Angie Alexandra Rincón-Real,Martha Cecilia Suárez-Alfonso
Carbapenem resistance in critically important human pathogens isolated from companion animals: a systematic literature review
Beta-lactam resistance,Carbapenem-resistant Enterobacteriaceae,Drug resistance,Gram-negative bacteria,One Health,Pets
This study aimed to describe the presence and geographical distribution of Gram-negative bacteria considered critical on the priority list of antibiotic-resistant pathogens published by the World Health Organization, including carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Acinetobacter spp., and carbapenem-resistant Pseudomonas aeruginosa. A systematic review of original studies published in 5 databases between 2010 and 2021 was conducted, including genotypically confirmed carbapenem-resistant isolates obtained from canines, felines, and their settings. Fifty-one articles met the search criteria. Carbapenem-resistant isolates were found in domestic canines and felines, pet food, and on veterinary-medical and household surfaces. The review found that the so-called “big five”—that is, the 5 major carbapenemases identified worldwide in Enterobacterales (New Delhi metallo-β-lactamase, active-on-imipenem, Verona integron-encoded metallo-β-lactamase, Klebsiella pneumoniae carbapenemase, and oxacillin [OXA]-48-like)—and the 3 most important carbapenemases from Acinetobacter spp. (OXA-23-like, OXA-40-like, and OXA-58-like) had been detected in 8 species in the Enterobacteriaceae family and 5 species of glucose non-fermenting bacilli on 5 continents. Two publications used molecular analysis to confirm carbapenem-resistant bacteria transmission between owners and dogs. Isolating critically important human carbapenem-resistant Gram-negative bacteria from domestic canines and felines highlights the importance of including these animal species in surveillance programs and antimicrobial resistance containment plans as part of the One Health approach.
Carbapenem resistance in critically important human pathogens isolated from companion animals: a systematic literature review

This study aimed to describe the presence and geographical distribution of Gram-negative bacteria considered critical on the priority list of antibiotic-resistant pathogens published by the World Health Organization, including carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Acinetobacter spp., and carbapenem-resistant Pseudomonas aeruginosa. A systematic review of original studies published in 5 databases between 2010 and 2021 was conducted, including genotypically confirmed carbapenem-resistant isolates obtained from canines, felines, and their settings. Fifty-one articles met the search criteria. Carbapenem-resistant isolates were found in domestic canines and felines, pet food, and on veterinary-medical and household surfaces. The review found that the so-called “big five”—that is, the 5 major carbapenemases identified worldwide in Enterobacterales (New Delhi metallo-β-lactamase, active-on-imipenem, Verona integron-encoded metallo-β-lactamase, Klebsiella pneumoniae carbapenemase, and oxacillin [OXA]-48-like)—and the 3 most important carbapenemases from Acinetobacter spp. (OXA-23-like, OXA-40-like, and OXA-58-like) had been detected in 8 species in the Enterobacteriaceae family and 5 species of glucose non-fermenting bacilli on 5 continents. Two publications used molecular analysis to confirm carbapenem-resistant bacteria transmission between owners and dogs. Isolating critically important human carbapenem-resistant Gram-negative bacteria from domestic canines and felines highlights the importance of including these animal species in surveillance programs and antimicrobial resistance containment plans as part of the One Health approach.

A systematic analysis of antimicrobial resistance (AMR) in 2019 found that bacteria-related AMR accounted for 4.5 million deaths worldwide. Moreover, the global burden of AMR will be responsible for 10 million deaths worldwide by 2050 [1]. AMR incurs increased healthcare system costs associated with length of hospital stay, additional follow-up visits, and using drugs of last resort (DoLR) [2−4]. Antimicrobial-resistant infections in clinical and community settings are frequently associated with β-lactam-resistant Gram-negative bacteria [5−7]. Resistance to β-lactams in Gram-negative bacteria occurs due to target site modification, decreased antibiotic concentration resulting from efflux pumps, changes in outer membrane permeability caused by the loss or modifications of porins, and enzymatic inactivation of the drug by β-lactamase production. More than 4.900 β-lactamases have been reported to date [6,8]. β-lactamases can be structurally [9] or functionally classified [10]. Four types are recognized structurally. Types A, C, and D are serine enzymes, and type B includes metallo-β-lactamases [6,8,9]. Carbapenemases are extremely relevant because they hydrolyze carbapenems; β-lactam DoLRs have broad-spectrum activity and stability [6]. Carbapenem resistance (CR) is considered a marker for extensively drug-resistant (XDR) and pandrug-resistant (PDR) Gram-negative bacteria because it is associated with a wide range of co-resistance to other antimicrobial drugs [11]. According to epidemiological factors related to the degree of global spread of carbapenem-hydrolyzing enzymes, 2 groups of carbapenemases have been proposed for Enterobacterales. The first group comprises the “big five” that are widespread worldwide, and the second group includes the minor or “rare” carbapenemases that have a limited geographical spread [8]. The “big five” carbapenemases include the class A enzyme Klebsiella pneumoniae carbapenemase (KPC), class B enzymes active-on-imipenem (IMP), Verona integron-encoded metallo-β-lactamase (VIM), New Delhi metallo-β-lactamase (NDM), and class D enzyme active on oxacillin [OXA]-48-like [8]. Three oxacillinases from the genus Acinetobacter (OXA-23-like, OXA-40-like, and OXA-58-like) have been reported as of concern due to their worldwide spread [12,13]. The World Health Organization (WHO) published a global priority list of antibiotic-resistant bacteria to guide the discovery, research, and development of new antibiotics in 2017. The most important category on the list (i.e., critical-priority microorganisms) includes the carbapenem-resistant Enterobacteriaceae (CRE) family and 2species of carbapeem-resistant, glucose-non-fermenting bacilli (CRGNFB), namely, carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas aeruginosa, due to their impact on mortality, disease burden, and circulation at the human-animal-environment interface [11]. AMR involves complex human-animal-environment interface-related microbial interactions. Carbapenem use is not recommended for companion animals [14]; however, CR isolates have been reported in these animals [15]. The interactions between owners and companion animals promote AMR dissemination and maintenance through bacterial bidirectional transmission [16]. Thus, a One Health-oriented approach to analyzing carbapenemase circulation in companion animals is essential—that is, an integrated, multisector approach seeking to balance and optimize the health of humans and animals, as well as environmental sustainability [17,18]. Accordingly, this study adopted a One Health perspective for describing the presence and geographic distribution of antibiotic-resistant Gram-negative bacteria classified as critical on the WHO priority list isolated from domestic canines and felines and the contexts associated with their presence, including CRE, carbapenem-resistant A. baumannii, and carbapenem-resistant P. aeruginosa.

A search for original articles that evaluated antimicrobial susceptibility to carbapenems in Enterobacteriaceae and glucose non-fermenting bacilli (GNFB), such as Acinetobacter spp. and P. aeruginosa obtained from domestic canines or felines, or both, and contexts associated with their presence (e.g., veterinary care, pet food, and/or the animal’s home) in which genotypic CR was detected was carried out. The databases consulted were Medline, PubMed, Web of Science, Scopus, Wiley Online Library, and CABI: VetMed Resource. Descriptors from the DeCS/MeSH thesauri were used by applying a specific search formula and combining the following terms: (carbapenemase OR CPE OR carbapenem-resistance) AND (Enterobacteriaceae OR Enterobacterales OR Escherichia coli OR Enterobacter cloacae OR Klebsiella pneumoniae OR Pseudomonas OR Pseudomonas aeruginosa OR Acinetobacter OR Acinetobacter baumannii) AND (companion animals OR pets OR dog OR cat) NOT review (Table S1). Articles in English, Spanish or Portuguese, published from January 1, 2010 to April 24, 2021 were included. Publications that informed only about isolates from human sources, animals other than canines or felines (origin or unrelated environmental origin), reports of phenotypic resistance without genotypic confirmation, review articles or meta-analyses, editorials, book chapters, proceedings of academic events, evaluation of diagnostic or therapeutic methods of CR bacteria, were excluded. Duplicates were removed using the Mendeley bibliographic manager ver. 1.19.8 (Elsevier, Amsterdam, The Netherlands). Two researchers carried out the search and selection independently in 2 phases. The first phase was based on the title and abstract, and the second was involved analyzing the full text. We conducted a manual search of the reference lists of included articles and selected publications according to the previous criteria to ensure better information coverage. Divergences in selection were resolved by consensus.

The information was compiled in an Excel sheet (Microsoft Corp., Redmond, WA, USA). It included the main author, year, country, place, sample collected, year of isolation, animal, population, health status, history of hospitalization or antimicrobial therapy in animals and/or owners, as well as the microorganism identified, number of isolates, sequence type (ST), epidemiological classification of AMR, susceptibility assessment, and the genetic mechanism of CR.

The initial search yielded 368 articles, 192 of which were duplicates. Of these, 91 were discarded according to their title and abstract, and 34 based on the full-text review (Figure 1). Fifty-one articles were included for analysis [19−69].

Of the 51 articles, 48 reported isolates from domestic canines and/or felines, 3 from veterinary medical care environments, 1 from home environments, and another from commercial food (wet food). Of the 48 publications, 26 included dogs, 4 involved cats, and 18 encompassed both species. Furthermore, of the 51 publications, 40 were original studies, and 11 were from epidemiological surveillance/monitoring programs. The publications included articles spanning 11 years (2010−2021); however, some CR isolates reported a longer period of analysis (Table 1) [19−69].

Of the 51 articles reporting CRE and CRGNFB, most (n=49) described the health status of animals. Four studies involved healthy animals, 38 included diseased animals, and 5 had animals of both statuses. In the reports of diseased animals, CRE isolates were obtained from different systems, including genitourinary, respiratory, gastrointestinal, cardiovascular, musculoskeletal, ear, skin, and soft tissues, as well as neoplasms and wounds of undescribed origin. The CRGNFB were obtained from respiratory, genitourinary, ocular and ear tissues, soft tissues, and systemic examinations (Table 2; Table S2) [19−69]. Twenty articles reported antimicrobial therapy close to the sampling date, previously, or both. Three articles reported the administration of carbapenems (meropenem) in South Korea, and 17 described the use of other antimicrobials, including tetracyclines, cephalosporins, and quinolones, β-lactams, β-lactams/β-lactamase inhibitors, aminoglycosides, lincosamides, sulfonamides, nitroimidazoles, and phosphonic acids (Table 2).

Twenty publications reported information on the frequency of animals with CR isolates. In dogs and cats, the proportions of CRE isolation ranged from 0.25% to 21.6%. The frequency of CRE was registered on 3 continents. In Europe, the highest frequency of CRE happened in an outbreak of CR E. coli in dogs in a veterinary hospital in Switzerland (21.65%). In Asia, the highest frequency of CRE was in dogs in veterinary hospitals in India (6.75%). In Africa, the highest frequency of CRE occurred in animals sampled at an official veterinary office in Algeria (2.5%) (Table 3) [19,20,24−28,30−33,44,45,48,56,59,61,63,67]. The frequency of companion animals from CRGNFB isolates ranged from 1.3% to 12.50%. The frequency of CRGNFB was reported on 2 continents. In Asia, the highest frequency of CRGNFB was registered in dogs in a university veterinary hospital in South Korea (12.50%). In Europe, the highest frequency of CRGNFB was in veterinary hospitals in Italy (5.34%) (Table 3).

Antimicrobial susceptibility in publications was evaluated using the agar diffusion (Kirby-Bauer) and minimum inhibitory concentration (MIC) techniques with standard and automated (Vitek bioMérieux, Marcy l'Étoile, Francia ; Wider (Francisco Soria Melguizo, SA, Madrid, Spain) and Sensitire (TREK Diagnostic Systems, Cleveland, OH, USA) broth microdilution and the epsilometry test (Epsilometer test (E test; AB Biodisk, Solna, Sweden). Two publications determined CR only with molecular techniques (Table S3). The MIC values of meropenem, imipenem, and ertapenem associated with CR isolates are presented in Table S3. Of 49 publications reporting CR phenotypes, 46 showed susceptibility data to antimicrobials other than carbapenems. CRE and CRGNFB exhibited resistance to penicillin; penicillin/β-lactamase inhibitors; cephamycins; first-, second-, third-, and fourth-generation cephalosporins; cephalosporins/β-lactamase inhibitors; monobactams; aminoglycosides; quinolones; sulfonamides; trimethoprim; tetracyclines; phenicols; nitrofurans; glycylcyclines; and polymyxins. Resistance to macrolides was evaluated and reported only in CRE. Susceptibility to phosphonic acids was evaluated, and resistance was reported in CRE and CRGNFB (Table S3). CRE and CRGNFB presented resistance to last-resort antimicrobials such as amikacin, colistin, fosfomycin, nitrofurantoin, and tigecycline in 14, 3, 8, 4, and 3 publications, respectively (Table S3). MDR isolates were reported in 20 articles in 4 CRE species (E. coli, K. pneumoniae, Salmonella enterica serovar Typhimurium, and E. cloacae) from companion animals and veterinary care surfaces. Four CRGNFB MDR species (P. aeruginosa, A. baumannii, Acinetobacter radioresistens, and Stenotrophomonas maltophilia) obtained from companion animals and domestic environments were reported in 7 articles (Table S3). The XDR phenotype was reported in 1 publication that highlighted 1 species of CRE (E. coli) from a canine, and 2 publications recording one species of CRGNFB (A. baumannii) obtained from dogs and cats. No isolates showed a PDR phenotype (Table S3).

Genotypic CR was confirmed in publications employing 3 single or combined techniques: polymerase chain reaction, microarrays, and whole-genome sequencing (Table S3). In CRE, resistance was associated only with the production of carbapenemases. In companion animals, the “big five” carbapenemases were detected, with higher frequencies of OXA-48-like and NDM, and smaller proportions of KPC, IMP, and VIM. In veterinary environments, only OXA-48-like and NDM were detected. Conversely, only OXA-48 was found in commercial feed. None of the minor carbapenemases were identified in the CRE reported (Table 2). CR Acinetobacter spp. found in dogs and cats showed carbapenemase production as the only mechanism of resistance, with 2 of the “big five” (i.e., IMP and NDM), and the 3 important members of the genus (i.e., OXA-23-like, OXA-40-like, and OXA-58-like). CR P. aeruginosa was detected in dogs and home environments (sofa) and produced 2 of the 5 major carbapenemases (i.e., IMP and VIM). A CR mechanism different from carbapenemases was detected in a P. aeruginosa isolate from a canine. This mechanism involved the loss of the OprD outer membrane porin. CR S. maltophilia isolated from dogs and cats was associated with the production of L1, a species-intrinsic metallo-carbapenemase (Table 3).

CRE and CRGNFB acquired from companion animals were reported in 19 countries on 5 continents: 5 countries from Asia, 2 from the Americas, 9 from Europe, 2 from Africa, and 1 from Oceania (Figures 2 and 3). In companion animal-associated contexts, CRE and CRGNFB were reported in 3 countries on 3 continents: 1 in the Americas, 1 in Africa, and 1 in Europe (Figure 4). The blaNDM-5 gene was reported in plasmids only in CRE in Asia, the Americas, Europe, and Africa. The blaNDM-1 gene in CRE was detected in plasmids and chromosomes in Asia and the Americas; and in CRGNFB, without reporting its genomic location, but only in Europe. The blaOXA-48, blaOXA-181, and blaOXA-244 genes were found in plasmids only in CRE in Europe, the Americas, Africa, and Asia. The blaOXA-23, blaOXA-58, and blaOXA-72 genes located on plasmids and chromosomes were reported only in CRGNFB in Europe and Asia. The blaVIM-1 and blaVIM-4 genes on plasmids were registered in CRE in Europe and Africa, while the blaVIM-2 gene located on chromosomes and integrons was present in CRGNFB in the Americas and Asia. The blaKPC-2 and blaKPC-4 genes in plasmids were found exclusively in CRE in North and South America. The blaIMP-4 gene located on plasmids was reported only in CRE in Oceania, while the blaIMP-1 gene, with no registry of its genomic location, and blaIMP-45 on chromosomes were detected in CRGNFB in Asia (Table 1; Figures 2–4).

In publications that established the STs of CR bacteria identified by the molecular epidemiology technique of multilocus sequence typing, 55 STs were reported in CR isolates from companion animals, and 4 had descriptions of the contexts associated with their presence. Eight publications registered no STs. In CRE carrying blaNDM genes, 4 STs (ST4656, ST167, ST410, and ST9437) were reported on all continents except Oceania. CRE carrying blaVIM genes were clustered into 3 STs (ST2090, ST493, and ST182) in Africa and Europe, and CRGNFB into 3 STs (ST1047, ST1203, and ST233) in Asia and the Americas. Bacteria carrying blaIMP had the ST19 sequence in Oceania and the ST308 sequence in Asia in CRE and CRGNFB, respectively. Two STs (ST11 and ST171) were identified in blaKPC-producing CRE in the Americas (ST11, ST171). Bacteria carrying blaOXA had the widest variety of STs, with 39 in CRE in Asia, the Americas, Europe, and Africa and 5 in CRGNFB (ST1, ST10, ST2, ST25, and ST93) in Europe and Asia (Table 1; Figures 2–4).

Eleven studies that reported CR isolates in dogs and cats also registered other animal species, including pigs, birds (poultry, pet, and wild), cattle, sheep, goats, guinea pigs, rats, mice, rabbits, horses, fish, and flies. Seven publications reported CR in humans. Two were from hospital and community settings not related to companion animals, 2 from owners and employees in veterinary care settings, and 1 from backyard swine farm residents. In addition, 2 articles molecularly confirmed human-animal transmission from owners to dogs. CRE in Finland and CRGNFB in Brazil showed confirmed transmission with the same types of sequences and CR genes found in dogs. Both owners reported previous hospitalization, and 1 of them had also traveled internationally [34,54] (Table S4).

The current review compiled evidence on the presence and spread on 5 continents of Gram-negative bacteria categorized as critical in the WHO priority list of antibiotic-resistant bacteria for research and development of new antibiotics, such as CRE and CRGNFB isolated from domestic canines and felines, and on the contexts associated with their presence. Only 20% of the studies originated from surveillance programs, all in high-income and upper-middle-income countries. Since 2018, the United States has included dogs and cats in AMR surveillance programs [70,71]. In Europe, some countries included both species in their specific AMR control programs, and the European Union plans to launch the European Antimicrobial Resistance Surveillance Network in Veterinary Medicine (EARS-Vet), in which dogs and cats will be included in the scope of surveillance [72,73]. However, the role of domestic canines and felines in CR bacterial transmission is often underestimated in local AMR containment programs [74,75]. Although in this study, the CR frequency in domestic canines and felines ranges from 0.4% to 6%, it is essential to integrate these animals into surveillance, control, and prevention strategies for CR, especially in low- and lower-middle-income countries where the problem may be underdiagnosed [76]. Of particular concern are reports of VIM-2-producing P. aeruginosa [61] and an outbreak of OXA-181-producing E. coli [20] in veterinary hospitals in South Korea and Switzerland, with frequencies close to 12% and 20%, respectively. Although neither of those 2 reports described the administration of carbapenems, the use of meropenem in veterinary hospitals in South Korea [22,32,68] and the use of β-lactams and quinolones in the Swiss veterinary hospital have been registered, favoring the co-selection of CR isolates [34,59,77]. Carbapenemase production was the most important mechanism of CR in Enterobacterales and GNFB of companion animals. The most frequent carbapenemase was OXA. Most OXA enzymes with carbapenemase activity have been identified in Acinetobacter spp. and P. aeruginosa [78]. The carbapenemases OXA-23-like (variant OXA-23), OXA-40-like (variant OXA-72), and OXA-58-like (variant OXA-58) were only identified in Acinetobacter spp. in companion animals from Europe and South Asia. However, the OXA-48-like enzyme in humans has been reported mainly in K. pneumoniae, E. coli, and E. cloacae [79], and it has been identified in pets, related environments, and food (variants OXA-48, OXA-181, and OXA-244). All were plasmid-encoded, confirming the risk posed by their rapid and easy transfer [79]. The blaOXA-48 gene detected in the European countries in animal feed might be associated with the components of the feed formulation. blaOXA-48 has also been reported on Enterobacteriaceae obtained from poultry and swine carcasses in Europe and Asia [80] and drinking water systems of industrialized countries (United States) [81]. Conversely, the most likely source in this case, is human intervention during manufacturing and before packing [50], suggesting the relevance of humans in contaminating animal feed with CR organisms. The second most frequent enzyme found in this review was NDM in E. coli, E. cloacae, Citrobacter freundii, and A. radioresistens in companion animals in North America, Asia, Europe, and North African veterinary settings. NDM is considered endemic in the Balkan countries, the Middle East, and India, although it has spread worldwide, mainly through E. coli, K. pneumoniae, and Acinetobacter spp. [81−83]. E. coli ST101 and ST131 and K. pneumoniae ST11 and ST147 have been reported as epidemic clones responsible for its dissemination [81,83,84]. In E. coli, NDM was localized on plasmids. Nevertheless, E. coli presented ST167 and ST410 sequences, suggesting different dynamics in the circulation of NDM-producing E. coli in companion animals. Domestic canines and felines are not considered the main sources of CR acquisition for humans [81]. However, the transmission of CRE and CRGNFB from humans to dogs, from which the same microorganisms were isolated, has been reported in Finland [34] and Brazil [54], respectively. In both cases, the owners had a history of international travel or prolonged hospitalization, which are risk factors associated with CR acquisition [78,81]. At the human-animal interface, the role of domestic canines and felines in CR dissemination in community settings should not be underestimated. In Brazil, the transmission of a CR bacterium between an owner and pet was confirmed, as well as its presence on shared household surfaces, such as the sofa [54]. The home can become a source of CR bacteria for companion animals, contributing to AMR spread in human environments. Canines, due to their generally more social behavior than felines, interact daily with other congeners and with people outside their family context, including at parks, daycare centers, shelters/kennels, and veterinary hospitals [85], favoring the spread/increase of the presence of CR bacteria in community settings. The presence of plasmid-borne IMP carbapenemase detected in the zoonotic bacterium Salmonella serovar Typhimurium in hospitalized cats in Australia [35] is of particular concern. Its presence could be attributed to human factors, considering that the circulation of the blaIMP gene has been demonstrated in Gram-negative bacteria in Australian human clinical settings and in migratory birds carrying CR Salmonella spp. acquired from human environments and genetically related to human isolates [86]. Companion animals may also be involved in the spread of AMR in rural settings. A Chinese backyard pig farm reported CRE circulation in humans, birds, and flies, predominantly originating from canine gene complexes [19]. Human and animal populations that coexist in small-scale agricultural productions with insufficient biosecurity measures have been reported to be more vulnerable to acquiring CR [76]. In rural settings, the use of carbapenems may be lower due to the associated cost. However, using other antimicrobials that may favor co-selection and the occurrence of CR cannot be ruled out [34,59,77]. The limitations of the current review include the fact that it was based only on studies published in electronic databases, and some valid reports of carbapenemases in companion animals in the gray literature may not have been identified unknown. Furthermore, low- and lower-middle-income countries may have been underrepresented due to the absence of publications in electronic databases. In addition, methods of isolation and detection of CR bacteria, phenotypic interpretation criteria, and the classification of resistant, intermediate, and susceptible isolates varied between the studies, and these factors may have influenced the collective results.

In conclusion, evidence of the presence of CRE and CRGNFB from companion animals and associated contexts in the 5 continents is compiled in this review. Domestic canines and felines are recognized as a possible source of dissemination and maintenance of carbapenemases for animals and humans. Thus, there is an urgent need for in-depth studies on the dynamics of CR circulation, including companion animals, under the concept of One Health in CR surveillance programs and plans for the containment of AMR, especially in low- and lower-middle-income countries, where the magnitude of the problem may be underestimated.