The Emergence of Novel Sequence Type Strains Reveals an Evolutionary Process of Intraspecies Clone Shifting in ICU-Spreading Carbapenem-Resistant Klebsiella pneumoniae

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an urgent public health problem worldwide, and its rapid evolution in the clinical environment has been a major concern. A total of 99 CRKP isolates spreading in the intensive care unit (ICU) setting were included and subjected to whole-genome sequencing, and their sequence types (STs), serotype loci, and virulence determinants were screened based on genome data. The phylogenetic structure was reconstructed based on the core genome multilocus sequence typing method. Regions of recombination were assessed. Biofilm formation, serum resistance assays, and a Galleria mellonella infection model were used to evaluate strain virulence. A novel ST, designated ST4496, emerged in the ICU and spread for 6 months before its disappearance. ST4496 was closely related to ST11, with only a single-allele variant, and ST11 is the most dominant clinical clone in China. Recombination events occurred at capsule biosynthesis loci and divided the strains of ST11 and its derivative ST4496 into three clusters, including ST11-KL47, ST11-KL64, and ST4496-KL47. The phylogenetic structure indicated that ST11-KL47 was probably the origin of ST11-related strain evolution and presented more diversity in terms of both sequence similarity and phenotypes. ST4496-KL47 cluster strains presented less virulence than ST11-KL64, which was probably one of the factors preventing the former from spreading widely. In conclusion, ST4496-KL47 was probably derived from ST11-KL47 via intraspecies shifting but was less competitive than ST11-KL64, which also evolved from ST11-KL47 and developed increased virulence via capsule biosynthesis locus recombination. ST11-KL64 has the potential to be the predominant CRKP clone in China.

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Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella

Pathogens and Disease ◽

10.1093/femspd/ftab009 ◽

2021 ◽

Author(s):

Sebastian Bruchmann ◽

Theresa Feltwell ◽

Julian Parkhill ◽

Francesca L Short

Keyword(s):

Klebsiella Pneumoniae ◽

Large Scale ◽

Galleria Mellonella ◽

Fitness Landscape ◽

Dominant Role ◽

Bacterial Pathogens ◽

Model Organism ◽

Multidrug Resistant ◽

Infection Model ◽

Virulence Determinants

Abstract Infections caused by Klebsiella pneumoniae are a major public health threat. Extensively drug-resistant and even pan-resistant strains have been reported. Understanding K. pneumoniae pathogenesis is hampered by the fact that murine models of infection offer limited resolution for non-hypervirulent strains which cause the majority of infections. The insect Galleria mellonella larva is a widely used alternative model organism for bacterial pathogens. We have performed genome-scale fitness profiling of a multidrug-resistant K. pneumoniae ST258 strain during infection of G. mellonella, to determine if this model is suitable for large-scale virulence factor discovery in this pathogen. Our results demonstrated a dominant role for surface polysaccharides in infection, with contributions from siderophores, cell envelope proteins, purine biosynthesis genes and additional genes of unknown function. Comparison with a hypervirulent strain, ATCC 43816, revealed substantial overlap in important infection-related genes, as well as additional putative virulence factors specific to ST258, reflecting strain-dependent fitness effects. Our analysis also identified a role for the metalloregulatory protein NfeR (YqjI) in virulence. Overall, this study offers new insight into the infection fitness landscape of K. pneumoniae, and provides a framework for using the highly flexible and easily scalable G. mellonella infection model to dissect molecular virulence mechanisms of bacterial pathogens.

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Identifying virulence determinants of multidrug-resistant Klebsiella pneumoniae in Galleria mellonella

10.1101/2020.10.30.362657 ◽

2020 ◽

Author(s):

Sebastian Bruchmann ◽

Theresa Feltwell ◽

Julian Parkhill ◽

Francesca L. Short

Keyword(s):

Klebsiella Pneumoniae ◽

Large Scale ◽

Galleria Mellonella ◽

Fitness Landscape ◽

Cell Envelope ◽

Dominant Role ◽

Multidrug Resistant ◽

Infection Model ◽

Virulence Determinants ◽

Genome Scale

AbstractInfections caused by Klebsiella pneumoniae are a major public health threat. Extensively drug-resistant and even pan-resistant strains have been reported. Understanding K. pneumoniae pathogenesis is hampered by the fact that murine models of infection offer limited resolution for the non-hypervirulent strains which cause the majority of infections. We have performed genome-scale fitness profiling of a multidrug-resistant K. pneumoniae ST258 strain during infection of the insect Galleria mellonella, with the aim to determine if this model is suitable for large-scale virulence factor discovery in this pathogen. Our results demonstrated a dominant role for surface polysaccharides in infection, with contributions from siderophores, cell envelope proteins, purine biosynthesis genes and additional genes of unknown function. Comparison with a hypervirulent strain, ATCC 43816, revealed substantial overlap in important infection-related genes, as well as additional putative virulence factors that may be specific to ST258. Our analysis also identified a role for the metalloregulatory protein NfeR (also called YqjI) in virulence. Overall, this study offers new insight into the infection fitness landscape of K. pneumoniae ST258, and provides a framework for using the highly flexible, scalable G. mellonella infection model to dissect the molecular virulence mechanisms of K. pneumoniae and other bacterial pathogens.

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Evolution of hypervirulence in carbapenem-resistant Klebsiella pneumoniae in China: a multicentre, molecular epidemiological analysis

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz446 ◽

2019 ◽

Vol 75 (2) ◽

pp. 327-336 ◽

Cited By ~ 13

Author(s):

Yawei Zhang ◽

Longyang Jin ◽

Pengwen Ouyang ◽

Qi Wang ◽

Ruobing Wang ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Virulence Genes ◽

Galleria Mellonella ◽

Infection Model ◽

Epidemiological Analysis ◽

Virulence Plasmids ◽

Carbapenem Resistant ◽

Resistance Plasmid ◽

Serum Killing ◽

Evolution Of Resistance

Abstract Objectives Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) have been increasingly reported in China. Here, a multicentre, longitudinal surveillance study on CR-hvKP is described. Methods We retrospectively investigated carbapenem-resistant K. pneumoniae (CRKP) in 56 centres across China during 2015–17 and screened the virulence genes (iucA, iroN, rmpA and rmpA2) for the presence of virulence plasmids. Hypermucoviscosity, serum killing and Galleria mellonella lethality experiments were conducted to identify CR-hvKP among strains with all four virulence genes. Capsule typing, fitness and plasmid features of CR-hvKP were also investigated. Results A total of 1052 CRKP were collected. Among these, 34.2% (360/1052) carried virulence genes and 72 of them had all four of the virulence genes tested. Fifty-five (76.4%) were considered to be CR-hvKP using the G. mellonella infection model, with KPC-2-producing K64-ST11 being the most common type (80%, 44/55). Prevalence of CR-hvKP differed greatly between regions, with the highest in Henan (25.4%, 17/67) and Shandong (25.8%, 25/97). A significant increase in CR-hvKP among KPC-2-producing ST11 strains was observed, from 2.1% (3/141) in 2015 to 7.0% (23/329) in 2017 (P=0.045). Alarmingly, compared with classic CRKP, no difference in growth was found among CR-hvKP (P=0.7028), suggesting a potential risk for dissemination. The hybrid virulence and resistance-encoding plasmid evolved from pLVPK and the resistance plasmid harbouring blaKPC-2, indicating evolution existed between the hypervirulence and hyper-resistance plasmid. Conclusions CR-hvKP were more frequently detected than previously assumed, especially among KPC-2-producing ST11. Dissemination of hypervirulence could be extremely rapid due to limited fitness cost. Also, the evolution of resistance genes into hypervirulence plasmids was identified, presenting significant challenges for public health and infection control.

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Yersiniabactin, Colibactin and Wider Resistome Contribute to Enhanced Virulence and Persistence of KPC-2-Producing Klebsiella pneumoniae CG258 in South America

10.1101/435750 ◽

2018 ◽

Cited By ~ 1

Author(s):

Louise T. Cerdeira ◽

Fernanda Esposito ◽

Margaret M. C. Lam ◽

Kelly L. Wyres ◽

Ryan R. Wick ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

South America ◽

Galleria Mellonella ◽

Genomic Analysis ◽

Public Health Issue ◽

Infection Model ◽

Veterinary Antibiotics ◽

Carbapenem Resistant ◽

Clonal Origin ◽

Sequence Types

AbstractThe emergence and dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) is a worrisome public health issue compromising the treatment and outcome of infections caused by this pathogen. We performed a detailed virulome and resistome analysis of representative KPC- and/or CTX-M-producing K. pneumoniae belonging to clonal group (CG) 258 (sequence types ST11, ST258, ST340, ST437), circulating in Argentina, Brazil, Chile, Colombia and Peru; with further evaluation of the virulence behavior using the Galleria mellonella infection model. Genomic analysis of K. pneumoniae strains recovered from the human-animal-environment interface revealed a wide resistome characterized by the presence of genes and mutations conferring resistance to human and veterinary antibiotics, quaternary ammonium compounds (QACs) and heavy metals. Plasmid Inc typing revealed the presence of a wide diversity of replicon types with IncF, IncN, IncR and Col-like being frequently detected. Moreover, KPC-2-producing K. pneumoniae belonging to ST11 (KL-64 andKL-105) and ST340 (KL-15) carried multiple variants of distinct yersiniabactin siderophore (ybt) and/or genotoxic colibactin (clb) genes. In this regard, ICEKp3, ICEKp4 and ICEKp12 were identified in strains belonging to ST11 and ST340, recovered from Argentina, Brazil, Chile and Colombia; whereas ybt 17 and a novel ybt sequence type (YbST346) were identified together with clb in ICEKp10 structures from ST11 and ST258, from Brazil and Colombia, respectively. K. pneumoniae ST11 (ICEKp10/YbST346 and ICEKp4/ybt 10) strains killed 100% of wax moth larvae, in a similar way to hypervirulent K1/ST23 strain (ybt- and clb-negative) carrying the pLVPK-like plasmid, indicating enhanced virulence. In summary, our results indicate that yersiniabactin, colibactin and an expanded resistome have contributed to enhanced virulence and persistence of KPC-2-producing K. pneumoniae CG258 in South America. Therefore, active surveillance of hospital-associated lineages of K. pneumoniae should not only focus on clonal origin and antimicrobial resistance, but also on the virulence factors ybt and clb.

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Phage Resistance Is Associated with Decreased Virulence in KPC-Producing Klebsiella pneumoniae of the Clonal Group 258 Clade II Lineage

Microorganisms ◽

10.3390/microorganisms9040762 ◽

2021 ◽

Vol 9 (4) ◽

pp. 762

Author(s):

Lucia Henrici De Angelis ◽

Noemi Poerio ◽

Vincenzo Di Pilato ◽

Federica De Santis ◽

Alberto Antonelli ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Parental Strain ◽

Bacterial Infections ◽

Capsular Polysaccharide ◽

Galleria Mellonella ◽

Bacterial Pathogen ◽

Phage Resistance ◽

Infection Model ◽

Lytic Phage ◽

Susceptible Host

Phage therapy is now reconsidered with interest in the treatment of bacterial infections. A major piece of information for this application is the definition of the molecular targets exploited by phages to infect bacteria. Here, the genetic basis of resistance to the lytic phage φBO1E by its susceptible host Klebsiella pneumoniae KKBO-1 has been investigated. KKBO-1 phage-resistant mutants were obtained by infection at high multiplicity. One mutant, designated BO-FR-1, was selected for subsequent experiments, including virulence assessment in a Galleria mellonella infection model and characterization by whole-genome sequencing. Infection with BO-FR-1 was associated with a significantly lower mortality when compared to that of the parental strain. The BO-FR-1 genome differed from KKBO-1 by a single nonsense mutation into the wbaP gene, which encodes a glycosyltransferase involved in the first step of the biosynthesis of the capsular polysaccharide (CPS). Phage susceptibility was restored when BO-FR-1 was complemented with the constitutive wbaP gene. Our results demonstrated that φBO1E infects KKBO-1 targeting the bacterial CPS. Interestingly, BO-FR-1 was less virulent than the parental strain, suggesting that in the context of the interplay among phage, bacterial pathogen and host, the emergence of phage resistance may be beneficial for the host.

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Emergence of ceftazidime/avibactam resistance in KPC-3-producing Klebsiella pneumoniae in vivo

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz330 ◽

2019 ◽

Vol 74 (11) ◽

pp. 3211-3216 ◽

Cited By ~ 13

Author(s):

Stephan Göttig ◽

Denia Frank ◽

Eleonora Mungo ◽

Anika Nolte ◽

Michael Hogardt ◽

...

Keyword(s):

Combination Therapy ◽

Klebsiella Pneumoniae ◽

Selection Pressure ◽

Galleria Mellonella ◽

Phenotypic Characterization ◽

Infection Model ◽

Development Of Resistance ◽

Time Kill

Abstract Objectives The β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam is active against KPC-producing Enterobacterales. Herein, we present molecular and phenotypic characterization of ceftazidime/avibactam resistance in KPC-3-producing Klebsiella pneumoniae that emerged in vivo and in vitro. Methods Sequence analysis of blaKPC-3 was performed from clinical and in vitro-generated ceftazidime/avibactam-resistant K. pneumoniae isolates. Time–kill kinetics and the Galleria mellonella infection model were applied to evaluate the activity of ceftazidime/avibactam and imipenem alone and in combination. Results The ceftazidime/avibactam-resistant clinical K. pneumoniae isolate revealed the amino acid change D179Y in KPC-3. Sixteen novel mutational changes in KPC-3 among in vitro-selected ceftazidime/avibactam-resistant isolates were described. Time–kill kinetics showed the emergence of a resistant subpopulation under selection pressure with either imipenem or ceftazidime/avibactam. However, combined selection pressure with imipenem plus ceftazidime/avibactam prevented the development of resistance and resulted in bactericidal activity. Concordantly, the G. mellonella infection model revealed that monotherapy with ceftazidime/avibactam is prone to select for resistance in vivo and that combination therapy with imipenem results in significantly better survival. Conclusions Ceftazidime/avibactam is a valuable antibiotic against MDR and carbapenem-resistant Enterobacterales. Based on time–kill kinetics as well as an in vivo infection model we postulate a combination therapy of ceftazidime/avibactam and imipenem as a strategy to prevent the development of ceftazidime/avibactam resistance in KPC-producing Enterobacterales in vivo.

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An Important Public Health Problem "Carbapenem Resistant Enterobacteriaceae Frequency": An Example From A Training And Research Hospital

Gevher Nesibe Journal, IESDR ◽

10.46648/gnj.256 ◽

2021 ◽

Vol 6 (14) ◽

pp. 51-55

Author(s):

Ülkü VERANYURT ◽

Betül AKALIN

Keyword(s):

Public Health ◽

Klebsiella Pneumoniae ◽

Klebsiella Oxytoca ◽

Public Health Problem ◽

Important Public Health Problem ◽

Klebsiella Species ◽

Research Hospital ◽

Control Programs ◽

Carbapenem Resistant ◽

Training And Research

Background: Carbapenem-resistant Enterobacterales (CRE) infections are a significant threat to public health due to the limited availability of antibiotics and the effect on mortality. This study was conducted retrospectively to determine the prevalence of CRE in a teaching and research hospital in Istanbul. Materials and Methods: In 2016, 2017 and in the first half of 2018 Klebsiella species were evaluated retrospectively in culture samples that were sent to Microbiology Laboratory in an educational hospital. The typing of Klebsiella species were performed with MALDITOF-MS device (Biomerieux, France). Imipenem, merapenem, ertapenem susceptibilities of the strains were evaluated with VITEC2 Compact (Biomerieux, France) according to EUCAST (European Committee on Antimicrobial Susceptibility Testing). If the strains were found to be resistant, the results were confirmed by the antibiotic gradient test. Results: In our study, 257 samples; Klebsiella oxytoca 9 (3.49%) and Klebsiella pneumoniae 248 (96.51%) were detected. 130 endotracheal aspirates, 57 wounds, 34 blood, 15 urine, 9 sputum, 3 catheters, 4 tissue biopsies, 2 mediastinum, 2 peritoneal fluid and 1 pleural fluid strains were identified. While none of the Klebsiella oxytoca strains were resistant to carbapenems, the percentages of Klebsiella pneumoniae resistance against imipenem, merapenem and ertapenem were found to be 23.29%, 16.94% and 29.44% respectively. Conclusion: The increasing problem of carbapenem-resistant (CR) Klebsiella pneumoniae in the last decade has been observed in our hospital for the last 2 years. CR strains often show increased resistance to other antibiotics and their treatment possibilities are limited. It increases the importance of controlling this factor. The application of effective infection control programs and the use of rational antibiotics are of great importance.

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An Outbreak of Carbapenem-Resistant Klebsiella Pneumoniae of K57 Capsular Serotype in an Emergency Intensive Care Unit of a Teaching Hospital in China

10.21203/rs.3.rs-96882/v1 ◽

2020 ◽

Author(s):

Chunhong Shao ◽

Yan Jin ◽

Shuang Liu ◽

Meijie Jiang ◽

Shuping Zhao

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

Klebsiella Pneumoniae ◽

Teaching Hospital ◽

Galleria Mellonella ◽

Pulse Field ◽

Carbapenem Resistant ◽

Surrounding Environment ◽

Antimicrobial Resistance Genes ◽

Broth Microdilution Method

Abstract Background: Klebsiella pneumoniae is a common causative pathogen of nosocomial infections. The emergence of carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) strains has further increased the threat posed by this bacterium. Here, we described an outbreak of 32 CR-hvKP isolates from the emergency intensive care unit (EICU) of a teaching hospital in China. Methods: From January 29, 2019 to March 11, 2019, 32 CRKp isolates were collected from 6 patients and their surrounding environment in EICU. Patient information including age, gender, length of EICU stay, diagnosis, treatment, and outcomes were obtained from electronic medical records. The isolates were identified using Vitek-MS system. The hypermucoviscosity phenotype was determined by the “string test”. Antimicrobial susceptibility testing was performed using VITEK 2 compact system, E-test or the broth microdilution method. All isolates were serotyped for K1, K2, K5, K20, K54, and K57 serotypes, antimicrobial resistance genes and twelve virulence-associated genes were screened using PCR and DNA sequencing. Multilocus sequence typing (MLST) and pulse-field gel electrophoresis (PFGE) were employed to characterize the genetic relationships among the CPKP isolates. The virulence capability of 11 CRKp isolates from 6 patients was evaluated through Galleria mellonella larva infection assay. Results: This outbreak involved 6 patients and lasted for 40 days. All 32 CR-hvKp isolates were obtained from 6 patients and their surrounding environment. PFGE showed that all 32 isolates belonged to one cluster, and MLST revealed that belonged to ST11. All isolates exhibited high resistance to β-lactam antibiotics, quinolones, and aminoglycosides. They were susceptible to ceftazidime/averbatan, tigecycline, and colistin. All 32 isolates harbored multiple resistance determinants, including blaKPC-2, blaSHV-11, blaTEM-1, rmtB, and qnrD. The serotype of all 32 isolates was K57 that was rarely reported. In the virulence gene analysis, all 32 isolates contained 6 virulence genes, namely, fimH, iucB, mrkD, rmpA, uge, and wabG. Infection assays demonstrated high mortality in the Galleria mellonella model. Following measures implemented by the hospital, the outbreak was controlled. The mortality rate was 83.3%.Conclusions: The epidemiology of CR-hvKP should be monitored closely to detect early indications of this emerging public health threat.

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Colistin Heteroresistance Is Largely Undetected among Carbapenem-Resistant Enterobacterales in the United States

mBio ◽

10.1128/mbio.02881-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Victor I. Band ◽

Sarah W. Satola ◽

Richard D. Smith ◽

David A. Hufnagel ◽

Chris Bower ◽

...

Keyword(s):

United States ◽

Klebsiella Pneumoniae ◽

Diagnostic Testing ◽

Cladistic Analysis ◽

The United States ◽

Clinical Diagnostics ◽

Infection Model ◽

Content Type ◽

Carbapenem Resistant ◽

Clinical Diagnostic

ABSTRACT Heteroresistance is a form of antibiotic resistance where a bacterial strain is comprised of a minor resistant subpopulation and a majority susceptible subpopulation. We showed previously that colistin heteroresistance can mediate the failure of colistin therapy in an in vivo infection model, even for isolates designated susceptible by clinical diagnostics. We sought to characterize the extent of colistin heteroresistance among the highly drug-resistant carbapenem-resistant Enterobacterales (CRE). We screened 408 isolates for colistin heteroresistance. These isolates were collected between 2012 and 2015 in eight U.S. states as part of active surveillance for CRE. Colistin heteroresistance was detected in 10.1% (41/408) of isolates, and it was more common than conventional homogenous resistance (7.1%, 29/408). Most (93.2%, 38/41) of these heteroresistant isolates were classified as colistin susceptible by standard clinical diagnostic testing. The frequency of colistin heteroresistance was greatest in 2015, the last year of the study. This was especially true among Enterobacter isolates, of which specific species had the highest rates of heteroresistance. Among Klebsiella pneumoniae isolates, which were the majority of isolates tested, there was a closely related cluster of colistin-heteroresistant ST-258 isolates found mostly in Georgia. However, cladistic analysis revealed that, overall, there was significant diversity in the genetic backgrounds of heteroresistant K. pneumoniae isolates. These findings suggest that due to being largely undetected in the clinic, colistin heteroresistance among CRE is underappreciated in the United States. IMPORTANCE Heteroresistance is an underappreciated phenomenon that may be the cause of some unexplained antibiotic treatment failures. Misclassification of heteroresistant isolates as susceptible may lead to inappropriate therapy. Heteroresistance to colistin was more common than conventional resistance and was overwhelmingly misclassified as susceptibility by clinical diagnostic testing. Higher proportions of colistin heteroresistance observed in certain Enterobacter species and clustering among heteroresistant Klebsiella pneumoniae strains may inform colistin treatment recommendations. Overall, the rate of colistin nonsusceptibility was more than double the level detected by clinical diagnostics, suggesting that the prevalence of colistin nonsusceptibility among CRE may be higher than currently appreciated in the United States.

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