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Carbapenem-resistant and virulence plasmid-harboring Klebsiella pneumoniae (pVir-CRKP) has emerged and spread globally, yet clinical investigations from the United States remain limited. We conducted a genomic analysis of 884 unique carbapenem-resistant K. pneumoniae isolates from a multicenter US cohort and identified 6 pVir-CRKP isolates, including 2 sequence type (ST) 23, 2 ST893, and 2 ST11 isolates. Patients infected with pVir-CRKP experienced high Pitt bacteremia scores and a 33% 30-day mortality rate. The pVir-CRKP isolates exhibited significant sequence variation in virulence genes and plasmids, along with differences in mucoviscosity, capsule production, survival in normal human serum, resistance to killing by human polymorphonuclear neutrophils, and in vivo pathogenicity. Phylogenetic analyses showed that most pVir-CRKP isolates were genetically similar to strains reported from other global regions. The emergence of pVir-CRKP with higher virulence potential and carbapenem resistance in the United States than the predominant carbapenem-resistant K. pneumoniae clone underscores the need for active global surveillance.

The incidence of salmonellosis in humans and animals is still high due to the occurrence of virulence factors in which play a role in the process of infection in the host and the spread of disease and most of the can infect humans and animals. The present study was aimed to identify and detect virulence genes related to pathogenicity islands (SPIs) and plasmid virulence (Spv). A total of 27 . archive isolates belonging to the National Veterinary Drug Assay Laboratory (NVDAL) were used in this study. The bacteria were collected in 2016 and 2017 from samples of the cloaca and fecal swabs from layer and broiler farms in five provinces of Java Island. Isolates were cultured in specific media, biochemical tests and Gram staining. Detection of . and virulence genes was done by polymerase chain reaction (PCR) method. Identification of serovar showed 100% (27/27) isolates were positive for the gene (304 bp). The result confirmed that all strains were . PCR based detection of virulence genes showed that 100% of isolates had virulence genes in SPI-1 to SPI-5, namely, and genes. All the isolates (27/27) were also positive to gene-based PCR. All the isolates of in this study carry virulence genes related to SPI-1 to SPI-5 and plasmid virulence. The existence of virulent genes indicates that the strain examined in this study is highly virulent and poses a potential threat of worse disease outcome in humans and animals.

Elucidating the adaptive strategies and plasticity of bacterial genomes is crucial for understanding the epidemiology and evolution of pathogens threatening human health. While much is known about the evolution of in controlled laboratory environments, less effort has been made to elucidate the genome dynamics of in its native settings. Here, we follow the genome dynamics of co-existing lineages of the infant gut during the first year of life. One lineage causes a urinary tract infection (UTI) and experiences several alterations of its genomic content during subsequent antibiotic treatment. Interestingly, all isolates of this uropathogenic strain carried a highly stable plasmid implicated in virulence of diverse pathogenic strains from all over the world. While virulence elements are certainly beneficial during infection scenarios, their role in gut colonization and pathogen persistence is poorly understood. We performed competitive fitness experiments to assess the role of this highly disseminated virulence plasmid in gut colonization, but found no evidence for a direct benefit of plasmid carriage. Through plasmid stability assays, we demonstrate that this plasmid is maintained in a parasitic manner, by strong first-line inheritance mechanisms, acting on the single-cell level, rather than providing a direct survival advantage in the gut. Investigating the ecology of endemic accessory genetic elements, in their pathogenic hosts and native environment, is of vital importance if we want to understand the evolution and persistence of highly virulent and drug resistant bacterial isolates.

Rhodococcus equi is an animal pathogen and zoonotic human opportunistic pathogen associated with immunosuppressive conditions. The pathogenicity of R. equi is linked to three animal host-associated virulence plasmids encoding a family of “Virulence Associated Proteins” (VAPs). Here, the PCR-based TRAVAP molecular typing system for the R. equi virulence plasmids was applied to 26 R. equi strains isolated between 2010 and 2016 at the Institute of Tropical Medicine “Pedro Kourí,” Cuba, from individuals living with HIV/AIDS. TRAVAP detects 4 gene markers, traA common to the three virulence plasmids, and vapA, vapB , and vapN specific to each of the host-associated plasmid types (equine pVAPA, porcine pVAPB, and ruminant pVAPN). Of the 26 isolates, six were positive to the vapB (porcine-type) marker, 4 (15.4%) to the vapA (equine-type) marker, and 1 (3.8%) to the vapN (ruminant-type) marker. Most of the isolates 14 (53.8%) were negative to all TRAVAP markers, suggesting they lacked a virulence plasmid. To our knowledge, this work is the first to report the molecular characterization of R. equi isolates from Cuba. Our findings provide insight into the zoonotic origin of R. equi infections in people and the potential dispensability of the virulence plasmid in immunosuppressed patients.

Background Klebsiella pneumoniae is a recognised agent of multidrug-resistant (MDR) healthcare-associated infections; however, individual strains vary in their virulence potential due to the presence of mobile accessory genes. In particular, gene clusters encoding the biosynthesis of siderophores aerobactin ( iuc ) and salmochelin ( iro ) are associated with invasive disease and are common amongst hypervirulent K . pneumoniae clones that cause severe community-associated infections such as liver abscess and pneumonia. Concerningly, iuc has also been reported in MDR strains in the hospital setting, where it was associated with increased mortality, highlighting the need to understand, detect and track the mobility of these virulence loci in the K . pneumoniae population. Methods Here, we examined the genetic diversity, distribution and mobilisation of iuc and iro loci amongst 2503 K . pneumoniae genomes using comparative genomics approaches and developed tools for tracking them via genomic surveillance. Results Iro and iuc were detected at low prevalence (< 10%). Considerable genetic diversity was observed, resolving into five iro and six iuc lineages that show distinct patterns of mobilisation and dissemination in the K . pneumoniae population. The major burden of iuc and iro amongst the genomes analysed was due to two linked lineages ( iuc1 / iro1 74% and iuc2 / iro2 14%), each carried by a distinct non-self-transmissible IncFIB K virulence plasmid type that we designate KpVP-1 and KpVP-2. These dominant types also carry hypermucoidy ( rmpA ) determinants and include all previously described virulence plasmids of K . pneumoniae . The other iuc and iro lineages were associated with diverse plasmids, including some carrying IncFII conjugative transfer regions and some imported from Escherichia coli ; the exceptions were iro3 (mobilised by ICE Kp1 ) and iuc4 (fixed in the chromosome of K . pneumoniae subspecies rhinoscleromatis ). Iro / iuc mobile genetic elements (MGEs) appear to be stably maintained at high frequency within known hypervirulent strains (ST23, ST86, etc.) but were also detected at low prevalence in others such as MDR strain ST258. Conclusions Iuc and iro are mobilised in K . pneumoniae via a limited number of MGEs. This study provides a framework for identifying and tracking these important virulence loci, which will be important for genomic surveillance efforts including monitoring for the emergence of hypervirulent MDR K . pneumoniae strains.

Since 2009, an emergent shrimp disease, acute hepatopancreatic necrosis disease (AHPND), has been causing global losses to the shrimp farming industry. The causative agent of AHPND is a specific strain of Vibrio parahaemolyticus . We present evidence here that the opportunistic V. parahaemolyticus becomes highly virulent by acquiring a unique AHPND-associated plasmid. This virulence plasmid, which encodes a binary toxin [ V. parahaemolyticus Photorhabdus insect-related toxins (PirA vp and PirB vp )] that induces cell death, is stably inherited via a postsegregational killing system and disseminated by conjugative transfer. The cytotoxicity of the PirA vp /PirB vp system is analogous to the structurally similar insecticidal pore-forming Cry toxin. These findings will significantly increase our understanding of this emerging disease, which is essential for developing anti-AHPND measures. Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirA vp and PirB vp ) proteins and found that the overall structural topology of PirA vp /PirB vp is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirAB vp heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirAB vp may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

Background Klebsiella pneumoniae , as a global priority pathogen, is well known for its capability of acquiring mobile genetic elements that carry resistance and/or virulence genes. Its virulence plasmid, previously deemed nonconjugative and restricted within hypervirulent K. pneumoniae (hvKP), has disseminated into classic K. pneumoniae (cKP), particularly carbapenem-resistant K. pneumoniae (CRKP), which poses alarming challenges to public health. However, the mechanism underlying its transfer from hvKP to CRKP is unclear. Methods A total of 28 sequence type (ST) 11 bloodstream infection-causing CRKP strains were collected from Ruijin Hospital in Shanghai, China, and used as recipients in conjugation assays. Transconjugants obtained from conjugation assays were confirmed by Xba I and S1 nuclease pulsed-field gel electrophoresis, PCR detection and/or whole-genome sequencing. The plasmid stability of the transconjugants was evaluated by serial culture. Genetically modified strains and constructed mimic virulence plasmids were employed to investigate the mechanisms underlying mobilization. The level of extracellular polysaccharides was measured by mucoviscosity assays and uronic acid quantification. An in silico analysis of 2608 plasmids derived from 814 completely sequenced K. pneumoniae strains available in GenBank was performed to investigate the distribution of putative helper plasmids and mobilizable virulence plasmids. Results A nonconjugative virulence plasmid was mobilized by the conjugative plasmid belonging to incompatibility group F (IncF) from the hvKP strain into ST11 CRKP strains under low extracellular polysaccharide-producing conditions or by employing intermediate E. coli strains. The virulence plasmid was mobilized via four modes: transfer alone, cotransfer with the conjugative IncF plasmid, hybrid plasmid formation due to two rounds of single-strand exchanges at specific 28-bp fusion sites or homologous recombination. According to the in silico analysis, 31.8% (242) of the putative helper plasmids and 98.8% (84/85) of the virulence plasmids carry the 28-bp fusion site. All virulence plasmids carry the origin of the transfer site. Conclusions The nonconjugative virulence plasmid in ST11 CRKP strains is putatively mobilized from hvKP or E. coli intermediates with the help of conjugative IncF plasmids. Our findings emphasize the importance of raising public awareness of the rapid dissemination of virulence plasmids and the consistent emergence of hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) strains.

Background Klebsiella pneumoniae is an opportunistic pathogen and a leading cause of healthcare-associated infections in hospitals, which are frequently antimicrobial resistant (AMR). Exacerbating the public health threat posed by K. pneumoniae , some strains also harbour additional hypervirulence determinants typically acquired via mobile genetic elements such as the well-characterised large virulence plasmid KpVP-1. The rmpADC locus is considered a key virulence feature of K. pneumoniae and is associated with upregulated capsule expression and the hypermucoid phenotype, which can enhance virulence by contributing to serum resistance. Typically such strains have been susceptible to all antimicrobials besides ampicillin; however, the recent emergence of AMR hypermucoid strains is concerning. Methods Here, we investigate the genetic diversity, evolution, mobilisation and prevalence of rmpADC , in a dataset of 14,000 genomes from isolates of the Klebsiella pneumoniae species complex, and describe the RmST virulence typing scheme for tracking rmpADC variants for the purposes of genomic surveillance. Additionally, we examine the functionality of representatives for variants of rmpADC introduced into a mutant strain lacking its native rmpADC locus. Results The rmpADC locus was detected in 7% of the dataset, mostly from genomes of K. pneumoniae and a very small number of K. variicola and K. quasipneumoniae . Sequence variants of rmpADC grouped into five distinct lineages ( rmp1 , rmp2 , rmp2A , rmp3 and rmp4 ) that corresponded to unique mobile elements, and were differentially distributed across different populations (i.e. clonal groups) of K. pneumoniae . All variants were demonstrated to produce enhanced capsule production and hypermucoviscosity. Conclusions These results provide an overview of the diversity and evolution of a prominent K. pneumoniae virulence factor and support the idea that screening for rmpADC in K. pneumoniae isolates and genomes is valuable to monitor the emergence and spread of hypermucoid K. pneumoniae , including AMR strains.

The epidemiological features of the newly emerged carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) and its potential threat to human health are currently unknown. In this study, a total of 784 bla KPC-2 -bearing CRKP strains collected from three hospitals located at different geographical locales in China during 2014-2017 were subjected to molecular typing, screening of virulence plasmid, string test and WGS (367/784 strains). The proportion of CRKP among all clinical K. pneumoniae strains increased sharply in China during 2014-2017. A large proportion (58%) of these CRKP strains were found to harbour a virulence-encoding plasmid, while only 13% of such strains exhibited a hypervirulent phenotype by string test and neutrophil assay. The lack of hypervirulent phenotype in virulent plasmid-bearing CRKP strains was found to be due to the mutation's presence on rmpA and rmpA2 genes, which rendered them non-functional, while some strains carrying wild type rmpA did not exhibit hypervirulent phenotype either suggesting that other factors might also contribute to the hypervirulence of CRKP. Phylogenetic and SNP analysis indicated that the transmission of these CRKP strains in China likely involved several major clones of ST11. Carriage of IncFII pSWU01-like, bla KPC-2 -bearing plasmid was found to be the major mechanism of carbapenem resistance in these CRKP strains. In conclusion, our data indicated that the prevalence of CRKP strains carrying the virulence plasmid has rapidly increased in China, while genetic markers were not correlated well with the hypervirulent phenotypes, which call for a better definition and screening for these truly hypervirulent CR-HvKP strains in clinical settings.

Virulence plasmids are key drivers of hypervirulence in Klebsiella pneumoniae . Here, we report a systematic analysis of chromosomal integration of a p17-15vir-derived fragment carrying virulence ( rmpA2 and iutA - iucABCD ) and resistance genes in nine bla KPC−2 -positive ST11-KL47 carbapenem-resistant K. pneumoniae (CRKP) isolates. Of these, seven exhibited a hypervirulent phenotype in a mouse infection model. Nanopore sequencing analysis revealed that these virulence-associated integration regions could be classified into three distinct groups based on their structural patterns. Notably, we investigated the mechanisms underlying the formation of the integration region and proposed an IS 26 -mediated model for the integration of virulence gene-carrying plasmid fragment into the chromosome. Besides, IS Kpn1 was identified for the first time as a preferred insertion hotspot. Both bla KPC−2 and rmpA2 demonstrated stable persistence in these isolates without antibiotic selection pressure, and group I integration regions displayed the capability to form circular intermediates. These findings provide critical insights into the virulence plasmid fragment integrated into their chromosomes and underscore the importance of surveillance for such hybrid threats.