scholarly journals Mobilization of the nonconjugative virulence plasmid from hypervirulent Klebsiella pneumoniae

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Yanping Xu ◽  
Jianfeng Zhang ◽  
Meng Wang ◽  
Meng Liu ◽  
Guitian Liu ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
In Silico ◽  
In Silico Analysis ◽  
Conjugative Plasmid ◽  
Virulence Plasmid ◽  
E Coli ◽  
Virulence Plasmids ◽  
Carbapenem Resistant ◽  
Helper Plasmids ◽  
Silico Analysis

Abstract 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 XbaI 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.

scholarly journals Acquisition of the Conjugative Virulence Plasmid From a CG23 Hypervirulent Klebsiella pneumoniae Strain Enhances Bacterial Virulence

2021 ◽  
Vol 11 ◽  
Author(s):  
Dongxing Tian ◽  
Weiwen Wang ◽  
Meng Li ◽  
Wenjie Chen ◽  
Ying Zhou ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Evidence Base ◽  
Serum Resistance ◽  
Virulence Plasmid ◽  
E Coli ◽  
Virulence Plasmids ◽  
Carbapenem Resistant ◽  
High Level

The emergence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has become a hot topic and confounding problem for clinicians and researchers alike. Conjugative virulence plasmids have the potential to cause more threatening dissemination of hv-CRKP strains. We previously identified K2606, a CG23 clinical hypervirulent strain of Klebsiella pneumoniae harboring a conjugative virulence plasmid designated pK2606. In this study we examined hypervirulence levels using assays of biofilm formation, serum resistance, and wax larvae and mouse in vivo infection models. Moreover, to define the transfer ability of pK2606 and whether this confers hypervirulence to other strains we performed plasmid transconjugation experiments between K2606 and the ST11 CRKP strain HS11286 along with E. coli J53. We found that although biofilm formation and serum resistance were not significantly increased, the transconjugants acquired the ability of produce high level of siderophores and also caused high mortality of wax larvae and mice. Furthermore, we identified pK2606-like conjugative virulence plasmids in GenBank, providing evidence that such plasmids may have begun to spread throughout China. These findings provide an evidence base for the possible mechanisms of the emergence of hv-CRKP strains and highlight the potential of pK2606-like conjugative virulence plasmids to spread worldwide.

scholarly journals Two Distinct Modes of Lysis Regulation in Campylobacter Fletchervirus and Firehammervirus Phages

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1247
Author(s):  
Athina Zampara ◽  
Stephen J. Ahern ◽  
Yves Briers ◽  
Lone Brøndsted ◽  
Martine Camilla Holst Sørensen
Keyword(s):  
Signal Peptide ◽  
In Silico ◽  
Protein Domains ◽  
In Silico Analysis ◽  
Cell Lysis ◽  
E Coli ◽  
Sec Pathway ◽  
Gene Functions ◽  
Lytic Genes ◽  
Silico Analysis

Campylobacter phages are divided into two genera; Fletchervirus and Firehammervirus, showing only limited intergenus homology. Here, we aim to identify the lytic genes of both genera using two representative phages (F352 and F379) from our collection. We performed a detailed in silico analysis searching for conserved protein domains and found that the predicted lytic genes are not organized into lysis cassettes but are conserved within each genus. To verify the function of selected lytic genes, the proteins were expressed in E. coli, followed by lytic assays. Our results show that Fletchervirus phages encode a typical signal peptide (SP) endolysin dependent on the Sec-pathway for translocation and a holin for activation. In contrast, Firehammervirus phages encode a novel endolysin that does not belong to currently described endolysin groups. This endolysin also uses the Sec-pathway for translocation but induces lysis of E. coli after overexpression. Interestingly, co-expression of this endolysin with an overlapping gene delayed and limited cell lysis, suggesting that this gene functions as a lysis inhibitor. These results indicate that Firehammervirus phages regulate lysis timing by a yet undescribed mechanism. In conclusion, we found that the two Campylobacter phage genera control lysis by two distinct mechanisms.

scholarly journals Co-existence of virulence factors and antibiotic resistance in new Klebsiella pneumoniae clones emerging in south of Italy

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Teresa Fasciana ◽  
Bernardina Gentile ◽  
Maria Aquilina ◽  
Andrea Ciammaruconi ◽  
Chiara Mascarella ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Virulence Factors ◽  
In Silico ◽  
Geographical Area ◽  
In Silico Analysis ◽  
University Hospital ◽  
Uptake System ◽  
Genes Encoding ◽  
Silico Analysis

Abstract Background Endemic presence of Klebsiella pneumoniae resistant to carbapenem in Italy has been due principally to the clonal expansion of CC258 isolates; however, recent studies suggest an ongoing epidemiological change in this geographical area. Methods 50 K. pneumoniae strains, 25 carbapenem-resistant (CR-Kp) and 25 susceptible (CS-Kp), collected from march 2014 to march 2016 at the Laboratory of Bacteriology of the Paolo Giaccone Polyclinic University hospital of Palermo, Italy, were characterized for antibiotic susceptibility and fully sequenced by next generation sequencing (NGS) for the in silico analysis of resistome, virulome, multi-locus sequence typing (MLST) and core single nucleotide polymorphism (SNP) genotypes Results MLST in silico analysis of CR-Kp showed that 52% of isolates belonged to CC258, followed by ST395 (12%), ST307 (12%), ST392 (8%), ST348 (8%), ST405 (4%) and ST101 (4%). In the CS-Kp group, the most represented isolate was ST405 (20%), followed by ST392 and ST15 (12%), ST395, ST307 and ST1727 (8%). The in silico β-lactamase analysis of the CR-Kp group showed that the most detected gene was blaSHV (100%), followed by blaTEM (92%), blaKPC (88%), blaOXA (88%) and blaCTX-M (32%). The virulome analysis detected mrk operon in all studied isolates, and wzi-2 was found in three CR-Kp isolates (12%). Furthermore, the distribution of virulence genes encoding for the yersiniabactin system, its receptor fyuA and the aerobactin system did not show significant distribution differences between CR-Kp and CS-Kp, whereas the Klebsiella ferrous iron uptake system (kfuA, kfuB and kfuC genes), the two-component system kvgAS and the microcin E495 were significantly (p < 0.05) prevalent in the CS-Kp group compared to the CR-Kp group. Core SNP genotyping, correlating with the MLST data, allowed greater strain tracking and discrimination than MLST analysis. Conclusions Our data support the idea that an epidemiological change is ongoing in the Palermo area (Sicily, Italy). In addition, our analysis revealed the co-existence of antibiotic resistance and virulence factors in CR-Kp isolates; this characteristic should be considered for future genomic surveillance studies.

scholarly journals Aerobactin Seems To Be a Promising Marker Compared With Unstable RmpA2 for the Identification of Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae: In Silico and In Vitro Evidence

2021 ◽  
Vol 11 ◽  
Author(s):  
Chaitra Shankar ◽  
Soumya Basu ◽  
Binesh Lal ◽  
Sathiya Shanmugam ◽  
Karthick Vasudevan ◽  
...  
Keyword(s):  
High Risk ◽  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
In Silico ◽  
Fitness Cost ◽  
Virulence Plasmid ◽  
Virulence Determinants ◽  
Carbapenem Resistant ◽  
Antimicrobial Resistance Genes

BackgroundThe incidence of hypervirulent (hv) carbapenem-resistant (CR) Klebsiella pneumoniae (Kp) is increasing globally among various clones and is also responsible for nosocomial infections. The CR-hvKp is formed by the uptake of a virulence plasmid by endemic high-risk clones or by the uptake of plasmids carrying antimicrobial resistance genes by the virulent clones. Here, we describe CR-hvKp from India belonging to high-risk clones that have acquired a virulence plasmid and are phenotypically unidentified due to lack of hypermucoviscosity.MethodsTwenty-seven CRKp isolates were identified to possess rmpA2 by whole-genome sequencing; and resistance and virulence determinants were characterized. By in silico protein modeling (and validation), protein backbone stability analysis, and coarse dynamics study, the fitness of RmpA, RmpA2, and aerobactin-associated proteins-IucA and IutA, were determined to establish a reliable marker for clinical identification of CR-hvKp.ResultsThe CR-hvKp belonged to multidrug-resistant (MDR) high-risk clones such as CG11, CG43, ST15, and ST231 and carried OXA-232 as the predominant carbapenemase followed by NDM. The virulence plasmid belonged to IncHI1B replicon type and carried frameshifted and truncated rmpA and rmpA2. This resulted in a lack of hypermucoviscous phenotype. However, functional aerobactin was expressed in all high-risk clones. In silico analysis portrayed that IucA and IutA were more stable than classical RmpA. Furthermore, IucA and IutA had lower conformational fluctuations in the functional domains than the non-functional RmpA2, which increases the fitness cost of the latter for its maintenance and expression among CR-hvKp. Hence, RmpA and RmpA2 are likely to be lost among CR-hvKp owing to the increased fitness cost while coding for essential antimicrobial resistance and virulence factors.ConclusionIncreasing incidence of convergence of AMR and virulence is observed among K. pneumoniae globally, which warrants the need for reliable markers for identifying CR-hvKp. The presence of non-functional RmpA2 among high-risk clones highlights the significance of molecular identification of CR-hvKp. The negative string test due to non-functional RmpA2 among CR-hvKp isolates challenges phenotypic screening and faster identification of this pathotype. This can potentially be counteracted by projecting aerobactin as a stable, constitutively expressed, and functional marker for rapidly evolving CR-hvKp.

Camelus dromedarius glucose transporter 4: in silico analysis, cloning, expression, purification and characterisation in E. coli

2017 ◽  
Vol 123 (4) ◽  
pp. 254-264
Author(s):  
Anwar Ahmed ◽  
Mohammed Arshad ◽  
Ajamaluddin Malik ◽  
Shama Parveen ◽  
Abdulrahman M. Alsenaidy
Keyword(s):  
In Silico ◽  
Glucose Transporter ◽  
In Silico Analysis ◽  
Camelus Dromedarius ◽  
Glucose Transporter 4 ◽  
E Coli ◽  
Silico Analysis

scholarly journals In Silico Analysis, Cloning and Expression of Recombinant CD166 in E. coli BL21 (DE3) as a Marker for Detection and Treatment of Colorectal Cancer

2017 ◽  
Vol 06 (01) ◽  
Author(s):  
Vahid Marmari ◽  
Habibollah Mahmoodzadeh ◽  
Hassan Dana ◽  
Ghanbar Mahmoodi Chalbatani ◽  
Ali Mazraeh ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
In Silico ◽  
In Silico Analysis ◽  
Cloning And Expression ◽  
E Coli ◽  
Silico Analysis

scholarly journals Prevalence of Carbapenem-Resistant Klebsiella pneumoniae Co-Harboring blaKPC-Carrying Plasmid and pLVPK-Like Virulence Plasmid in Bloodstream Infections

2021 ◽  
Vol 10 ◽  
Author(s):  
Fang-ling Du ◽  
Qi-sen Huang ◽  
Dan-dan Wei ◽  
Yan-fang Mei ◽  
Dan Long ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Galleria Mellonella ◽  
Bloodstream Infections ◽  
Multi Drug Resistance ◽  
Virulence Plasmid ◽  
E Coli ◽  
Plasmid Gene ◽  
Carbapenem Resistant ◽  
Large Plasmids ◽  
Serotype K2

This study aimed to characterize carbapenem-resistant Klebsiella pneumoniae (CR-KP) co-harboring blaKPC-2-carrying plasmid and pLVPK-like virulence plasmid. Between December 2017 and April 2018, 24 CR-KP isolates were recovered from 24 patients with bacteremia. The mortality was 66.7%. Pulsed-field gel electrophoresis and multilocus sequence typing results indicated four clusters, of which cluster A (n = 21, 87.5%) belonged to ST11 and the three remaining isolates (ST412, ST65, ST23) had different pulsotypes (cluster B, C, D). The blaKPC-2-carrying plasmids all belonged to IncFIIK type, and the size ranged from 100 to 390 kb. Nineteen strains (79.2%) had a 219-kb virulence plasmid possessed high similarity to pLVPK from CG43 with serotype K2. Two strains had a 224-kb virulence plasmid resembled plasmid pK2044 from K. pneumoniae NTUH-K2044(ST23). Moreover, three strains carried three different hybrid resistance- and virulence-encoding plasmids. Conjugation assays showed that both blaKPC-2 and rmpA2 genes could be successfully transferred to E. coli J53 in 62.5% of the strains at frequencies of 4.5 × 10−6 to 2.4 × 10−4, of which three co-transferred blaKPC-2 along with rmpA2 in large plasmids. Infection assays in the Galleria mellonella model demonstrated the virulence level of these isolates was found to be consistently higher than that of classic Klebsiella pneumoniae. In conclusion, CR-KP co-harboring blaKPC-2-carrying plasmid and pLVPK-like virulence plasmid were characterized by multi-drug resistance, enhanced virulence, and transferability, and should, therefore, be regarded as a real superbug that could pose a serious threat to public health. Hence, heightened efforts are urgently needed to avoid its co-transmission of the virulent plasmid (gene) and resistant plasmid (gene) in clinical isolates.

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