During a surveillance study conducted to assess the occurrence and genomic landscape of critical priority pathogens circulating at the human-animal-environment interface in Brazil, as part of the Grand Challenges Explorations-New Approaches to Characterize the Global Burden of Antimicrobial Resistance program, two multidrug-resistant (MDR) Citrobacter portucalensis carrying blaCTX-M-15 extended-spectrum β-lactamase (ESBL) genes, isolated from green sea turtles, were characterized. Genomic and phylogeographical analysis of C. portucalensis genomes available in public databases revealed the intercontinental dissemination of clades carrying different arrays of clinically relevant genes conferring resistance to carbapenems, broad-spectrum cephalosporins, cephamycins, aminoglycosides and fluoroquinolones, disinfectants, and heavy metals. Our observations suggest that C. portucalensis could be emerging as critical priority bacteria of both public and One Health importance worldwide. IMPORTANCE The global spread of antibiotic-resistant priority pathogens beyond the hospital setting is a critical issue within a One Health context that integrates the human-animal-environment interfaces. On the other hand, next-generation sequencing technologies along with user-friendly and high-quality bioinformatics tools have improved the identification of bacterial species, and bacterial resistance surveillance. The novel Citrobacter portucalensis species was proposed in 2017 after taxonomic reclassification and definition of the strain A60T isolated in 2008. Here, we presented genomic data showing the occurrence of multidrug-resistant C. portucalensis isolates carrying blaCTX-M-15 ESBL genes in South America. Additionally, we observed the intercontinental dissemination of clades harboring a broad resistome to clinically relevant antibiotics. Therefore, these findings highlight that C. portucalensis is a global MDR bacteria that carries intrinsic blaCMY- and qnrB-type genes and has become a critical priority pathogen due to the acquisition of clinically relevant resistance determinants, such as ESBL and carbapenemase-encoding genes.

Convergence of resistance and virulence in Klebsiella pneumoniae is a critical public health issue worldwide. A multidrug-resistant CTX-M-15-producing K. pneumoniae (TIES-4900 strain) was isolated from a highly impacted urban river, in Brazil. The genome was sequenced by MiSeq Illumina platform and de novo assembled using Unicycler. In silico prediction was accomplished by bioinformatics tools. The size of the genome is 5.4 Mb with 5145 protein-coding genes. TIES-4900 strain belonged to the sequence type ST15, yersiniabactin sequence type YbST10, ICEKp4, KL24 (wzi-24) and O1v1 locus. Phylogenomics confirmed genomic relatedness with ST15 clones from human and animal hosts. Convergence of broad resistome (antibiotics, heavy-metals and biocides) and virulome, including the Kpi pilus system involved in host-pathogen interaction and persistence of ST15 clone to hospital environments, were predicted. Virulent behavior was confirmed in the Galleria mellonella infection model. This study may give genomic insights on the spread of critical-priority WHO pathogens beyond hospital settings.

Klebsiella pneumoniae is an opportunistic pathogen that can cause several infections, mainly in hospitalised or immunocompromised individuals. The spread of K. pneumoniae emerging virulent and multidrug-resistant clones is a worldwide concern and its identification is crucial to control these strains especially in hospitals. This article reports data related to multi-resistant K. pneumoniae strains, isolated from inpatients in the city of Manaus, Brazil, harbouring virulence and antimicrobial-resistance genes, including high-risk international clones belonging to clonal group (CG) 258. Twenty-one strains isolated from different patients admitted to four hospitals in the city of Manaus, located in the state of Amazonas, Northern Brazil (Amazon Rainforest region) were evaluated. The majority of strains (61.9% n = 13) were classified as multidrug-resistant (MDR), and five strains (23.8%) as extensively drug-resistant (XDR). Several virulence and antimicrobial-resistance genes were found among the strains and eight strains (38.1%) presented the hyper-mucoviscous phenotype. MLST analysis demonstrated a great diversity of STs among the strains, totaling 12 different STs (ST11, ST23, ST198, ST277, ST307, ST340, ST378, ST462, ST502, ST3991, ST3993 and ST5209). Three of these (ST11, ST23 and ST340) belong to CG258.

The dissemination of antibiotic-resistant priority pathogens beyond hospital settings is both a public health and an environmental problem. In this regard, high-risk clones exhibiting a multidrug-resistant (MDR) or extensively drug-resistant (XDR) phenotype have shown rapid adaptation at the human-animal-environment interface. In this study, we report genomic data and the virulence potential of the carbapenemase, São Paulo metallo-β-lactamase (SPM-1)-producing Pseudomonas aeruginosa strains (Pa19 and Pa151) isolated from polluted urban rivers, in Brazil. Bioinformatic analysis revealed a wide resistome to clinically relevant antibiotics (carbapenems, aminoglycosides, fosfomycin, sulfonamides, phenicols, and fluoroquinolones), biocides (quaternary ammonium compounds) and heavy metals (copper), whereas the presence of exotoxin A, alginate, quorum sensing, types II, III, and IV secretion systems, colicin, and pyocin encoding virulence genes was associated with a highly virulent behavior in the Galleria mellonella infection model. These results confirm the spread of healthcare-associated critical-priority P. aeruginosa belonging to the MDR sequence type 277 (ST277) clone beyond the hospital, highlighting that the presence of these pathogens in environmental water samples can have clinical implications for humans and other animals.

Even though antimicrobial-resistant bacteria have begun to be detected in wildlife, raising important issues related to their transmission and persistence of clinically important pathogens in the environment, little is known about the role of these bacteria on wildlife health, especially on endangered species. The Brazilian merganser (Mergus octosetaceus) is one of the most threatened waterfowl in the world, classified as Critically Endangered by the International Union for Conservation of Nature. In 2019, a fatal case of sepsis was diagnosed in an 8-day-old Brazilian merganser inhabiting a zoological park. At necropsy, major gross lesions were pulmonary and hepatic congestion. Using microbiologic and genomic methods, we identified a multidrug-resistant (MDR) extended-spectrum β-lactamase (ESBL) CTX-M-8-producing Escherichia coli (designed as PMPU strain) belonging to the international clone ST58, in coelomic cavity, oesophagus, lungs, small intestine and cloaca samples. PMPU strain harboured a broad resistome against antibiotics (cephalosporins, tetracyclines, aminoglycosides, sulphonamides, trimethoprim and quinolones), domestic/hospital disinfectants and heavy metals (arsenic, mercury, lead, copper and silver). Additionally, the virulence of E. coli PMPU strain was confirmed using a wax moth (Galleria mellonella) infection model, and it was supported by the presence of virulence genes encoding toxins, adherence factors, invasins and iron acquisition systems. Broad resistome and virulome of PMPU contributed to therapeutic failure and death of the animal. In brief, we report for the first time a fatal colibacillosis by MDR ESBL-producing E. coli in critically endangered Brazilian merganser, highlighting that besides colonization, critical priority pathogens are threatening wildlife. E. coli ST58 clone has been previously reported in humans, food-producing animals, wildlife and environment, supporting broad adaptation and persistence at human-animal-environment interface.

Wild birds have emerged as novel reservoirs and potential spreaders of antibiotic-resistant priority pathogens, being proposed as sentinels of anthropogenic activities related to the use of antimicrobial compounds. The aim of this study was to investigate the occurrence and genomic features of extended-spectrum β-lactamase (ESBL)-producing bacteria in wild birds in South America. In this regard, we have identified two ESBL (CTX-M-55 and CTX-M-65)-positive Escherichia coli (UNB7 and GP188 strains) colonizing Creamy-bellied Thrush (Turdus amaurochalinus) and Variable Hawk (Geranoaetus polyosoma) inhabiting synanthropic and wildlife environments from Brazil and Chile, respectively. Whole-genome sequence (WGS) analysis revealed that E. coli UNB7 and GP188 belonged to the globally disseminated clone ST602, carrying a wide resistome against antibiotics (β-lactams), heavy metals (arsenic, copper, mercury), disinfectants (quaternary ammonium compounds), and pesticides (glyphosate). Additionally, E. coli UNB7 and GP188 strains harbored virulence genes encoding hemolysin E, type II and III secretion systems, increased serum survival, adhesins and siderophores. SNP-based phylogenomic analysis, using an international genome database, revealed genomic relatedness (19-363 SNP differences) of GP188 with livestock and poultry strains, and genomic relatedness (61-318 differences) of UNB7 with environmental, human and livestock strains (Table S1), whereas phylogeographical analysis confirmed successful expansion of ST602 as a global clone of One Health concern. In summary, our results support that ESBL-producing E. coli ST602 harboring a wide resistome and virulome have begun colonizing wild birds in South America, highlighting a potential new reservoir of critical priority pathogens.

Untreated wastewater is a reservoir for multidrug-resistant bacteria, but its role in the spread of antibiotic resistance in the human population remains poorly investigated. In this study, we isolated a KPC-2-producing ST2787 Klebsiella quasipneumoniae subsp. quasipneumoniae (WW14A), recovered from raw sewage at a wastewater treatment plant in Argentina in 2018 and determined its complete genome sequence. Strain WW14A was resistant to all β-lactams, ciprofloxacin and amikacin. A core genome phylogenetic analysis indicated that WW14A was closely related to a GES-5-producing Taiwanese strain isolated from hospital wastewater in 2015 and it was clearly distinct from strains isolated recently in Argentina and Brazil. Interestingly, blaKPC-2 was harbored by a recently described IncP-6 broad-spectrum plasmid which was sporadically reported worldwide and had never been reported before in Argentina. We investigated the presence of the IncP-6 replicon in isolates obtained from the same sampling and found a novel non-typable/IncP-6 hybrid plasmid in a newly assigned ST1407 Enterobacter asburiae (WW19C) also harboring blaKPC-2. Nanopore sequencing and hybrid assembly of strains WW14A and WW19C revealed that both IncP-6 plasmids shared 72% of coverage (~20 kb), with 99.99% of sequence similarity and each one also presented uniquely combined regions that were derived from other plasmids recently reported in different countries of South America, Asia, and Europe. The region harboring the carbapenem resistance gene (~11 kb) in both plasmids contained a Tn3 transposon disrupted by a Tn3-ISApu-flanked element and the core sequence was composed by ΔISKpn6/blaKPC-2/ΔblaTEM-1/ISKpn27. Both strains also carried genes conferring resistance to heavy metals (e.g., arsenic, mercury, lead, cadmium, copper), pesticides (e.g., glyphosate), disinfectants, and several virulence-related genes, posing a potential pathogenic risk in the case of infections. This is the first study documenting blaKPC-2 associated with IncP-6 plasmids in K. quasipneumoniae and Enterobacter cloacae complex from wastewater in Argentina and highlights the circulation of IncP-6 plasmids as potential reservoirs of blaKPC-2 in the environment.

The dissemination of carbapenem-resistant and third generation cephalosporin-resistant pathogens is a critical issue that is no longer restricted to hospital settings. The rapid spread of critical priority pathogens in Brazil is notably worrying, considering its continental dimension, the diversity of international trade, livestock production, and human travel. We conducted a nationwide genomic investigation under a One Health perspective that included Escherichia coli strains isolated from humans and nonhuman sources, over 45 years (1974-2019). One hundred sixty-seven genomes were analyzed extracting clinically relevant information (i.e., resistome, virulome, mobilome, sequence types [STs], and phylogenomic). The endemic status of extended-spectrum β-lactamase (ESBL)-positive strains carrying a wide diversity of blaCTX-M variants, and the growing number of colistin-resistant isolates carrying mcr-type genes was associated with the successful expansion of international ST10, ST38, ST115, ST131, ST354, ST410, ST648, ST517, and ST711 clones; phylogenetically related and shared between human and nonhuman hosts, and polluted aquatic environments. Otherwise, carbapenem-resistant ST48, ST90, ST155, ST167, ST224, ST349, ST457, ST648, ST707, ST744, ST774, and ST2509 clones from human host harbored blaKPC-2 and blaNDM-1 genes. A broad resistome to other clinically relevant antibiotics, hazardous heavy metals, disinfectants, and pesticides was further predicted. Wide virulome associated with invasion/adherence, exotoxin and siderophore production was related to phylogroup B2. The convergence of wide resistome and virulome has contributed to the persistence and rapid spread of international high-risk clones of critical priority E. coli at the human-animal-environmental interface, which must be considered a One Health challenge for a post-pandemic scenario. IMPORTANCE A One Health approach for antimicrobial resistance must integrate whole-genome sequencing surveillance data of critical priority pathogens from human, animal and environmental sources to track hot spots and routes of transmission and developing effective prevention and control strategies. As part of the Grand Challenges Explorations: New Approaches to Characterize the Global Burden of Antimicrobial Resistance Program, we present genomic data of WHO critical priority carbapenemase-resistant, ESBL-producing, and/or colistin-resistant Escherichia coli strains isolated from humans and nonhuman sources in Brazil, a country with continental proportions and high levels of antimicrobial resistance. The present study provided evidence of epidemiological and clinical interest, highlighting that the convergence of wide virulome and resistome has contributed to the persistence and rapid spread of international high-risk clones of E. coli at the human-animal-environmental interface, which must be considered a One Health threat that requires coordinated actions to reduce its incidence in humans and nonhuman hosts.