Plasmids are extracromosomal circular DNA molecules that can replicate autonomously. They are globally described as primary promoters for the dissemination of antimicrobial resistance genes. Escherichia coli and Klebsiella pneumoniae have been described as critical propriety microorganisms due to their increasing detection as multiresistant bacteria. In these bacteria, beta-lactamases constitute one of the most disseminated resistance-providing mechanisms through plasmids. Through the study and comparison of the plasmid content in the commensal microbiota from healthy individuals and patients with bacteriemia, the aim was to determine the differences and similarities of the plasmid content between these two populations, with the purpose to shed light on the origin and evolution of bacteria-causing infection. To achieve that goal, E. coli (n=244) and K. pneumoniae (n=115) strains from 150 faecal samples from healthy individuals and 202 patients with bacteraemia where isolated. An antimicrobial susceptibility testing was performed through the disc diffusion technique, the multiresistant strains were identified and, the blaESBL, blaAmpC and blacarbapenemase genes were characterised by PCR and sequencing approaches. The plasmid study was performed by using the PBRT technique. Plasmids IncF, Incl1 and IncN plasmids, were subtyped by pMLST, to differentiate each of them within the same incompatibility group. The location of the resistance genes was performed by using the Southern blot and hybridisation techniques. The same methodology was used to generate the FAB formula of the IncF plasmids. From the clinical samples, 29.2% of the strains turned out to be multiresistant against 10.8% from healthy individuals. The prevalence of clinical ESBL-producing and AmpC-producing E. coli strains was 17.2% and 5%, respectively. The prevalence of clinical ESBL-producing, AmpC-producing and carbapenemase-producing K. pneumoniae strains was 16.5%, 1% and 1.9%, respectively. Furthermore, 4.7% of the healthy individuals were carriers of ESBL-producing E. coli and 2.7% were carriers of AmpC-producing E. coli. Both populations have suffered an increased detection of strains that produce those enzymes and, in both cases, the beta-lactamases CTX-M-15 and CMY-2 were the most frequent. The PBRT results showed how the plasmid content in both populations kept the same trend without significant differences. The exception was observed in the replicons L, M, A/C and N, which were only detected in strains from clinical samples. What is more, some of these plasmids with L or N repliclon were detected in association with blaESBL genes. The plasmid subtyping, allowed us to observe a great diversity of sequence types (ST) within the same group of incompatibility. Within the Incl1-plasmids, a huge difference between the identified ST in healthy individuals and patient strains was observed. Only ST12 and ST36 were identified in both populations of E. coli. Noticeably, all the ST12 were found in association with the blaCMY-2 gene. In contrast, even though a great diversity of RSTs was detected within the IncF plasmids, those detected at higher rates were present in both populations. Furthermore, no formula was distinguishable by its association with bla genes. In conclusion, the replicon content of clinical strains mirrored that of comensal strains. Nevertheless, there are certain plasmids only detected in infection-causing strains, suggesting a better adaptation to an enviroment subjected to selective presure. IncI1 sequencetypes such as ST12 can be considered epidemic plasmids due to their high prevalence and frequency in association with blaCMY-2. On the other hand, the IncF plasmids have acquired multiple antimicrobial resistace genes randomly, with no evidence that the persitance of a single IncF is responsible for their dissemination. Therfore, the entire IncF family of plasmids is potentially capable of acquiring and propagating resitance genes.
Diversidad plasmídica en cepas de Escherichia coli y Klebsiella pneumoniae: Comparación entre aislados comensales y clínicos
Rodriguez Navarro, J. (Author). 20 Nov 2020
Student thesis: Doctoral thesis
Student thesis: Doctoral thesis