Abstract
The global spread of antibiotic-resistant bacteria and their resistance genes is a critical issue that is no longer restricted to hospital settings, but also represents a growing problem involving environmental and food safety. In this study, we have performed a microbiological and genomic investigation of critical priority pathogens resistant to broad-spectrum cephalosporins and showing endophytic lifestyles in fresh vegetables sold in a country with high endemicity of extended-spectrum β-lactamases (ESBLs). We report the isolation of international high-risk clones of CTX-M-15-producing Escherichia coli, belonging to clonal complexes CC38 and CC648, and Klebsiella pneumoniae of complex CC307 from macerated tissue of surface-sterilized leaves of spinach, cabbage, arugula, and lettuce. Regardless of species, all ESBL-positive isolates were able to endophytically colonize common bean (Phaseolus vulgaris) seedlings, showed resistance to acid pH, and had a multidrug-resistant (MDR) profile to clinically relevant antibiotics (i.e., broad-spectrum cephalosporins, aminoglycosides, and fluoroquinolones). Genomic analysis of CTX-M-producing endophytic Enterobacterales revealed a wide resistome (antibiotics, biocides, disinfectants, and pesticides) and virulome, and genes for endophytic fitness and for withstanding acidic conditions. Transferable IncFIB and IncHI2A plasmids carried bla CTX-M-15 genes and, additionally, an IncFIB plasmid (named pKP301cro) also harbored genes encoding resistance to heavy metals. These data support the hypothesis that fresh vegetables marketed for consumption can act as a figurative Trojan horse for the hidden spread of international clones of critical WHO priority pathogens producing ESBLs, and/or their resistance genes, to humans and other animals, which is a critical issue within a food safety and broader public and environmental health perspective.IMPORTANCE Extended-spectrum β-lactamases (ESBL)-producing Enterobacterales are a leading cause of human and animal infections, being classified as critical priority pathogens by the World Health Organization. Epidemiological studies have shown that spread of ESBL-producing bacteria is not a problem restricted to hospitals, but also represents a growing problem involving environmental and food safety. In this regard, CTX-M-type β-lactamases have become the most widely distributed and clinically relevant ESBLs worldwide. Here, we have investigated the occurrence and genomic features of ESBL-producing Enterobacterales in surface-sterilized fresh vegetables. We have uncovered that international high-risk clones of CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae harboring a wide resistome and virulome, carry additional genes for endophytic fitness and resistance to acidic conditions. Furthermore, we have demonstrated that these CTX-M-15-positive isolates are able to endophytically colonize plant tissues. Therefore, we believe that fresh vegetables can act as a figurative Trojan horse for the hidden spread of critical priority pathogens exhibiting endophytic lifestyles.
Highlights
The global spread of antibiotic-resistant bacteria and their resistance genes is a critical issue that is no longer restricted to hospital settings, and represents a growing problem involving environmental and food safety
The presence of endophytic extended-spectrum b-lactamases (ESBLs)-producing Enterobacterales was confirmed in 10.4% of 48 fresh vegetables samples screened after surface sterilization, including spinach (2 positive samples for ESBL-producing E. coli strain ESP110 and E. cloacae strain ESP151), cabbage (1 positive sample for 2 ESBL-producing E. coli strains [REP215 and REP237]), lettuce (1 positive sample for ESBL-producing K. pneumoniae [strain ALF301]), and arugula (1 positive sample for ESBL-producing K. pneumoniae [strain RUC232])
ESBL-producing Enterobacterales, including CTX-M-15 producers, have been reported in vegetables, representing a risk of human exposure to MDR critical priority pathogens through this food source [7, 13, 14]
Summary
The global spread of antibiotic-resistant bacteria and their resistance genes is a critical issue that is no longer restricted to hospital settings, and represents a growing problem involving environmental and food safety. Transferable IncFIB and IncHI2A plasmids carried blaCTX-M-15 genes and, an IncFIB plasmid (named pKP301cro) harbored genes encoding resistance to heavy metals These data support the hypothesis that fresh vegetables marketed for consumption can act as a figurative Trojan horse for the hidden spread of international clones of critical WHO priority pathogens producing ESBLs, and/or their resistance genes, to humans and other animals, which is a critical issue within a food safety and broader public and environmental health perspective. Extended-spectrum b-lactamase (ESBL)-producing Gram-negative bacteria are a leading cause of human and animal infection, being categorized as critical priority pathogens by the World Health Organization [1] In this regard, plasmid-mediated ESBLs of the CTX-M family have been widely identified in different genera of Enterobacterales, with CTX-M-15 being the most clinically relevant ESBL worldwide [2]. A potential threat to human health would be associated with future endogenous infections, mainly in immunosuppressed patients, where therapeutic failure could occur
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