Characterization of Extended-Spectrum β-Lactamase-Producing and AmpC β-Lactamase-Producing Enterobacterales Isolated from Companion Animals in Korea

The emergence of extended-spectrum cephalosporin (ESC)-resistant Gram-negative bacteria is of great concern in both human and veterinary medicine. The aim of this study was to investigate ESC-resistant bacterial isolates from companion animals in South Korea between 2017 and 2019. Isolates with ESC resistance genes, which were identified by PCR, were assessed for genetic relatedness by multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). In total, 91 ESC-resistant Escherichia coli, Klebsiella spp., Serratia spp., and Enterobacter cloacae isolates harbored the blaTEM gene. Among other ESC resistance genes, blaCTX-M-15, blaCIT, and blaCTX-M-55 were predominantly detected in E. coli isolates, whereas blaSHV and blaDHA were more frequently detected in Klebsiella pneumoniae isolates. In addition, all blaEBC-positive isolates were classified as E. cloacae. From the MLST results, blaCTX-M-9-carrying ST131, blaCIT-carrying ST405, and blaCTX-M-1-carrying ST3285 strains were dominant among E. coli isolates. ST273 and ST275 strains harboring blaSHV were frequently detected in K. pneumoniae isolates. Various sequence types were obtained in E. cloacae and Klebsiella oxytoca isolates. All isolates demonstrated unique PFGE profiles (<57–98% similarity) and were unlikely to be derived from a single clone. The present study reveals the presence and wide genetic distribution of ESC-resistant bacterial species in South Korean companion animals.


Introduction
The emergence and prevalence of β-lactam resistance in Gram-negative bacteria has increased consistently over the past few decades [1,2]. Resistance to β-lactams is mostly caused by bacterially produced β-lactamases that hydrolyze and inactivate extendedspectrum cephalosporins (ESCs), such as third and fourth generation cephalosporins [1]. ESC resistance is mainly caused by the expression of extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase (AmpC) genes that are normally encoded on mobile genetic elements, mostly plasmids [1].
The first ESBLs have evolved from the native β-lactamases TEM and SHV via genetic mutations [3]. CTX-M β-lactamases, a new group of plasmid-mediated ESBLs, were first reported in Japan in 1986 [4]. However, since 2000, CTX-M β-lactamases have increasingly been reported in both human and animal populations and are now the dominant type of ESBL, replacing classical TEM-and SHV-type ESBLs in most areas of the world [5]. Currently, there are >120 different CTX-M β-lactamases that are clustered into five groups (CTX-M-1, 2, 8, 9, and 25) [6]. Among CTX-M-type enzymes, the presence of CTX-M-15 and CTX-M-14 has increasingly been reported in most areas of the world, including South Korea [5,7].
Since the detection of CMY-1, the first reported AmpC type β-lactamases, in 1989 [6], various types of AmpCs have been identified in clinical isolates of Enterobacterales around the world [8]. Among AmpCs, CIT-and DHA-type enzymes are the most prevalent [2]. Especially, DHA-producing Klebsiella spp. isolates and CIT-producing Escherichia coli isolates have been repeatedly reported for Enterobacterales in South Korea [2,9]. Despite many studies on ESBL-and AmpC-producing bacteria from human or livestock isolates, studies concerning antimicrobial resistance (AMR) bacteria associated with companion animals are lacking, especially with respect to Serratia spp. and Enterobacter spp. [10][11][12].
The popularity of companion animals in South Korea has been growing, which provides a potential reservoir of AMR bacteria, as pets are closely associated with humans, living in their homes and near their food [9]. Thus, the importance of profiling AMR bacteria was emphasized in the "One Health" initiative, which integrates veterinary medicine, human health, animal-production systems, and the environment [13]. Systematic control and prevention, through implementation of a national AMR surveillance program, is greatly needed and should be applied in both human and veterinary clinical medicine. The goal of the current study was to investigate AMR among bacterial isolates belonging to Enterobacterales in companion animals within the province of South Korea, with an emphasis on ESC resistance genes in E. coli, Klebsiella spp., Serratia spp., and Enterobacter cloacae.
Hospital-admitted cats

Genetic Relatedness
In E. coli PFGE analysis, the e1 group consisted of three ST3285 strains containing bla CTX-M-55 , bla TEM , and bla CIT from hospital-admitted and stray dogs and showed high similarity (>90%) (Figure 1a). Seven ST131 strains of E. coli isolates from five hospitaladmitted dogs and two hospital-admitted cats were identified. PFGE results involved only five hospital-admitted dogs and one hospital-admitted cat, because the banding pattern for one hospital-admitted cat isolate was not defined. Two of them, which belonged to the e18 group, showed more than 93% similarity, compared with the remaining isolates in which low similarity was observed (<85%) (Figure 1a). e3 and e25 groups contained two ST448 strains with bla CTX-M-55 and bla TEM and two ST457 strains with bla TEM and bla CIT from the same shelter and showed high similarity (>87%) (Figure 1a).
All bla TEM -positive K. pneumoniae isolates from hospital-admitted dogs harbored the bla SHV gene and exhibited 60-95% similarity (Figure 1b). One K. pneumoniae ST273 strain from stray cats belonged to the kp8 group and showed 87% similarity with hospitaladmitted dog isolates (Figure 1b). Two ST275 strains co-carrying bla SHV and bla TEM , which belonged to the kp5 group, showed high similarity (>90%) (Figure 1b). The same pulsotype belonged to both ST273 and ST275, which were single-locus variants at the tonB allele; for example, kp1 and kp8. The remaining strains were independent of the groups obtained. Various STs and PFGE profiles were obtained for K. oxytoca and E. cloacae isolates, and genetic relatedness was not revealed for those strains (Figure 1c,d). isolates demonstrated unique PFGE profiles (57-95% similarity), indicating genetic heterogeneity in ESBL-or AmpC-producing strains (Figure 1).

Genetic Relatedness
In E. coli PFGE analysis, the e1 group consisted of three ST3285 strains containing blaCTX-M-55, blaTEM, and blaCIT from hospital-admitted and stray dogs and showed high similarity (>90%) (Figure 1a). Seven ST131 strains of E. coli isolates from five hospital-admitted dogs and two hospital-admitted cats were identified. PFGE results involved only five hospital-admitted dogs and one hospital-admitted cat, because the banding pattern for one hospital-admitted cat isolate was not defined. Two of them, which belonged to the e18 group, showed more than 93% similarity, compared with the remaining isolates in which low similarity was observed (<85%) (Figure 1a). e3 and e25 groups contained two ST448 strains with blaCTX-M-55 and blaTEM and two ST457 strains with blaTEM and blaCIT from the same shelter and showed high similarity (>87%) (Figure 1a).

Discussion
This study presents the characteristics of 91 Enterobacterales isolates harboring ESC resistance genes, including E. coli, Klebsiella spp., Serratia spp., and E. cloacae, collected from South Korean companion animals between 2017 and 2019. All isolates harbored the bla TEM gene and demonstrated unique PFGE profiles. Similarly, a study by Shin et al. revealed that all E. coli isolates from beef cattle harbored the bla TEM gene [12]. Recent reports have identified CTX-M-type β-lactamases as the most widespread ESBL type, replacing classical TEM and SHV-type ESBLs [14]; however, TEM-type β-lactamases remained the most prevalent ESBL type identified in the current study. We observed varying predominant βlactamase gene types in different Enterobacterales species, summarized as follows: bla CTX-M and bla CIT in E. coli isolates, bla SHV and bla DHA in K. pneumoniae isolates, and bla EBC in E. cloacae. β-Lactamase gene distribution for each Enterobacterales species was similar to that described in a previous study with human samples [2]. These findings reveal that ESC resistance gene variants are not limited to certain hosts, emphasizing the need for coordinated control in both humans and animals. In E. coli isolates in this study, bla TEM and bla CTX-M were most frequently detected, followed by bla CIT . Among bla CTX-M positive isolates, bla CTX-M-15 was the most commonly detected gene followed by bla CTX-M-55 in both dogs and cats. A previous study investigating E. coli isolates from dogs reported that bla CTX-M-15 , bla CTX-M-14 , and bla CIT were the most prevalent β-lactamase genes, whereas bla CTX-M-55 was rarely detected in South Korea [15]. However, bla CTX-M-55 -carrying E. coli has become increasingly prevalent in dogs in South Korea [9]. The present study revealed that bla CTX-M-55 was predominantly detected rather than bla CTX-M-14 in E. coli from companion animals, which concurred with the results of the study by Hong et al. [9]. All five E. coli ST405 strains investigated in the current study harbored both bla TEM and bla CIT and were collected from stray dogs in the same shelter. The spread of bla CIT -carrying E. coli ST405 was described in a previous study, which suggested the possibility of direct transmission between humans and companion animals [9]. The spread of E. coli ST405 is usually described in humans harboring bla CTX-M-15 [16]. However, E. coli ST405 did not harbor bla CTX-M-15 in the current study. The increasing prevalence of E. coli ST131 carrying bla CTX-M-15 has been described in humans and animals [15,17]. Unexpectedly, only one bla CTX-M-15 -carrying E. coli ST131 strain was detected from hospitaladmitted cats in this study. From E. coli PFGE results, the two ST3285 strains showing the same PFGE pattern were both from the same shelter and isolated on the same date ( Figure 1a). In this case, it could be the result of contaminated samples during sampling or transmission of a same clone between the two stray dogs in a shared place.
Among the seven K. pneumoniae and four K. oxytoca isolates harboring bla CTX-M from hospital-admitted dogs in this study, the CTX-M-15 genotype accounted for a large proportion. A recent study also reported that K. pneumoniae isolates from companion animals producing CTX-M-15 either alone or in combination with DHA were frequently detected in South Korea [9]. The present MLST results revealed that ST275 and ST273 strains carrying both bla SHV and bla TEM were most commonly identified among K. pneumoniae isolates from hospital-admitted dogs. ST275 and ST273 are differentiated by one allele of the seven housekeeping genes, indicating that they are genetically close sequence types. Recently, the bla SHV -and bla TEM -co-carrying K. pneumoniae ST273 strain has emerged in human patients in Italy and is being disseminated, whereas ST273 and ST275 Klebsiella spp. isolates carrying both bla SHV and bla TEM have not yet been reported in South Korea [18,19]. Moreover, ST11, ST15, ST307, and ST392 strains have been globally identified as β-lactamase-producing Klebsiella spp. [9,20,21]. β-Lactamase-producing ST275 or ST273 strains among Klebsiella spp. isolates were newly discovered in South Korean companion animals in the current study.
All E. cloacae isolates in this study harbored bla TEM and some carried bla EBC , whereas none carried bla CTX-M and bla SHV . E. cloacae isolates from companion animals combined with more than two ESBL-type genes were described in Germany [22]. Meanwhile, E. cloacae isolates investigated in the present study harbored only one type of ESBL gene. In Serratia spp. isolates, only bla TEM was detected, except one S. marcescens isolate that harbored both bla TEM and bla SHV . An S. marcescens isolate carrying bla TEM was previously identified in South Korea that caused urinary infections in humans [23]. However, in many countries including South Korea, the status of emerging AMR among Serratia spp. and E. cloacae in companion animals remains unknown. To our knowledge, this is the first report of ESC-resistant Serratia spp. and E. cloacae isolates from companion animals in South Korea.
In conclusion, we illustrated the presence and genetic heterogeneity of ESC-resistant Gram-negative bacteria in companion animals in South Korea, providing a potential reservoir of ESC-resistant bacteria and a transmission pathway. More organized surveillance is required to prevent and control the spread of ESC-resistant bacteria between companion animals and humans, in accordance with the "One Health" initiative.

Characterization of β-Lactamase Genes
PCR amplification of entire bla CTX-M , bla TEM , and bla SHV genes was performed as previously described [24,25]. For bla CTX-M -positive isolates, PCR and DNA sequencing were carried out for CTX-M-subtype detection. bla CTX-M group-specific primers for five clusters (CTX-M-1, 2, 8, 9, and 25) were used following Kor-GLASS (Korea Global Antimicrobial Resistance Surveillance System) guidelines and previously published protocols [26,27]. DNA sequencing was performed by Intron Biotechnology (Seongnam, South Korea) and homologous sequences were searched against the GenBank database using the BLAST tool of the National Center for Biotechnology Information website (http://www.ncbi.nlm. nih.gov/BLAST (accessed on 15 December 2020)). In ESBL-positive strains, six groups of AmpC β-lactamases (MOX, CIT, DHA, ACC, EBC, and FOX) were screened by PCR amplification [28].

Pulsed-Field Gel Electrophoresis
PFGE of XbaI (Takara Bio Inc., Shiga, Japan)-digested genomic DNA was carried out for E. coli, K. pneumoniae, K. oxytoca, and E. cloacae isolates according to the CDC PulseNet standardized procedure using the Chef Mapper system (Bio-Rad Laboratories, Hercules, CA, USA) [32]. PFGE analysis for Serratia spp. was ignored because different digested genomic DNA samples were employed for Serratia spp. Similarities between restriction frag-ment length polymorphisms were analyzed using GelCompar II software v. 6.5 (Applied Maths NV, St-Martens-Latem, Belgium) to produce a dendrogram. The unweighted-pair group method using average linkages (UPGMA) cluster analysis was conducted based on an 85% similarity cut-off with 0.5% optimization and 2.0% band tolerance.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.