Esposito, Eliana Pia (2020) Molecular epidemiology of antimicrobial resistance and virulence in epidemic Klebsiella pneumoniae clonal lineages. [Tesi di dottorato]

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Tipologia del documento:	Tesi di dottorato
Lingua:	English
Titolo:	Molecular epidemiology of antimicrobial resistance and virulence in epidemic Klebsiella pneumoniae clonal lineages.
Autori:	Autore Email Esposito, Eliana Pia elianapia.esposito@unina.it
Data:	12 Marzo 2020
Numero di pagine:	68
Istituzione:	Università degli Studi di Napoli Federico II
Dipartimento:	Sanità Pubblica
Dottorato:	Sanità pubblica e medicina preventiva
Ciclo di dottorato:	32
Coordinatore del Corso di dottorato:	nome email Troncone, Giancarlo giancarlo.troncone@unina.it
Tutor:	nome email Zarrilli, Raffaele [non definito]
Data:	12 Marzo 2020
Numero di pagine:	68
Parole chiave:	Klebsiella pneumoniae, antimicrobial resistance, virulence, clonal lineages
Settori scientifico-disciplinari del MIUR:	Area 06 - Scienze mediche > MED/07 - Microbiologia e microbiologia clinica Area 06 - Scienze mediche > MED/42 - Igiene generale e applicat
Depositato il:	23 Mar 2020 10:45
Ultima modifica:	10 Nov 2021 09:45
URI:	http://www.fedoa.unina.it/id/eprint/13092

Abstract

Klebsiella pneumoniae is a Gram-negative, encapsulated, nonmotile bacterium belonging to the Enterobacteriaceae family. K. pneumoniae is one of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) recognized as the most common opportunistic pathogens in nosocomial infections. However, over the past three decades, the notoriety of K. pneumoniae is due to the emergence of strains that have acquired additional genetic traits and become either antibiotic resistant or hypervirulent, associated with hospital outbreaks and severe community-acquired infections, respectively. The global dissemination of K. pneumoniae has been largely attributed to the increasing incidence of extended spectrum beta-lactamase (ESBL) and carbapenem-resistant (CRE) isolates, because of their capability to acquire antimicrobial resistance (AMR) genes primarily due to horizontal gene transfer (HGT) aided by plasmids and mobile genetic elements. The first study of this thesis analyzes the mechanism of carbapenem resistance acquisition of multi-drug resistant K. pneumoniae isolates from 20 neonates in the neonatal intensive care unit (NICU) of the V. Monaldi Hospital in Naples, Italy. Genotype analysis by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) identified PFGE type A and subtypes A1 and A2 in 17, 2, and 1 isolates, respectively, and assigned all isolates to sequence type (ST) 104. K. pneumoniae isolates were resistant to all classes of beta-lactams, including carbapenems, fosfomycin, gentamicin, and trimethoprim–sulfamethoxazole, but susceptible to quinolones, amikacin, and colistin. Conjugation experiments demonstrated that resistance to third-generation cephems and imipenem could be transferred along with an IncA/C plasmid containing the ESBL blaSHV-12 and carbapenem-hydrolyzing metallo-beta-lactamase blaVIM-1 genes. The plasmid that we called pIncAC_KP4898 was 156,252 bp in size and included a typical IncA/C backbone, which was assigned to ST12 and core genome (cg) ST12.1 using the IncA/C plasmid MLST (PMLST) scheme. pIncAC_KP4898 showed a mosaic structure with blaVIM-1 into a class I integron, blaSHV-12 flanked by IS6 elements, a mercury resistance and a macrolide 20-phosphotransferase clusters, ant(3”), aph(3”), aacA4, qnrA1, sul1, and dfrA14 conferring resistance to aminoglycosides, quinolones, sulfonamides, and trimethoprim, respectively, and several genes predicted to encode transfer functions and proteins involved in DNA transposition. The acquisition of pIncAC_KP4898 carrying blaVIM-1 and blaSHV-12 contributed to the spread of ST104 K. pneumoniae in the NICU of V. Monaldi Hospital in Naples. The growing epidemic of infections caused by multidrug-resistant (MDR) Gram-negative bacteria, including CRE strains, has led to the revival of polymyxins worldwide as the last-resort treatment option. Unfortunately, recent data reported a raising trend of colistin-resistant K. pneumoniae isolates collected in the nosocomial setting worldwide. These data also showed how antibiotic resistant and hypervirulent phenotypes are overlapping in clinical isolates, with the increasing scarcity of effective treatments. The second study of the thesis focuses on the molecular epidemiology and virulence profiles of 25 colistin-resistant K. pneumoniae blood isolates from the Hospital Agency “Ospedale dei Colli,” Naples, Italy. The inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, was the most frequent mechanism of colistin resistance found in 22 out of 25 isolates. Of these, ten isolates assigned to ST512 and PFGE types A and A4 showed identical frameshift mutation and premature termination of mgrB gene; four isolates assigned to ST258 and PFGE types A1 showed non-sense frameshift mutation and premature termination; three and one isolates assigned to ST258 and PFGE A2 and ST512 and PFGE A3, respectively, had insertional inactivation of mgrB gene due to IS5-like mobile element; two isolates assigned to ST101 and 1 to ST392 had missense mutations in the mgrB gene; one isolate assigned to ST45 showed insertional inactivation of mgrB gene due to IS903-like mobile element. phoQ missense mutations were found in 2 isolates assigned to ST629 and ST101, respectively, which also showed a missense mutation in pmrA gene. Colistin-resistant K. pneumoniae isolates showed variable virulence profiles in Galleria mellonella infection assays, with the infectivity of two isolates assigned to ST45 and ST629 being significantly higher than that of all other strains (P < 0.001). Interestingly, colistin MIC values proved to make a significant contribution at predicting lethal doses values (LD50 and LD90) of studied isolates in G. mellonella. Our data show that MgrB inactivation is a common mechanism of colistin resistance among K. pneumoniae in our clinical setting. The presence of identical mutations/insertions in isolates of the same ST and PFGE profile suggests the occurrence of clonal expansion and cross-transmission. Although virulence profiles differ among isolates irrespective of their genotypes, our results suggest that high colistin MIC could predict lower infectivity capability of the isolates. The success of K. pneumoniae as a nosocomial pathogen correlates with high genome plasticity and understanding high-risk clones’ ability to adapt and survive in the hospital environment is important to contain the spread of antibiotic resistance. The global dissemination of K. pneumoniae carbapenemase KPC-producing K. pneumoniae has been largely attributed to the spread of few high-risk STs (ST258, ST11, ST512) associated with human disease. ST101 is an emerging clone, which was identified in different parts of the world with the potential to become a global, persistent public health threat. Recent research suggests the ST101 lineage is associated with an 11% increase in mortality rate in comparison to non-ST101 infections. The third manuscript of the current thesis is a study on the genomic features of ST101 emerging clone to understand what allowed it to succeed and became an epidemic clone. A high-quality, near-finished genome assembly of a MDR K. pneumoniae isolate from Italy (isolate 4743) was generated, that is a single locus variant of ST101 (ST1685). We demonstrated that the 4743 genome contains virulence features such as an integrative conjugative element carrying the yersiniabactin siderophore (ICEKp3), the mannose-resistant Klebsiella-like (type III) fimbriae cluster (mrkABCDFHIJ), the ferric uptake system (kfuABC), the yersiniabactin receptor gene fyuA, a capsular K type K17, and an O antigen type of O1. K. pneumoniae 4743 carries the blaKPC-2 carbapenemase gene along with genes conferring resistance to aminoglycosides, beta-lactams, fluoroquinolones, fosfomycin, macrolides, lincosamides, and streptogramin B. A comparative genomics analysis of 44 ST101 genomes as well as newly sequenced isolate 4743 identified variable AMR profiles and incompatibility plasmid types, but similar virulence factor profiles. Using Bayesian methodologies, we estimate that the common ancestor for the ST101 lineage emerged in 1990 (95% HPD: 1965 to 2007) and isolates within the lineage acquired blaKPC after the divergence from its parental clonal group and dissemination. The identification of virulence factors and antibiotic resistance genes acquired by this newly emerging clone provides insight into the reported increased mortality rates and highlights its potential success as a persistent nosocomial pathogen. With a combination of both colistin resistance, carbapenem resistance, and several known virulence factors, the ST101 genetic repertoire may be a “perfect storm” allowing for a newly emerging, high-risk, extensively antibiotic resistant clone. This high-risk clone appears adept at acquiring resistance and may perpetuate the dissemination of extensive AMR. Greater focus on the acquisition of virulence factors and antibiotic resistance genes is crucial for understanding the spread of antibiotic resistance in K. pneumoniae.

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