Companion Animals: Reservoir of Carbapenem Resistance

Companion Animals: Reservoir of Carbapenem Resistance: Comparison

Please note this is a comparison between Version 1 by Joana Cabral Moreira da Silva and Version 2 by Vivi Li.

The dissemination of antimicrobial-resistance is a major global threat affecting both human and animal health. Carbapenems are human use β-lactams of last resort; thus the dissemination of carbapenemase-producing (CP) bacteria creates severe limitations for the treatment of multidrug-resistant bacteria in hospitalized patients. Even though carbapenems are not routinely used in veterinary medicine, reports of infection or colonization by carbapenemase-producing Enterobacterales in companion animals are being reported. NDM-5 and OXA-48-like carbapenemases are among the most frequently reported in companion animals. Like in humans, Escherichia coli and Klebsiella pneumoniae are the most represented CP Enterobacterales found in companion animals, alongside with Acinetobacter baumannii. Considering that the detection of carbapenemase-producing Enterobacterales presents several difficulties, misdiagnosis of CP bacteria in companion animals may lead to important animal and public-health consequences. It is of the upmost importance to ensure an adequate monitoring and detection of CP bacteria in veterinary microbiology in order to safeguard animal health and minimise its dissemination to humans and the environment.

companion animals
Enterobacterales
carbapenemase detection methods

1. Introduction

Carbapenems are β-lactam antibiotics with broad antimicrobial spectrum. With the emergence of Extended Spectrum β-lactamases (ESBLs), carbapenems became the antibiotics of last resort for treatment of human patients with ESBL-producing Enterobacterales infections [1]. Although carbapenems are not hydrolysed by most β-lactamases, their effectiveness was seriously compromised by the emergency of carbapenem-hydrolysing enzymes, the carbapenemases ^[1][2][1,2]. The most important carbapenemases belong to three different Amber classes [2]: (i) class A, including the KPC, IMI/NMC, SFC, GES type enzymes ^[1][2][1,2]; (ii) class B, including VIM, IMP, and NDM metallo-β-lactamases (MBL) [3]; and (iii) class D, including OXA-48-like type enzymes [4].

Regulation on the use of carbapenems in animals varies worldwide and they do not belong to the OIE List of Antimicrobial Agents of Veterinary Importance [5]. According to the European Medicine Agency categorization of antibiotics for animal use, carbapenems are included in category A (“Avoid”), meaning they are not authorized for use in veterinary medicine in the European Union (EU), except in exceptional clinical cases in companion animals, under the cascade according to Article 112 of the veterinary medicinal products Regulation 2019 of the European Union Legislation [6]. Reports of carbapenemase-producing (CP) and carbapenem-resistant Enterobacterales (CRE) detection among companion animals are emerging worldwide (Table 1). The identification of CP bacteria in companion animals, which have significant direct contact with humans, has raised public health concern as animals may constitute an important reservoir of carbapenems resistance genes and contribute to its dissemination [7]. Very recently, the building of an European Antimicrobial Resistance Surveillance network in veterinary medicine (EARS-Vet) has been reported [8]. However, carbapenem resistance epidemiology remains quite unknow, as, unlike in human medicine, no global surveillance protocol is currently in place for companion animal veterinary medicine. Furthermore, the detection of CP bacteria relying on antimicrobial susceptibility testing alone (AST) presents several pitfalls leading to its possible miss detection in veterinary medicine. Appropriate monitoring and detection of antimicrobial resistance against these critically important antimicrobials in veterinary medicine is of the utmost importance to avoid treatment failure and prevent its dissemination to humans and the environment. However, there is a lack of recommendations directed specifically to the veterinary medicine needs in the published literature.

2. Carbapenemase-Producing Bacteria in Companion Animals

To ouresearchers' best knowledge, more than 25 reports of CP bacteria in dogs and cats have been published worldwide. These include, both infection and colonization CP isolates harbouring KPC, VIM, IMP, NDM, or OXA β-lactamases (Table 1). Briefly, three studies detected KPC-producing Escherichia coli and Klebsiella pneumoniae from dogs in Brazil and in Enterobacter xiangfangensis from a dog in the United States ^[9][10][11][9,10,11]. A IMP-4-enzyme in Salmonella isolates was recovered from a cat’s faecal samples in Australia [12], VIM-2 in Pseudomonas aeruginosa from dogs with pyoderma and otitis in South Korea [13] and VIM-1 in K. pneumoniae from dogs in Spain [14]. A number of NDM-5-producing E. coli have been found in dogs and cats ^{[15][16][17][18][19][20][21][22]}[15,16,17,18,19,20,21,22], one NDM-1-producing Acinetobacter radioresistens was detected in a dog, six NDM-1-producing E. coli from dogs and cats in the United States, two NDM-1-producing E. coli from a dog in China, and finally one NDM-9 from a farm dog in China ^[23][24][25][23,24,25]. Several OXA-48-like carbapenemase-producing E. coli, K. pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae isolates were recovered from dogs, cats, and horses, representing one of the most frequent carbapenemases detected in companion animals alongside with NDM-5 (Table 1) ^{[17][26][27][28][29][30][31][32]}[17,26,27,28,29,30,31,32]. In addition, OXA-23- and OXA-66-producing Acinetobacter baumannii were isolated from clinical samples from dogs and cats ^[23][33][34][23,33,34]. Interestingly, although the detection of CP bacteria in companion animals dates to at least 2009, detection methods vary widely between studies, with the use of selective culture media being the most frequent for the detection of commensal CP isolates, while antimicrobial susceptibility testing alone (AST) is the main method used for the detection of CP isolates in infection cases (Table 1). Another important finding is that most CP bacterial species isolated from companion animals belong to the priority 1 (“critical”) category within the WHO priority pathogens list [35], thus highlighting the importance of properly monitoring and effectively detecting these carbapenem resistance mechanisms in companion animals.

Table 1.

Carbapenemases found in companion animals across the world.

Enzyme	Year	Country	Host	Source	Bacterial Species

Table 2.

Common β-lactam hydrolysis profile of carbapenemases.

Amber Class	Representative Carbapenemase	Detection Methods	Refs.
Amber Class	Representative Carbapenemase	Type	Hydrolysis Profile				Refs.
Narrow Spectrum Cephalosporins	Extended Spectrum Cephalosporins	Type	Imipenem *	Meropenem *			Refs.
IMP-4	2016	Australia	Cats	Commensal	Salmonella enterica serovar Typhimurium	AST	[12]
KPC-2
Class A	KPC	+	+	+	+	^[2]^[9][2,9]
2018	Brazil	Dog	Infection	(UTI)	Escherichia coli	Imipenem synergy test, modified Hodge testing, PCR
Class B	IMP, VIM, NDM,	[	+	9	]
+	+	+	[	3	]	KPC-2	2021	Brazil	Dog	Infection (UTI)	Klebsiella pneumoniae
Class D	Imipenem synergy test, AST	OXA-48-like	+	-	Variable ¹	-	^[4]^[38]^[39][4,38,39][10]
Class D	KPC-4	2018	USA	Dog	Infection (UTI, SSTI)	Enterobacter xiangfangensis	Biochemical Tests	[11]
OXA-23-like	+	+	+	+	[4]	NDM-1	2013	United States	Dogs, Cats	Infection (SSTI, UTI)	Escherichia coli	AST	[24]
NDM-1	2017	China	Dogs	Commensal	Escherichia coli	Selective culture media	^[16]^[25][16,25]
NDM−1	2018	Italy	Dog	Commensal	Acinetobacter radioresistens	Selective culture media	[23]
NDM-5	2016	Algeria	Dogs	Commensal	Escherichia coli	PCR	[17]
NDM-5	2017	China	Dogs	Commensal	Escherichia coli	Selective culture media	[16]
NDM-5	2019	United Kingdom	Dog	Infection (SSTI)	Escherichia coli	AST	[19]
NDM-5	2018	Finland	Dogs	Infection (Otitis externa)	Escherichia coli	AST followed by modified Hodge testing, UV spectrometric detection of imipenem hydrolysis	[18]
NDM-5	2021	Italy	Dog	Infection (UTI)	Escherichia coli	Meropenem synergy test	[15]
NDM-5	2018	United States	Dog	Infection (URTI)	Escherichia coli	AST	[20]
NDM-5	2018	United States	Dogs, Cats	Infection (UTI, URTI)	Escherichia coli	AST	[22]
NDM-5	2018	South Korea	Dog, Cat	Commensal	Escherichia coli	AST, PCR	[21]
NDM-9	2017	China	Dog	Commensal	Escherichia coli	Selective culture media	[16]
OXA-48	2009–2010	Germany	Dogs, Cats, Horses	Infection	Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae	Selective culture media for cephalosporin resistance, PCR	[36]
OXA-48	2013	Germany	Dog	Commensal, Infection (UTI, SSTI, URTI, CRBSI)	Klebsiella pneumoniae, Escherichia coli	AST	[29]
OXA-48	2016	United States	Dogs, Cats	Infection (UTI, SSTI, Genital tract)	Escherichia coli	AST	[31]
OXA-48	2016	Algeria	Dogs	Commensal	Escherichia coli	PCR	[17]
OXA-48	2017	Algeria	Dogs, Cat, Horses, Pet birds	Commensal	Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae	Selective culture media	[32]
OXA-48	2017	France	Dog	Commensal	Escherichia coli	Selective culture media	[30]
OXA-48	2018	Germany	Dogs, Cats, Horses	Infection (UTI, SSTI, genital tract, otitis, URTI)	Klebsiella pneumoniae, Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca	Selective culture media	[28]
OXA-181	2018	Switzerland	Dogs, Cats	Commensal	Escherichia coli	Selective culture media	[26]
OXA-181	2020	Portugal	Dog	Commensal	Escherichia coli	Selective culture media and AST	[27]
OXA-181	2021	Portugal	Cat	Infection (SSTI)	Klebsiella pneumoniae	Selective culture media and AST	[37]
OXA-23	2014	Portugal	Cat	Infection (UTI)	Acinetobacter baumannii	AST	[33]
OXA-23	2017	Germany	Dogs, Cats	Infection (UTI, suppurate inflammation)	Acinetobacter baumannii	Selective culture media	[34]
OXA−23	2018	Italy	Dogs, Cats	Commensal	Acinetobacter baumanni	Selective culture media	[23]
OXA-66	2017	Germany	Dogs, Cats	Infection (UTI, SSTI, URTI, CRBSI, suppurate inflammation)	Acinetobacter baumannii	Selective culture media	[34]
VIM-1	2016	Spain	Dog	Commensal	Klebsiella pneumoniae	Selective culture media, Meropenem synergy test	[14]
VIM-2	2018	South Korea	Dog	Infection (SSTI)	Pseudomonas aeruginosa	AST	[13]

AST, antimicrobial susceptibility testing; CRBSI, catheter-related bloodstream infection; SSTI, skin soft tissue infection; URTI, upper respiratory tract infections; UTI, urinary tract infection.

3. Phenotypic Characteristics of Carbapenemases and Their Genetic Background in Isolates from Companion Animals

The β-lactam resistance phenotype of CP isolates can vary depending on the type of carbapenemase and its hydrolysing activity (Table 2

* Imipenem and meropenem representative MIC values for carbapenemase-producing isolates from companion animals are listed in Table S1. ¹ Imipenem susceptible in OXA-48-like has been reported.

3.1. Serine Carbapenemases

Serine carbapenemases of molecular (Ambler) class A corresponds to the KPC, IMI/NMC, SFC, and GES enzymes that have a hydrolytic mechanism involving an active site serine at position 70 (Ambler numbering of class A β-lactamases), conferring resistance to first-, second-, and third-generation cephalosporins, imipenem, and meropenem [2]. Class A carbapenemases have been rarely detected in companion animals, the KPC enzyme being the only one reported until now from dogs with UTI and SSTI (Table 1). In K. pneumoniae and E. coli from dogs, the bla_KPC-2 gene was found in Tn4401 transposons contained in IncN plasmids ^[9][10][9,10] and the bla_KPC-4 gene was detected in an IncHI2 plasmid in the context of Tn4401b transposon in E. xiangfangensis isolated from a dog’s clinical samples [11].

3.2. Metallo-β-Lactamases

Class B carbapenemases have a critical clinical significance due to their ability to hydrolyse all β-lactams (Table 2) ^[3][40][3,40]. So far, more than 50 allelic β-lactamase-conferring imipenem resistance (IMP) variants are listed at GenBank DNA sequence database. However, only IMP-4 has been reported among companion animals, namely, cats, in Salmonella enterica serovar Typhimurium (Table 1) [12]. The IMP-4 coding gene was located on a gene cassette (bla_IMP-4-qacG-aacA4-catB3) in a class 1 integron, associated with a conjugative plasmid IncHI2, also carrying other resistance genes, such as tetA (mediating resistance to tetracycline), aac (resistance to aminoglycosides), cat (chloramphenicol resistance), sul (sulphonamide resistance), bla_OXA (different serine oxacillinases), and bla_TEM-1 (narrow-spectrum β-lactamases) [12]. Verona Integron-encoded Metallo-β-Lactamase (VIM) enzymes are the second most common Class B carbapenemase detected in companion animals (Table 1). VIM-1 and VIM-2 were described in K. pneumoniae and P. aeruginosa isolates from dogs, respectively; both located in class 1 integrons incorporated on untyped plasmids ^[13][14][13,14]. The bla_NDM genes pose a serious public health concern, since most common plasmids associated with its spread often have various antibiotic resistance genes resulting in multidrug resistance phenotypes ^[18][19][41][18,19,41]. Until now, 28 variants have been described, with resistance against all β-lactams expect monobactams [42]. In companion animals only NDM-1 and NDM-5 have been described so far (Table 1), the latter being more frequent. For one metallo-β-lactamase NDM-1, the encoding gene was located in a transposon Tn125 (composed of bla_NDM-1-ble_MBL-trpF-TAT-cutA1-groES-groEL-insE-Δpac genes between a pair of ISAba125), integrated in the chromosome of an A. radioresistens isolated from a dog in Italy [23]. This Tn125 transposon usually encompasses bla_NDM genes with two flanking ISAba125 elements, and in companion animals it was also found in bla_NDM-5 carrying strains ^[16][23][16,23]. A NDM-1-producing E. coli isolate harboured bla_NDM-1 in another genetic region, which was not flanked by ISAba125 elements downstream of the resistance gene [25]. NDM-5 metallo-β-lactamase differs from NDM-1 by four amino acids and has been found in the chromosome of an integrated IncF plasmid, from an E .coli isolate causing skin and soft tissue infection on a dog in the United Kingdom [19]. In the United States, the bla_NDM-5-encoding gene has been found on IncFII-type plasmids ^[20][22][20,22], whereas in South Korea it was described in an IncX3-type plasmid [21] with the surrounding genetic environment of ISAba125-bla_NDM-5-ble_MBL-trpF-TAT-ISCR26.

3.3. Oxacillinases

The class D, carbapenem-hydrolysing OXA-48 and its variants, namely, OXA-181, are one of the most common in veterinary settings (Table 1). The OXA-181 variant weakly hydrolyses both carbapenem and extended-spectrum cephalosporins and differs from OXA-48 at four amino acid substitution, yet its kinetic properties appear broadly similar to OXA-48 ^[43][44][45][43,44,45]. These enzymes can be associated more with different β-lactam hydrolysis profiles than the other serine-metallo-β-lactamases, making its accurate detection difficult. By possibly being susceptible in vitro to meropenem and imipenem (Table 2), two widely used surrogates to identify carbapenem resistance in clinical microbiology, carbapenem-resistant bacteria harbouring OXA-48-like carbapenemases may easily be misdiagnosed as ESBL-producers, which may lead to treatment failure. OXA-48-coding genes in CP isolates have been associated with no other resistance genes; or with extended-spectrum β-lactamases coding genes, thus conferring either low or high MIC against carbapenems [46]. High-level resistance to carbapenems has also been observed [31] that may be associated with the combination of these carbapenemases with outer membrane lack of permeability [47]. Importantly, regardless of the carbapenem susceptibility profile detected in vitro, carbapenem therapy is not reliable against OXA-48-like-producing bacteria [45]. In companion animals, the bla_OXA-48 gene has been commonly observed on pOXA-48a plasmid, a self-conjugative IncL/M plasmid ^[28][29][30][28,29,30]. This plasmid has a high conjugation rate, therefore, it can be transferred at a very high frequency across Gram-negative bacteria ^[41][48][41,48]. Flanking the bla_OXA-48 gene is the Tn1999 composite transposon, which cooperates in mobilizing pOXA-48a or closely related plasmids ^[44][48][44,48]. The bla_OXA-181 gene was found to be part of the transposon Tn2013, inserted at the downstream region of ISEcp1, which is a very efficient genetic vehicle for spreading ESBL genes, namely, the bla_CTX-M-15 gene [49]. The bla_OXA-181 gene has been frequently identified in IncX3 plasmids ^[26][27][26,27]. The frequency of OXA-48-like-producing bacteria in companion animals (Table 1) and its frequent association with mobile genetic determinants that facilitate its dissemination, highlight the importance of monitoring this resistance mechanism in companion animals. Furthermore, the possible misdiagnosis of OXA-48-like-producing bacteria when using meropenem and imipenem as surrogates may lead to underestimating its frequency and the epidemiological role of companion animals as reservoirs. The bla_OXA-23 gene has been reported coming from A. baumannii isolates (Table 1). This gene is often located on transposon Tn2006, but has also been identified in transposon Tn2008 in animals isolates ^[23][34][23,34]. The bla_OXA-23 is usually flanked between ISAba1 insertion sequences, known to promote the expression of bla_OXA-23 and bla_OXA-51-like genes in A. baumannii for an elevated level sufficient to display carbapenem resistance ^[23][50][23,50]. In addition to carbapenems, the OXA-23 enzymes can hydrolyse cephalosporins, aminopenicillins, piperacillin, oxacillin, and aztreonam (Table 2) [4].