Klebsiella pneumoniae species complex (KpSC) refers to a group of five Gram-negative bacteria that form the broader Klebsiella genus. KpSC is composed of opportunistic bacteria that are found widespread in the environment and have consequently been isolated from foods and associated environments. These species are of concern due to the multidrug-resistant (MDR) phenotypes expressed by isolates from One Health (OH) origins, along with their ability to form biofilms on both biotic and abiotic surfaces, further increasing their non-susceptibility to antibiotics. Moreover, it has been reported previously that environmental contaminants (including pesticides, microplastics and others) may lead to an increase in the bacterial tolerance/resistance to antibiotics. Therefore, the aim of this thesis was to investigate the effect of two such environmental contaminants on the antimicrobial-resistant (AMR) phenotypes expressed by two different Klebsiella species identified from a larger collection. Furthermore, the study also explored the impact of sub-inhibitory exposure to the two contaminants on biofilm formation and the microbiota composition contained therein. From an initial set of 112 strains obtained from the laboratory culture collection, strains were identified based on the use of selective and differential bacteriological media, and they were further confirmed with MALDI-TOF mass spectral analysis. The strains of interest were characterised by whole genome sequencing (WGS) and biofilm morphotypes were assessed following incubation on Congo red-containing agar. Susceptibilities of all study isolates to a panel of antibiotics were tested using Sensititer™ Gram Negative GN4F AST plates (Thermo Fisher Scientific) and the hypermucoviscous phenotype was determined using the string test. Two of these isolates were of particular interest. One was Klebsiella pneumoniae, while the second was Klebsiella variicola, both originated from an iced tea machine. These isolates were tested for their susceptibilities to two selected herbicides (namely glyphosate, a commonly used chemical, and MCPA [2-methyl-4-chlorophenoxyacetic acid]) along with a synthetic compound used to produce plastic (denoted as bisphenol A). Furthermore, Sensititer™ tests were performed in the presence and absence of sub-inhibitory concentrations of the two herbicides. Moreover, using a laboratory scale trap model, OH relevant biofilms were studied to describe their bacterial abundances using 16S rRNA gene amplicon melting curve analysis and shotgun sequencing, and the effects of the two contaminants on the microbiota were established. By adopting a One Health approach, this project provides insights into the effects that the exposure to glyphosate and MCPA can have on the antimicrobial resistance of Klebsiella isolates and on the effect of glyphosate on the bacterial composition of natural biofilms.
Il termine Klebsiella pneumoniae species complex (KpSC) fa riferimento ad un gruppo di cinque batteri Gram-negativi che fanno parte del genere Klebsiella. Il complesso è formato da batteri opportunisti che sono ampiamente diffusi nell’ambiente e che di conseguenza sono stati isolati dagli alimenti e dagli ambienti ad essi connessi. Queste specie sono fonte di preoccupazione a causa della presenza di fenotipi resistenti a diversi antibiotici (MDR; multidrug-resistant) in ceppi di interesse per il modello One Health (OH) e per la loro abilità di formare biofilm in superfici abiotiche e biotiche, causando un aumento della loro non-suscettibilità agli antibiotici. In aggiunta, è stato riportato che i contaminanti ambientali (ad esempio pesticidi, microplastiche ed altri composti) possano portare ad un aumento nella resistenza/tolleranza dei batteri agli antibiotici. Per queste ragioni, lo scopo di questa tesi è quello di investigare gli effetti di due di questi contaminanti sulla resistenza agli antimicrobici (AMR) di due ceppi di Klebsiella spp. identificati a partire da una collezione di ceppi. In aggiunta, è stato studiato l’impatto di concentrazioni sub-inibitorie di un contaminante nella formazione di un biofilm e della composizione del microbiota contenuto in esso. Lo studio è partito da un’iniziale identificazione di 112 ceppi che erano presenti nella collezione del laboratorio utilizzando terreni batteriologici selettivi e differenziali, seguita da una conferma avvenuta con lo strumento di spettrometria di massa MALDI-TOF. I ceppi d’interesse per lo studio sono stati caratterizzati tramite il sequenziamento dell’intero genoma (WGS) e il loro morfotipo di biofilm è stato valutato tramite l’utilizzo di un terreno microbiologico contenente Congo-red. La suscettibilità dei ceppi a diversi antibiotici è stata valutata utilizzando Sensititer™ Gram Negative GN4F AST plates (Thermo Fisher Scientific), mentre la mucoviscoità dei ceppi è stata determinata tramite lo string test. Due dei ceppi identificati sono stati riscontrati come d’interesse per la tesi. Un ceppo appartiene a Klebsiella pneumoniae, mentre l’altro a Klebsiella variicola, ed entrambi sono stati isolati da campioni prelevati da macchine per la distribuzione di thè freddo. Questi due ceppi sono stati testati per la loro suscettibilità ai due erbicidi (glifosato e MCPA [acido 2-Metil-4-clorofenossiacetico]) ed a un componente sintetico utilizzato per la produzione di plastiche (bisfenolo A). In aggiunta, dei test in cui si è fatto utilizzo delle Sensititer™ sono stati svolti in presenza e assenza di concentrazioni sub-inibitorie dei due erbicidi. Inoltre, biofilm rilevanti dal punto di vista dell’approccio OH sono stati analizzati utilizzando un modello di sifone del lavandino creato in laboratorio con lo scopo di descrivere la composizione del biofilm presente tramite l’analisi della curva di dissociazione di una porzione del gene 16S rRNA amplificato e il sequenziamento shotgun del genoma. È stato inoltre descritto come il microbiota del biofilm viene modificato dalla presenza del glifosato. Tramite l’adozione di un approccio One Health questo progetto fornisce approfondimenti sugli effetti che il glifosato e l’MCPA hanno sull’antibiotico resistenza di due ceppi di Klebsiella e sull’effetto che il glifosato ha sulla composizione batterica di un biofilm naturale.
A study of the effect of two herbicides- impact on antimicrobial resistance of two Klebsiella spp. strains and on microbiota in a natural biofilm
BENETTI, SARA
2022/2023
Abstract
Klebsiella pneumoniae species complex (KpSC) refers to a group of five Gram-negative bacteria that form the broader Klebsiella genus. KpSC is composed of opportunistic bacteria that are found widespread in the environment and have consequently been isolated from foods and associated environments. These species are of concern due to the multidrug-resistant (MDR) phenotypes expressed by isolates from One Health (OH) origins, along with their ability to form biofilms on both biotic and abiotic surfaces, further increasing their non-susceptibility to antibiotics. Moreover, it has been reported previously that environmental contaminants (including pesticides, microplastics and others) may lead to an increase in the bacterial tolerance/resistance to antibiotics. Therefore, the aim of this thesis was to investigate the effect of two such environmental contaminants on the antimicrobial-resistant (AMR) phenotypes expressed by two different Klebsiella species identified from a larger collection. Furthermore, the study also explored the impact of sub-inhibitory exposure to the two contaminants on biofilm formation and the microbiota composition contained therein. From an initial set of 112 strains obtained from the laboratory culture collection, strains were identified based on the use of selective and differential bacteriological media, and they were further confirmed with MALDI-TOF mass spectral analysis. The strains of interest were characterised by whole genome sequencing (WGS) and biofilm morphotypes were assessed following incubation on Congo red-containing agar. Susceptibilities of all study isolates to a panel of antibiotics were tested using Sensititer™ Gram Negative GN4F AST plates (Thermo Fisher Scientific) and the hypermucoviscous phenotype was determined using the string test. Two of these isolates were of particular interest. One was Klebsiella pneumoniae, while the second was Klebsiella variicola, both originated from an iced tea machine. These isolates were tested for their susceptibilities to two selected herbicides (namely glyphosate, a commonly used chemical, and MCPA [2-methyl-4-chlorophenoxyacetic acid]) along with a synthetic compound used to produce plastic (denoted as bisphenol A). Furthermore, Sensititer™ tests were performed in the presence and absence of sub-inhibitory concentrations of the two herbicides. Moreover, using a laboratory scale trap model, OH relevant biofilms were studied to describe their bacterial abundances using 16S rRNA gene amplicon melting curve analysis and shotgun sequencing, and the effects of the two contaminants on the microbiota were established. By adopting a One Health approach, this project provides insights into the effects that the exposure to glyphosate and MCPA can have on the antimicrobial resistance of Klebsiella isolates and on the effect of glyphosate on the bacterial composition of natural biofilms.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/52101