Bacterias gram negativas resistentes a los antibióticos y patógenas transmitidas a través de vegetales frescos

Abstract

Fresh produce has traditionally been less monitored for the presence of pathogenic bacteria and antibiotic-resistant strains. Information on this matter is scarce, and available data suggests that these products could be significant reservoirs of bacteria with such characteristics. Moreover, vegetables are often eaten raw, exposing consumers directly to the microorganisms. In the current context of the antibiotic crisis, the lack of proper monitoring of vegetables might contribute to a silent spread of these resistant strains, posing a serious threat to public health. This thesis assesses the overall microbiological quality of fresh vegetables and the presence of isolates producing -lactamases, including ESBL and AmpC (Chapter 1), as well as carbapenemases, strains resistant to colistin and pathogenic E. coli. To achieve this, 145 samples of fresh vegetables and 90 samples from the production environment were collected. First of all, enterobacterial load was evaluated through plate counting. Additionally, an enrichment of these samples was cultured using chromogenic media to select isolates producing ESBL, AmpC, carbapenemases, and E. coli. Colonies displaying the desired characteristics were selected, identified by MALDI-TOF and characterized regarding their -lactamase production and resistance to colistin. Enterobacteriaceae counts showed significant differences, with leafy and root vegetables exhibiting counts significantly higher than those in fruit vegetables. The overall prevalence of ESBL in vegetables was 12.4 %, while for constitutive BlAmpCproducing isolates, it was 7.6 %. In general, higher values were observed among leafy products compared to carrots or fruit vegetables. Concerning the production environment, irrigation water showed high prevalence values for both ESBL (12.5 %) and cAmpC (33.0 %) isolates, suggesting its role as a reservoir for these bacteria in the agricultural environment. Among ESBL-producing isolates, there was a predominance of environmental species Serratia fonticola and Rahnella aquatilis. Strains of these species were characterized (Chapter 2) based on their susceptibility to several antibiotics, revealing five S. fonticola isolates with multidrug-resistant patterns. Genetic characterization of ESBL enzymes by PCR amplification and sequencing identified the blaFONA5 enzyme in three S. fonticola isolates and blaRAHN2 in 2 R. aquatilis isolates. Although these enzymes are naturally encoded in the chromosome of these species, the mobilization of FONA family genes from S. fonticola to clinical isolates has been reported, highlighting their potential role as reservoirs of ESBL determinants. Methods that allow for the rapid detection of ESBL-producing isolates are a crucial aspect in clinical settings and have a high potential for acquiring large-scale data in environmental studies. A promising technique for this purpose is MALDI-TOF mass spectrometry, which was validated against a collection of 161 bacterial isolates from clinical and environmental origins (Chapter 3). For this, isolates were incubated in the presence of cefotaxime for 30 minutes. Establishing a threshold value of NlogRQ > 0.210 based on the relationship between the intensity of mass peaks associated with antibiotic hydrolysis and non-hydrolysis accurately classified all included ESBL isolates, highlighting the method's excellent sensitivity. Regarding BlAmpC-producing isolates, these were more diverse, mainly including genera naturally carrying these enzymes such as Citrobacter, Enterobacter, and Aeromonas. Within this group, the clinical implications associated with the Enterobacter cloacae complex (ECC, Chapter 4) and the genus Aeromonas (Chapter 5) led to a in deep characterization of these bacterial groups regarding their antibiotic resistance mechanisms and pathogenicity. Antibiotic susceptibility Testing was performed for both groups of isolates, including agents from different families, and the MIC value was established for those antibiotics to which resistance patterns were observed. In vitro assays were also conducted to determine their potential pathogenicity by infecting different cell lines and using confocal microscopy. In the case of Aeromonas, potential pathogenicity was also studied through in vivo assays in Galleria mellonella larvae. The genetic basis for antibiotic resistance and pathogenicity shown by the isolates was studied through whole genome sequencing. Some ECC isolates showed significant resistance patterns to third and even fourth generation cephalosporins and colistin. The colistin resistance pattern observed in E. kobei AG07E isolate was associated with the mcr-9.1 which was confirmed as transferable through conjugation. Regarding their potential pathogenicity, three ECC isolates showed ability to adhere to human colon cells and/or fibroblasts. Isolate E. kobei ZA03E was particularly noteworthy, as it exhibited a highly pronounced adherence pattern to both cell types. Both E. kobei AG07E (ST56) and E. kobei ZA03E (ST125) could be related to isolates from clinical and animal production environments through their sequence type (ST). As for Aeromonas strains, A. dhakensis AG29E1 and A. salmonicida CI21E, isolated from coriander and an irrigation water sample, respectively, showed high cytotoxicity against human colon cells, vero cells, mouse fibroblasts, and even macrophages. Additionally, these Aeromonas strains exhibited much higher lethality in G. mellonella in the case of A. dhakensis AG29E1 occurred in combination with resistance to third generation cephalosporins and colistin, increasing awareness about the transmission of these isolates due to their potential involvement in infections. These results indicate the potential public health risk posed by the transmission of Aeromonas strains through fresh vegetables.