The prevalence of carbapenem-resistant Enterobacterales and the emergence of novel ST11-KL30 carbapenem-resistant Klebsiella pneumoniae in Xinjiang, China.

OBJECTIVES: To address the lack of research on the prevalence of carbapenem-resistant Enterobacterales (CREs) in Xinjiang, China, and elucidate the genomic characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) ST11-KL30. METHODS: CREs were collected in Xinjiang from 2021 to 2023. The antimicrobial susceptibility testing of carbapenems was performed via agar dilution method. Whole-genome sequencing was completed on the Illumina platform, and subsequent genomic analyses of CRKP, such as sequencing typing, K-locus and O-locus identification, virulence score assessment, and phylogenetic analysis, were performed. The virulence of CRKP isolates was determined in vitro and in vivo, and biofilm formation was assessed by crystal violet staining. Additionally, the virulence plasmid was reconstructed, and the formation of CRKP ST11-KL30 was revealed based on genome data from public database. RESULTS: Eighty-five CRE isolates were collected, among which CRKP was most prevalent (68/85). KPC was the most dominant carbapenemase (60/68) in CRKP, while NDM-type carbapenemase was more prevalent in other species. ST11 was the dominant CRKP clone, and was phylogenetically divided into three clusters: ST11-KL64, ST11-KL47, and ST11-KL30. CRKP ST11-KL30 is a novel recombinant clone that harbours a pK2044-like virulence plasmid and can be derived from ST11-KL64 by obtaining an ∼57 kb region from ST29-KL30. Compared to ST11-KL47 and ST11-KL64, ST11-KL30 had lower virulence, but had enhanced biofilm formation. CONCLUSIONS: We describe the prevalence of CRE prevalence southern Xinjiang and report the emergence of a region-specific clone. Our findings underscore the potential dissemination of ST11-KL30, which warrants increased monitoring in the future.

Virulence plasmid with IroBCDN deletion promoted cross-regional transmission of ST11-KL64 carbapenem-resistant hypervirulent Klebsiella pneumoniae in central China.

BACKGROUND: Carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKP) caused infections of high mortality and brought a serious impact on public health. This study aims to evaluate the epidemiology, resistance and virulence characteristics of CR-hvKP and to identify potential drivers of cross-regional transmission in different regions of China, in order to provide a basis for developing targeted prevention measures. METHODS: Clinical K. pneumoniae strains were collected from Jiujiang and Nanchang in Jiangxi province between November 2021 to June 2022. Clinical data of patients (age, sex, source of infection, and diagnosis) were also gathered. We characterized these strains for their genetic relatedness using PFGE, antimicrobial and virulence plasmid structures using whole-genome sequencing, and toxicity using Galleria mellonella infection model. RESULTS: Among 609 strains, 45 (7.4%) CR-hvKP were identified, while the strains. isolated from Nanchang and Jiujiang accounted for 10.05% (36/358) and 3.59% (9/251). We observed that ST11-KL64 CR-hvKP had an overwhelming epidemic dominance in these two regions. Significant genetic diversity was identified among all ST11-KL64 CR-hvKP cross-regional transmission between Nanchang and Jiujiang and this diversity served as the primary driver of the dissemination of clonal groups. Virulence genes profile revealed that ST11-KL64 CR-hvKP might harbour incomplete pLVPK-like plasmids and primarily evolved from CRKP by acquiring the hypervirulence plasmid. We found the predominance of truncated-IncFIB/IncHI1B type virulence plasmids with a 25 kb fragment deletion that encoded iroBCDN clusters. CONCLUSION: ST11-KL64 is the most cross-regional prevalent type CR-hvKPs in Jiangxi province, which mainly evolved from CRKPs by acquiring a truncated-IncHI1B/IncFIB virulence plasmid with the deletion of iroBCDN. Stricter surveillance and control measures are urgently needed to prevent the epidemic transmission of ST11-KL64 CR-hvKP.

The Putative Virulence Plasmid pYR4 of the Fish Pathogen Yersinia ruckeri Is Conjugative and Stabilized by a HigBA Toxin-Antitoxin System.

The bacterium Yersinia ruckeri causes enteric redmouth disease in salmonids and hence has substantial economic implications for the farmed fish industry. The Norwegian Y. ruckeri outbreak isolate NVH_3758 carries a relatively uncharacterized plasmid, pYR4, which encodes both type 4 pili and a type 4 secretion system. In this study, we demonstrate that pYR4 does not impose a growth burden on the Y. ruckeri host bacterium, nor does the plasmid contribute to twitching motility (an indicator of type 4 pilus function) or virulence in a Galleria mellonella larval model of infection. However, we show that pYR4 is conjugative. We also reveal, through mutagenesis, that pYR4 encodes a functional post-segregational killing system, HigBA, that is responsible for plasmid maintenance within Y. ruckeri. This is the first toxin-antitoxin system to be characterized for this organism. Whilst further work is needed to elucidate the virulence role of pYR4 and whether it contributes to bacterial disease under non-laboratory conditions, our results suggest that the plasmid possesses substantial stability and transfer mechanisms that imply importance within the organism. These results add to our understanding of the mobile genetic elements and evolutionary trajectory of Y. ruckeri as an important commercial pathogen, with consequences for human food production.

Conjugative transmission of virulence plasmid in Klebsiella pneumoniae mediated by a novel IncN-like plasmid.

Klebsiella pneumoniae (Kp) is increasingly recognized as a reservoir for a range of antibiotic resistance genes and a pathogen that frequently causes severe infections in both hospital and community settings. In this study, we have identified a novel mechanism of conjugative transfer of a non-conjugative virulence plasmid through the formation of a fusion plasmid between the virulence plasmid and a novel 59,162 bp IncN- plasmid. This plasmid was found to be a multidrug-resistance (MDR) plasmid and carried a T4SS cluster, which greatly facilitated the efficient horizontal transfer of the fusion plasmid between Kp strains. The fused virulence plasmid conferred the resistance of serum killing and macrophage phagocytosis to the transconjugants. Importantly, this plasmid was shown to be essential for Kp virulence in a mouse model. Mechanistic analysis revealed that the virulence factors encoded by this virulence plasmid contributed to resistance to in vivo clearance and induced a high level of proinflammatory cytokine IL-1ß, which acts as an inducer for more neutrophil recruitment. The transmission of the fusion plasmid in Kp has the potential to convert it into both MDR and hypervirulent Kp, accelerating its evolution, and posing a serious threat to human health. The findings of this study provide new insights into the rapid evolution of MDR and hypervirulent Kp in recent years.

Deciphering the relative importance of genetic elements in hypervirulent Klebsiella pneumoniae to guide countermeasure development.

BACKGROUND: Quantitating the contribution of phenotype-responsible elements in hypervirulent Klebsiella pneumoniae is needed. METHODS: Isogenic mutants of four hypervirulent clinical isolates that produced K1 (ST23), K2 (ST86), K20 (ST1544), or K54 (ST29) capsules (mean 2.2 log10 LD50 (range 1.5-2.9)) were created to measure the effects on LD50 in a murine model of the hypervirulence-associated plasmid (pVir), iucA, prmpA, prmpA2 (truncated), irp2, and clbBC. FINDINGS: Curing pVir had the greatest increase in survival (mean LD50 to 7.6 (range 7.0-9.0, p ≤ 0.0001), a dosage comparable to classical K. pneumoniae. Results also showed increased mean LD50s for ΔprmpA (5.9, p ≤ 0.0001), ΔiucA (3.6, p ≤ 0.0001), Δirp2 (3.4), ΔrmpAΔiucA (6.3, p ≤ 0.0001), and ΔpVirΔirp2 (8.7, p ≤ 0.0001). Notably ΔpVir had an additional mean LD50 increase of 1.3 compared to the pVir-encoded ΔprmpAΔiucA (p ≤ 0.01), suggesting presence of additional pVir-virulence genes. Truncated pRmpA2 did not contribute to virulence. Odd ratios in the absence of pVir/yersiniabactin, pVir, pRmpA/aerobactin, pRmpA, aerobactin, yersiniabactin, and colibactin demonstrated a 250-fold, 67-fold, 20-fold, 16.7-fold, 9.6-fold, and 1.7-fold decrease in lethality respectively. INTERPRETATION: These data can guide countermeasure development. FUNDING: This work was supported by NIH R21 AI123558-01 and 1R21AI141826-01A1 (Dr. Russo) and the Department of Veterans Affairs VA Merit Review (I01 BX004677-01) (Dr. Russo). This study was also partially funded by the U.S. Defense Health Program (DHP) Operations and Maintenance.

Molecular characteristics and pathogenic mechanisms of KPC-3 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (ST23-K1).

Objective: This study aimed to comprehensively investigate hypervirulent carbapenem-resistant Klebsiella pneumoniae (CR-hvKP) in the Ningbo region. Importantly, we sought to elucidate its molecular characteristics and pathogenic mechanisms. This information will provide evidence-based insights for preventing and controlling nosocomial infections and facilitate improved clinical diagnosis and treatment in this region. Methods: 96 carbapenem-resistant Klebsiella pneumoniae strains were collected from the Ningbo region between January 2021 and December 2022. Whole genome sequencing and bioinformatic methods were employed to identify and characterize CR-hvKP strains at the molecular level. The minimum inhibitory concentrations (MICs) of common clinical antibiotics were determined using the VITEK-2 Compact automatic microbiological analyzer. Plasmid conjugation experiments evaluated the transferability of resistance plasmids. Finally, mouse virulence assays were conducted to explore the pathogenic mechanisms. Results: Among the 96 strains, a single CR-hvKP strain, designated CR-hvKP57, was identified, with an isolation frequency of 1.04%. Whole-genome sequencing revealed the strain to be ST23 serotype with a K1 capsule. This strain harbored three plasmids. Plasmid 1, a pLVPK-like virulence plasmid, carried multiple virulence genes, including rmpA, rmpA2, iroB, iucA, and terB. Plasmid 2 contained transposable element sequences such as IS15 and IS26. Plasmid 3, classified as a resistance plasmid, harbored the bla KPC-3 carbapenem resistance gene. Mouse virulence assays demonstrated a high mortality rate associated with CR-hvKP57 infection. Additionally, there was a significant increase in IL-1ß, IL-6, and TNF-α levels in response to CR-hvKP57 infection, indicating varying degrees of inflammatory response. Western blot experiments further suggested that the pathogenic mechanism involves activation of the NF-κB signaling pathway. Conclusion: This study confirms the emergence of hypervirulent CR-hvKP in the Ningbo region, which likely resulted from the acquisition of a pLVPK-like virulence plasmid and a bla KPC-3 resistance plasmid by the ST23-K1 type Klebsiella pneumoniae. Our findings highlight the urgent need for more judicious use of antibiotics to limit the emergence of resistance. Additionally, strengthening infection prevention and control measures is crucial to minimize the spread of virulence and resistance plasmids.

Insights into the Evolution of IncR Plasmids Found in the Southern European Clone of the Monophasic Variant of Salmonella enterica Serovar Typhimurium.

Salmonella enterica subspecies enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium which has emerged as a world-wide distributed pathogen in the last decades. Several clones have been identified within this variant, the European clone, the Spanish clone, the Southern European clone and the U.S./American clone. The present study focused on isolates of the Southern European clone that were obtained from clinical samples at Spanish hospitals. The selected isolates were multidrug resistant, with most resistance genes residing on IncR plasmids that also carried virulence genes. These plasmids had a mosaic structure, comprising a highly reduced IncR backbone, which has acquired a large amount of exogenous DNA mostly derived from pSLT and IncI1-I(alfa) plasmids. Although composed of approximately the same elements, the investigated plasmids displayed a high diversity, consistent with active evolution driven by a wealth of mobile genetic elements. They comprise multiple intact or truncated insertion sequences, transposons, pseudo-compound transposons and integrons. Particularly relevant was the role of IS26 (with six to nine copies per plasmid) in generating insertions, deletions and inversions, with many of the rearrangements uncovered by tracking the patterns of eight bp target site duplications. Most of the resistance genes detected in the analyzed isolates have been previously associated with the Southern European clone. However, erm(B), lnu(G) and blaTEM-1B are novel, with the last two carried by a second resistance plasmid found in one of the IncR-positive isolates. Thus, evolution of resistance in the Southern European clone is not only mediated by diversification of the IncR plasmids, but also through acquisition of additional plasmids. All isolates investigated in the present study have the large deletion affecting the fljBA region previously found to justify the monophasic phenotype in the Southern European and U.S./American clones. An SNP-based phylogenetic analysis revealed the close relationship amongst our isolates, and support that those sharing the large fljBA deletion could be more heterogeneous than previously anticipated.

Molecular characterization of hybrid virulence plasmids in ST11-KL64 KPC-2-producing multidrug-resistant hypervirulent Klebsiella pneumoniae from China.

Introduction: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains combining virulence and multidrug resistance (MDR) features pose a great public health concern. The aim of this study is to explore the evolutionary characteristics of virulence in CR-HvKP by investigating the genetic features of resistance and virulence hybrid plasmids. Methods: The resistance and virulence phenotypes were determined by using antimicrobial susceptibility testing and the mouse bacteremia infection model, respectively. Plasmid profiles were investigated by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting, conjugation assay, and whole genome sequencing (WGS). Bioinformatics tools were used to uncover the genetic features of the resistance and virulence hybrid plasmids. Results: Two ST11-KL64 CRKP clinical isolates (KP18-3-8 and KP18-2079), which exhibited enhanced virulence compared with the classic CRKP, were detected positive for blaKPC-2 and rmpA2. The virulence level of the hypermucoviscous strain KP18-3-8 was higher than that of KP18-2079. S1-PFGE, Southern hybridization and WGS analysis identified two novel hybrid virulence plasmids in KP18-3-8 (pKP1838-KPC-vir, 228,158 bp) and KP18-2079 (pKP1838-KPC-vir, 182,326 bp), respectively. The IncHI1B/repB-type plasmid pKP1838-KPC-vir co-harboring blaKPC-2 and virulence genes (rmpA2, iucABCD and iutA) but lacking type IV secretion system could transfer into non-hypervirulent ST11 K. pneumoniae with the assistance of a helper plasmid in conjugation. The IncFII/IncR-type virulence plasmid pKP18-2079-vir may have been generated as a result of recombination between a typical pLVPK-like virulence plasmid and an MDR plasmid. Conclusion: Our studies further highlight co-evolution of the virulence and resistance plasmids in ST11-CRKP isolates. Close surveillance of such hybrid virulence plasmids in clinical K. pneumoniae should be performed.

Epidemiology and molecular characterization of carbapenem-resistant Klebsiella pneumoniae isolated from neonatal intensive care units in General Hospital of Ningxia Medical University, China, 2017-2021.

OBJECTIVES: This study aimed to retrospectively investigate the epidemiology and molecular characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from neonatal intensive care units (NICU) between 2017 and 2021. METHODS: The antibacterial susceptibility of all strains was assessed using the VITEK 2 compact system. The presence of antibiotic resistance, virulence genes, sequence types (STs), capsular (K) types, and the wzi genes was determined through polymerase chain reaction (PCR). Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI. Additionally, the virulence potential of peg344-positive strains was evaluated using the string test and mouse intraperitoneal infection models. Whole-genome sequencing was conducted on the DNB system and PacBio platforms. RESULTS: A total of 46 CRKP isolates were collected during the study period. Out of these, 93.47% (43/46) were identified as CRKP strains belonging to the ST76-K10 type carrying blaNDM-5. It was observed that CRKP infection resulted in more severe clinical symptoms compared to CRKP colonization. Among the CRKP strains, a hypervirulent CRKP strain called KP-63, belonging to the ST23 type, was identified. This strain exhibited high mortality in the mouse infection model and was found to possess virulence genes. Genomic alignment analysis revealed a significant similarity between the virulence plasmid from KP-63 strain (pKP-63) and pK2044 from the hypervirulent K. pneumoniae strain NTUH-2044. CONCLUSIONS: There has been a potential dissemination of ST76-K10 type CRKP carrying blaNDM-5 in the NICU at Ningxia Hospital. Neonatal CRKP infection has been found to cause more severe clinical symptoms than colonization. Furthermore, we have discovered a CR-hvKP strain of ST23 with serotype K1, which exhibits a significant resemblance in its virulent plasmid to pK2044. Therefore, it is crucial to enforce effective measures to restrict the spread and hinder the evolution of CRKP within the hospital.

RES-Xre toxin-antitoxin locus knaAT maintains the stability of the virulence plasmid in Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae isolates have been increasingly reported worldwide, especially hypervirulent drug-resistant variants owing to the acquisition of a mobilizable virulence plasmid by a carbapenem-resistant strain. This pLVPK-like mobilizable plasmid encodes various virulence factors; however, information about its genetic stability is lacking. This study aimed to investigate the type II toxin-antitoxin (TA) modules that facilitate the virulence plasmid to remain stable in K. pneumoniae. More than 3,000 TA loci in 2,000 K. pneumoniae plasmids were examined for their relationship with plasmid cargo genes. TA loci from the RES-Xre family were highly correlated with virulence plasmids of hypervirulent K. pneumoniae. Overexpression of the RES toxin KnaT, encoded by the virulence plasmid-carrying RES-Xre locus knaAT, halts the cell growth of K. pneumoniae and E. coli, whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. knaA and knaT were co-transcribed, representing the characteristics of a type II TA module. The knaAT deletion mutation gradually lost its virulence plasmid in K. pneumoniae, whereas the stability of the plasmid in E. coli was enhanced by adding knaAT, which revealed that the knaAT operon maintained the genetic stability of the large virulence plasmid in K. pneumoniae. String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of K. pneumoniae. These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in K. pneumoniae.

Clinical use of tigecycline may contribute to the widespread dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae strains.

The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) poses grave threats to human health. These strains increased dramatically in clinical settings in China in the past few years but not in other parts of the world. Four isogenic K. pneumoniae strains, including classical K. pneumoniae, carbapenem-resistant K. pneumoniae (CRKP), hypervirulent K. pneumoniae (hvKP) and CR-hvKP, were created and subjected to phenotypic characterization, competition assays, mouse sepsis model and rat colonization tests to investigate the mechanisms underlying the widespread nature of CR-hvKP in China. Acquisition of virulence plasmid led to reduced fitness and abolishment of colonization in the gastrointestinal tract, which may explain why hvKP is not clinically prevalent after its emergence for a long time. However, tigecycline treatment facilitated the colonization of hvKP and CR-hvKP and reduced the population of Lactobacillus spp. in animal gut microbiome. Feeding with Lactobacillus spp. could significantly reduce the colonization of hvKP and CR-hvKP in the animal gastrointestinal tract. Our data implied that the clinical use of tigecycline to treat carbapenem-resistant K. pneumoniae infections facilitated the high spread of CR-hvKP in clinical settings in China and demonstrated that Lactobacillus spp. was a potential candidate for anticolonization strategy against CR-hvKP.

Emergence of an ST1934:KL121 hypervirulent Klebsiella pneumoniae carrying a novel virulence-resistance hybrid plasmid with chromosomal integration of ICEKp1.

To identify the phenotypic and genomic characteristics of K. pneumoniae KP43 from bloodstream infection. KP43 was resistant to ticarcillin and tetracycline and was hypervirulent in the Galleria mellonella larvae infection model, positive for string test, and possessed high-level macrophage killing resistance. The hypervirulence phenotype was associated with the chromosome integration of ICEKp1 carrying iroBCDN-iroP, rmpADC, and peg-344, and a novel plasmid pKP43_vir_amr harboring iutAiucABCD. pKP43_vir_amr was an IncFIBκ/FII virulence-resistance hybrid conjugative plasmid which also carried antibiotic resistance genes. The emergence of such a strain and the spread of the novel virulence-resistance plasmid might pose a potential threat to public health.

Genomic epidemiology of hypervirulent carbapenem-resistant Klebsiella pneumoniae at Jinshan local hospital, Shanghai, during 2014-2018.

BACKGROUND: Hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) triggered a significant public health challenge. This study explored the prevalence trends and key genetic characteristics of Hv-CRKP in one Shanghai suburbs hospital during 2014-2018. METHODS: During five years, Hv-CRKP strains identified from 2579 CRKP by specific PCR, were subjected to performed short- and long-read sequencing technology; epidemiological characteristics, antimicrobial-resistance genes (ARGs), virulence determinants, detailed plasmid profiles and conjugation efficiency were comprehensively investigated. RESULTS: 155 Hv-CRKP and 31 non-Hv-CRKP strains were sequenced. Hv-CRKP strains exhibited significant resistance to six common antibiotic classes (>92%). ST11 steadily increased and became the most prevalent ST (85.2%), followed by ST15 (8.5%), ST65 (2.6%), ST23 (1.9%), and ST86 (0.6%). ST11-KL64 (65.2%) rapidly increased from 0 in 2014 to 93.9% in 2018. blaKPC-2 was the primary carbapenemase gene (97.4%). Other ARGs switched from aac(3)-IId to aadA2 in aminoglycoside and from sul1 to sul2 in sulfanilamide. The time-dated phylogenetic tree was divided into four independent evolutionary clades. Clade 1 and 3 strains were mostly limited in the ICU, whereas Clade 2 strains were distributed among multiple departments. Compared to ybt14 in ICEKp12 in Clade 1, Clade 3 strains harbored ybt9 in ICEKp3 and blaCTX-M-65. Hv-CRKP infected more wards than non-Hv-CRKP and showed greater transmission capacity. Three plasmids containing crucial carbapenemase genes demonstrated their early transmission across China. CONCLUSION: The Hv-CRKP ST11-KL64 has rapidly replaced ST11-KL47 and emerged as the predominant epidemic subtype in various hospital wards, highlighting the importance of conducting comprehensive early surveillance for Hv-CRKP, especially in respiratory infections.

Short review: Geographical distribution of equine-associated pVAPA plasmids in Rhodococcus equi in the world.

Virulent Rhodococcus equi strains expressing virulence-associated 15-17 kDa protein (VapA) and having a large virulence plasmid (pVAPA) of 85-90 kb containing vapA gene are pathogenic for horses. In the last two decades, following pVAPA, two host-associated virulence plasmid types of R. equi have been discovered: a circular plasmid, pVAPB, associated with porcine isolates in 1995, and a recently detected linear plasmid, pVAPN, related to bovine and caprine isolates. Molecular epidemiological studies of R. equi infection in foals on horse-breeding farms in Japan and many countries around the world have been conducted in the last three decades, and the epidemiological studies using restriction enzyme digestion patterns of plasmid DNAs from virulent isolates have shown 14 distinct pVAPA subtypes and their geographical preference. This short review summarizes previous reports regarding equine-associated pVAPA subtypes in the world and discusses their geographic distribution from the standpoint of horse movements.

A novel virulence plasmid encoding yersiniabactin, salmochelin, and RmpADC from hypervirulent Klebsiella pneumoniae of distinct genetic backgrounds.

Hypervirulent Klebsiella pneumoniae (hvKP) is increasingly reported worldwide as a major clinical and public health threat. The virulence of hvKP is attributed largely to the carriage of virulence plasmids (KpVPs). To date, two dominant types of KpVP have been identified, namely, KpVP-1 and KpVP-2. In this study, we reported two hvKP strains from bloodstream infections that carry highly identical virulence plasmids that exhibited <40% coverage compared with KpVP-1 and KpVP-2. This novel virulence plasmid was designated KpVP-3. The two hvKP have different genetic backgrounds, which belonged to ST29-K54 and ST111-K63, respectively. They were both positive for the string test, highly virulent on the Galleria mellonella infection model, and possess high-level macrophage-killing resistance in vitro. Apart from the intrinsic non-susceptibility to ampicillin, both strains were susceptible to commonly used antibiotics. The virulence plasmid carried virulence genes rmpADC, iroBCDN (iro1), and the ybt locus (ybt4) which was not present on either KpVP-1 or KpVP-2. It did not carry antimicrobial resistance genes but carried an incomplete conjugation machinery containing only the traH and traF genes. The KpVP-3 plasmid was stably maintained in both hvKP strains and could not be eliminated with SDS treatment or by serial passage on stress-free agar plates. KpVP-3 was non-self-transmissible under experimental conditions. Data mining suggested KpVP-3-type plasmids have emerged in different countries including China, Australia, and the USA. The emergence of this novel virulence plasmid might pose a potential threat to public health. Heightened efforts are required to study its dissemination mechanism.

Construction and characterization of a saturated Tn-seq library of Salmonella Typhimurium ATCC 14028.

Salmonella enterica is an important foodborne pathogen. Here, we present the construction and characterization of a high-density transposon sequencing library of the Salmonella Typhimurium ATCC 14028 strain. Essential, advantageous, and disadvantageous genes for growth in rich culture medium were identified on the chromosome and the pSLT plasmid.

Plasmid-mediated virulence in Chlamydia.

Chlamydia trachomatis infection of ocular conjunctiva can lead to blindness, while infection of the female genital tract can lead to chronic pelvic pain, ectopic pregnancy, and/or infertility. Conjunctival and fallopian tube inflammation and the resulting disease sequelae are attributed to immune responses induced by chlamydial infection at these mucosal sites. The conserved chlamydial plasmid has been implicated in enhancing infection, via improved host cell entry and exit, and accelerating innate inflammatory responses that lead to tissue damage. The chlamydial plasmid encodes eight open reading frames, three of which have been associated with virulence: a secreted protein, Pgp3, and putative transcriptional regulators, Pgp4 and Pgp5. Although Pgp3 is an important plasmid-encoded virulence factor, recent studies suggest that chlamydial plasmid-mediated virulence extends beyond the expression of Pgp3. In this review, we discuss studies of genital, ocular, and gastrointestinal infection with C. trachomatis or C. muridarum that shed light on the role of the plasmid in disease development, and the potential for tissue and species-specific differences in plasmid-mediated pathogenesis. We also review evidence that plasmid-associated inflammation can be independent of bacterial burden. The functions of each of the plasmid-encoded proteins and potential molecular mechanisms for their role(s) in chlamydial virulence are discussed. Although the understanding of plasmid-associated virulence has expanded within the last decade, many questions related to how and to what extent the plasmid influences chlamydial infectivity and inflammation remain unknown, particularly with respect to human infections. Elucidating the answers to these questions could improve our understanding of how chlamydia augment infection and inflammation to cause disease.

Whole genome sequence of carbapenem-resistant hypermucoviscous Klebsiella pneumoniae K2-ST375 with blaNDM-harbouring conjugative IncX3 and pLVPK-like virulence plasmids from a patient in China.

OBJECTIVES: Carbapenem-resistant hypermucoviscous Klebsiella pneumoniae (CR-HMKP) poses unprecedented public health challenges. However, genomic information regarding the CR-HMKP K2-ST375 strain is scarce. The aim of this study was to characterize the whole genome sequence of the CR-HMKP K2-ST375 strain Kp0179 isolated from a male patient in China. METHODS: The whole genome of Kp0179 was sequenced using the DNBSEQ and Pacific Biosciences RSII platforms. The capsular serotype, multilocus sequence typing (MLST), antimicrobial resistance genes, and virulence factors were determined using available databases and bioinformatics tools. Conjugation experiments were performed using rifampicin-resistant Escherichia coli C600 as the recipient. RESULTS: The Kp0179 strain with hypermucoviscous phenotype was resistant to almost all ß-lactams, including ertapenem and imipenem. Whole genome sequencing revealed that Kp0179 belonged to K2-ST375 and contained blaNDM-IncX3 and a virulence plasmid ca. 121 kb. Kp0179 contained 5146 coding genes, 88 tRNAs, 25 rRNAs and 38 non-coding RNA genes. Among the six acquired antibiotic resistance genes, blaSHV-99, fosA, oqxAB were located on the chromosome, whereas blaNDM-1, qnrS1 and blaSHV-12 were located on the conjugative plasmid pNDM-Kp0179 (IncX3 type). Virulence gene analysis indicated that pLVPK-Kp0179 carried multiple virulence-encoding genes, such as iroBCDN, iucABCDiutA, rmpA and rmpA2. In addition to carrying a virulence plasmid, capsule formation (kvgA) and the type 3 fimbriae operon (mrkABCDFHIJ) were located on the chromosome of Kp0179. CONCLUSION: To our knowledge, this is the first report of a CR-HMKP K2-ST375 strain with a blaNDM-harboured conjugative IncX3 plasmid and a pLVPK-like virulence plasmid from a patient in China. Therefore, the spread of CR-HMKP K2-ST375 isolates in China should be closely monitored.

Acquisition of regulator on virulence plasmid of hypervirulent Klebsiella allows bacterial lifestyle switch in response to iron.

Hypervirulent Klebsiella pneumoniae causes liver abscess and potentially devastating metastatic complications. The majority of Klebsiella-induced liver abscess are caused by the CG23-I sublineage of hypervirulent Klebsiella pneumoniae. This and some other lineages possess a >200-kb virulence plasmid. We discovered a novel protein IroP nestled in the virulence plasmid-encoded salmochelin operon that cross-regulates and suppresses the promoter activity of chromosomal type 3 fimbriae (T3F) gene transcription. IroP is itself repressed by iron through the ferric uptake regulator. Iron-rich conditions increase T3F and suppress capsule mucoviscosity, leading to biofilm formation and cell adhesion. Conversely, iron-poor conditions cause a transcriptional switch to hypermucoid capsule production and T3F repression. The likely acquisition of iroP on mobile genetic elements and successful adaptive integration into the genetic circuitry of a major lineage of hypervirulent K. pneumoniae reveal a powerful example of plasmid chromosomal cross talk that confers an evolutionary advantage. Our discovery also addresses the conundrum of how the hypermucoid capsule that impedes adhesion could be regulated to facilitate biofilm formation and colonization. The acquired ability of the bacteria to alternate between a state favoring dissemination and one that favors colonization in response to iron availability through transcriptional regulation offers novel insights into the evolutionary success of this pathogen. IMPORTANCE Hypervirulent Klebsiella pneumoniae contributes to the majority of monomicrobial-induced liver abscess infections that can lead to several other metastatic complications. The large virulence plasmid is highly stable in major lineages, suggesting that it provides survival benefits. We discovered a protein IroP encoded on the virulence plasmid that suppresses expression of the type 3 fimbriae. IroP itself is regulated by iron, and we showed that iron regulates hypermucoid capsule production while inversely regulating type 3 fimbriae expression through IroP. The acquisition and integration of this inverse transcriptional switch between fimbriae and capsule mucoviscosity shows an evolved sophisticated plasmid-chromosomal cross talk that changes the behavior of hypervirulent K. pneumoniae in response to a key nutrient that could contribute to the evolutionary success of this pathogen.

Corrigendum: Epidemiological characteristics an outbreak of ST11 multidrug-resistant and hypervirulent Klebsiella pneumoniae in Anhui, China.

[This corrects the article DOI: 10.3389/fmicb.2022.996753.].