Vigilancia, prevención, diagnóstico y tratamiento de las resistencias a antibióticos b-lactámicos desde la perspectiva one health

Resumen

Before the outbreak of the pandemic Covid-19, one of the greatest threats to medicine in the 21st century was and continues to be the antimicrobial resistances (AMR). In order to address this problem with a global perspective and taking into account the strategic lines proposed by the Spanish National Antibiotic Resistance Plan (PRAN), the general objective of this work has been to approach the surveillance, prevention, diagnosis and treatment of resistance to antibiotics, with special emphasis on the extended spectrum β-lactamase-producing Enterobacteriaceae (ESBL). The first objective focused on the surveillance of the resistances in different environments. In Chapter 1 it was reported the isolation and characterization of priority pathogens according to the WHO list from rivers, wastewater treatment plants (WWTPs) and collectors in northern Spain and southern France (POCTEFA area). The results showed that 100% of the WWTPs and collectors and 96.4% of the rivers carried resistant bacteria against at least one of the following antibiotic families: β-lactams, carbapenems, vancomycin and colistin. More than a half of the 55 isolated strains come from wastewater environments and multidrug resistances (MDR) were observed in 96.4% of them, with penicillin/cephalosporin resistance being the most widespread. In agreement with that, in Chapter 2 we performed the phylogenetic characterization of ESBL-producing E. coli strains, isolated from animal, environmental and human environment in the present and previous studies. Important clonal complexes (23CC, 10CC, 131CC and 38CC) related to the spread of β-lactam antibiotic resistance genes were found, but none of them was present in all sources. However, wastewater environments housed these 4 CCs and showed identical profiles to strains isolated from other environments, showing the importance that water acquire in the dissemination of these resistances. Abstract 24 The second objective addressed the study of the diffusion of resistance among the different ecosystems. Therefore, in Chapter 3 the characterization of integrons and insertion sequences was carried out. Results showed the wide presence and dissemination of intI1 (92%), IS26 (99.4%) as well as the genetic pattern IS26-ISEcp1 (related with the pathogenic clone 131CC) that was present in 22% of the strains. The coexistence of various types of integrons and insertion sequences suggests a possible risk of spread of resistance between different environments. Meanwhile, in Chapter 4 it was analysed the genetic profiles associated with virulence factors, as well as the conjugative capacity presented by these E. coli strains. Virulence genes fimA, papC, and aer were detected in all environments, papG III was mainly associated with clinical strains, and wastewater was a point of diffusion for cnf1 and hlyA genes. In addition, isolated strains from aquatic environments showed a significantly higher conjugation frequency than those coming from farms and food, pointing one more time the importance that the aquatic environment acquires for the exchange of genes and resistance to antibiotics. The third objective consisted of evaluating a proposal for the prevention of AMR in animal environment. Thus, in Chapter 5 it was assessed the antimicrobial activity of silver nanomaterials to be added in feed as an alternative to the use of antibiotics in animal production. The results showed that C3 product has antimicrobial activity against a wide spectrum of bacteria (including sensitive and resistant ones), with a higher bactericidal activity against Gram negative bacteria (lower concentration needed and faster reduction of viable microorganisms than in the case of S. aureus). Finally, in the fourth objective, diagnostic and treatment alternatives for infections caused by ESBL-producing Enterobacteriaceae were addressed. In Chapter 6 a cefotaxime hydrolysis protocol has been defined that allows rapid detection of ESBL- Abstract 25 producing strains (CTX-M1 type) by using the VITEK®-MS RUO (bioMérieux). Secondly, in Chapter 7 two treatment alternatives have been tested; on the one hand, the activity of two antimicrobial peptides derived from lactoferricin (P4-1 and P2-15) was evaluated against ESBL-producing E. coli. Despite subinhibitory concentrations of both peptides in the presence of 1 µg/ml of clavulanic acid managed to reduce the MIC of the antibiotic, none of them achieved sensitization to amoxicillin. On the other hand, the antibacterial activity of the metabolites produced by 3 lactic acid bacteria (C1, A1 and C34) of animal origin was studied, confirming that they were capable of inhibiting the growth of ESBL-producing E. coli, being L. plantarum C1 the most active one, showing a genetic cluster compatible with the plantaricin-type bacteriocin. The data obtained in this Doctoral Thesis have shown that it is necessary to carry out a more effective control of the presence of MDR bacteria in the aquatic environment, implementing surveillance of relevant clonal complexes that act as markers for the presence of resistant bacteria as well as more effective wastewater treatment programs to stop the dispersion. In relation to the animal environment, a silver nanomaterial was positively evaluated as possible feed additive to replace or reduce the use of antibiotics in animal production. Finally, with regard to human health, a rapid diagnostic protocol for the detection of blaCTX-M1-producing Enterobacteriaceae was developed using the VITEK®-MS RUO equipment. In addition, the antimicrobial activity of the three tested LAB was confirmed, but further studies are necessary before considering their possible therapeutic application.