The experimental thesis work was aimed at the phenotypic and molecular characterization of a total of n = 8 Proteus mirabilis strains, isolated in February-March 2019 at the "Ospedale Maggiore di Lodi" both from the hospital environment and from two patients affected from chronic obstructive pulmonary disease and pneumonia, respectively. The patients, both catheterized and admitted to the Internal Medicine ward, shared the same room for a short time. All the isolates were positive, using the XpertCarba-R System (Cepheid), for the presence of a blaNDM type gene, coding for a New Delhi Metal-β-lactamase (class B carbapenemase). The aim of the work was also characterizing the genome and plasmid sequences of a representative NDM‐1 positive (IBCRE14) strain. Species identification and antibiotic sensitivity were confirmed with Microscan AutoScan‐4 (Beckman-Coulter) and broth microdilution. The results, interpreted in accordance with the EUCAST guidelines (http://www.eucast.org), indicated multi-antibiotic resistance. The conjugation was performed in liquid medium using Escherichia coli J62 as a receptor. Microarray, PCR targeting blaNDM and subsequent gene sequencing, confirmed the presence of a blaNDM-1 determinant. Typing, conducted on all eight isolates using Pulsed Field Gel Electrophoresis (PFGE), confirmed the presence of a single clone. The genome of the IBCRE14 isolate was subjected to Whole Genome Sequencing (WGS) with the Sequel I (Pacific Biosciences) platform, using the long reads sequencing technology. Genomic analysis was performed by uploading the contigs to the ResFinder and PlasmidFinder databases, of the Center for Genomic Epidemiology. Molecular results: the genome size of IBCRE14 harbored genes for resistance to aminoglycosides (aadA1), phenicol (cat), tetracycline (tetJ) and trimethoprim (dfrA1), was of 4,018,329 bp. A large plasmid (pIB_NDM_1) was found to harboured resistance genes to sulfonamides (sul1), trimethoprim (dfrA14), tetracycline (tetB), rifampicin (arr - 2), aminoglycosides (aadA1, aph3-VI) and β-lactams (blaOXA-10, blaNDM-1). Furthermore, a small plasmid (pIB_COL3M) was found to host a qnrD1 gene that encodes quinolone resistance. Conclusions: the ability to conjugate and the presence of a composite antibiotic resistance island suggests that pIB_NDM_1 could acquire additional resistance genes, as well as spread easily. This experimental work represents the first report on a positive atypical blaNDM- 1 plasmid in P. mirabilis, in Italy. This confirms the need to apply accurate infection control and cohorting measures in the clinical setting, to avoid the onset of outbreaks.
Il lavoro di tesi sperimentale è stato volto alla caratterizzazione fenotipica e molecolare di un totale di n = 8 ceppi di Proteus mirabilis, isolati nel febbraio-marzo 2019 presso l’“Ospedale Maggiore di Lodi” sia dall’ambiente nosocomiale che da due pazienti affetti rispettivamente da malattia polmonare cronica ostruttiva e polmonite. I pazienti, entrambi cateterizzati e ricoverati presso il reparto di Medicina Interna, hanno condiviso per breve periodo la stessa stanza. Tutti gli isolati sono risultati positivi, mediante sistema XpertCarba‐R System (Cepheid), per la presenza di un gene di tipo blaNDM, codificante per una New Delhi Metallo‐β‐lattamasi (carbapenemasi) di classe B. Scopo del lavoro è stato inoltre caratterizzare le sequenze di genoma e plasmidi di un ceppo NDM‐1‐positivo (IBCRE14) rappresentativo. Identificazione di specie ed antibiotico-sensibilità sono stati confermati con Microscan AutoScan‐4 (Beckman‐Coulter) e brodo microdiluizione. I risultati, interpretati in accordo alle linee guida EUCAST (http://www.eucast.org), hanno indicato multi-antibiotico-resistenza. La coniugazione è stata eseguita in terreno liquido utilizzando Escherichia coli J62 come recettore. Microarray, PCR con target blaNDM e successivo sequenziamento genico, hanno confermato la presenza di un determinante blaNDM-1. La tipizzazione, condotta su tutti gli otto isolati mediante Pulsed Field Gel Electrophoresis (PFGE), ha confermato l’appartenenza ad un unico clone. Il genoma dell’isolato IBCRE14 è stato sottoposto a Whole Genome Sequencing (WGS) con l’apparecchio Sequel I (Pacific Biosciences), usando la tecnologia long reads sequencing. L'analisi genomica è stata condotta caricando i contig sui database ResFinder e PlasmidFinder del Center for Genomic Epidemiology. Risultati molecolari: le dimensioni del genoma di IBCRE14 contenente i geni per la resistenza ad aminoglicosidi (aadA1), fenicolo (cat), tetraciclina (tetJ) e trimetoprim (dfrA1), sono risultate pari 4.018.329 bp. Un grande plasmide (pIB_NDM_1) è risultato albergare geni di resistenza a sulfonamidi (sul1), trimetoprim (dfrA14), tetraciclina (tetB), rifampicina (arr‐2), aminoglicosidi (aadA1, aph3-VI) e β-lattamici (blaOXA‐10, blaNDM-1). Inoltre, un piccolo plasmide (pIB_COL3M) è risultato ospitare un gene qnrD1 che codifica per la resistenza ai chinoloni. Conclusioni: la capacità di coniugare e la presenza di una composita antibiotic resistance island suggerisce che pIB_NDM_1 potrebbe acquisire ulteriori geni di resistenza, oltre che diffondersi facilmente. Il presente lavoro sperimentale risulta la prima segnalazione di un plasmide atipico blaNDM-1 positivo in P. mirabilis, in Italia. Ciò conferma la necessità di applicare accurate misure di infection control, e cohorting, per evitare l’insorgenza di outbreak in ambito clinico.
Caratterizzazione fenotipica e molecolare di isolati clinici di Proteus mirabilis NDM-1 produttori
DE VITA, DARIO
2018/2019
Abstract
The experimental thesis work was aimed at the phenotypic and molecular characterization of a total of n = 8 Proteus mirabilis strains, isolated in February-March 2019 at the "Ospedale Maggiore di Lodi" both from the hospital environment and from two patients affected from chronic obstructive pulmonary disease and pneumonia, respectively. The patients, both catheterized and admitted to the Internal Medicine ward, shared the same room for a short time. All the isolates were positive, using the XpertCarba-R System (Cepheid), for the presence of a blaNDM type gene, coding for a New Delhi Metal-β-lactamase (class B carbapenemase). The aim of the work was also characterizing the genome and plasmid sequences of a representative NDM‐1 positive (IBCRE14) strain. Species identification and antibiotic sensitivity were confirmed with Microscan AutoScan‐4 (Beckman-Coulter) and broth microdilution. The results, interpreted in accordance with the EUCAST guidelines (http://www.eucast.org), indicated multi-antibiotic resistance. The conjugation was performed in liquid medium using Escherichia coli J62 as a receptor. Microarray, PCR targeting blaNDM and subsequent gene sequencing, confirmed the presence of a blaNDM-1 determinant. Typing, conducted on all eight isolates using Pulsed Field Gel Electrophoresis (PFGE), confirmed the presence of a single clone. The genome of the IBCRE14 isolate was subjected to Whole Genome Sequencing (WGS) with the Sequel I (Pacific Biosciences) platform, using the long reads sequencing technology. Genomic analysis was performed by uploading the contigs to the ResFinder and PlasmidFinder databases, of the Center for Genomic Epidemiology. Molecular results: the genome size of IBCRE14 harbored genes for resistance to aminoglycosides (aadA1), phenicol (cat), tetracycline (tetJ) and trimethoprim (dfrA1), was of 4,018,329 bp. A large plasmid (pIB_NDM_1) was found to harboured resistance genes to sulfonamides (sul1), trimethoprim (dfrA14), tetracycline (tetB), rifampicin (arr - 2), aminoglycosides (aadA1, aph3-VI) and β-lactams (blaOXA-10, blaNDM-1). Furthermore, a small plasmid (pIB_COL3M) was found to host a qnrD1 gene that encodes quinolone resistance. Conclusions: the ability to conjugate and the presence of a composite antibiotic resistance island suggests that pIB_NDM_1 could acquire additional resistance genes, as well as spread easily. This experimental work represents the first report on a positive atypical blaNDM- 1 plasmid in P. mirabilis, in Italy. This confirms the need to apply accurate infection control and cohorting measures in the clinical setting, to avoid the onset of outbreaks.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/23196