

This observation is in line with Lam and colleagues where this globally distributed sub-lineage is driven by ICEKp10 with ybt1. The sub-lineage II where two study isolates clustered were predominantly associated with sepsis and other invasive infection. In addition, when growth of the two study isolates was compared with ST K. However, after 25h, the ST Kp showed a rapid decline in growth while no decline in the growth of two hvKp was observed.

The acquisition of antimicrobial resistance including colistin resistance in hvKp has been associated with increased fitness cost Choi and Ko, ; Lee et al. Based on the genetic composition of the virulence plasmids, which had multiple insertion elements, we speculate further plasmid recombination events will occur leading to emergence of novel resistance-virulence encoding plasmids. Our findings are relevant in understanding the future risk of the emergence of individual K.

Specifically, our data indicate that MDR clones pose the greatest risk because they are more likely to acquire virulence genes than the hypervirulent clones acquiring resistance genes. Written informed consent for participation was not required for this study since the bacterial isolates were used without patient identifier. CS: Laboratory methods, data analysis and interpretation, manuscript writing.

JJ: Data analysis, interpretation and manuscript writing. KV: Hybrid genome assembly and other bioinformatics methods.

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