Detecting KPC-2 and NDM-1 Coexpression in Klebsiella pneumoniae Complex from Human and Animal Hosts in South America.

Reports of Gram-negative bacteria harboring multiple carbapenemase genes have increased in South America, leading to an urgent need for appropriate microbiological diagnosis. We evaluated phenotypic methods for detecting Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-ß-lactamase-1 (NDM-1) coexpression in members of the K. pneumoniae complex (i.e., K. pneumoniae, K. quasipneumoniae, and K. variicola) isolated from human and animal hosts, based on inhibition of ceftazidime-avibactam (CZA) and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, or avibactam (AVI). While the presence of blaKPC-2 and blaNDM-1 genes was confirmed by whole-genome sequencing, PCR, and/or GeneXpert, coexpression was successfully detected based on the following: (i) a ≥5-mm increase in the zone diameter of ATM (30 µg) disks plus AVI (4 or 20 µg) and ≥4-mm and ≥10-mm increases in the zone diameters for "CZA 50" (30 µg ceftazidime [CAZ] and 20 µg AVI) and "CZA 14" (10 µg CAZ and 4 µg AVI) disks, respectively, when we added DPA (1 mg/disk) or EDTA (5 mM) in a combined disk test (CDT); (ii) a positive ghost zone (synergism) between ATM (30 µg) and CZA 50 disks and between CZA 50 and DPA (1 mg) disks, using the double-disk synergy test (DDST) at a disk-disk distance of 2.5 cm; (iii) ≥3-fold MIC reductions of ATM and CZA in the presence of AVI (4 µg/mL), DPA (500 µg/mL), or EDTA (320 µg/mL); and (iv) immunochromatography. Although our results demonstrated that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex, additional studies are necessary to confirm the accuracy of these methodologies by testing other Gram-negative bacterial species and other KPC and NDM variants coexpressed by WHO critical priority pathogens detected worldwide. IMPORTANCE Alerts regarding the emergence and increase of combinations of carbapenemases in Enterobacterales in Latin America and the Caribbean have recently been issued by PAHO and WHO, emphasizing the importance of appropriate microbiological diagnosis and the effective and articulated implementation of infection prevention and control programs. In this study, we evaluated methods based on inhibition of ceftazidime (CAZ), ceftazidime-avibactam (CZA), and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, and avibactam (AVI) inhibitors for the identification of KPC-2- and NDM-1-coexpression in members of the K. pneumoniae complex recovered from human and animal hosts. Our results demonstrate that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex.

Endophytic Lifestyle of Global Clones of Extended-Spectrum ß-Lactamase-Producing Priority Pathogens in Fresh Vegetables: a Trojan Horse Strategy Favoring Human Colonization?

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 ß-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 ß-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 ß-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.