Extended-Spectrum β-Lactamases (ESBL) Producing Bacteria in Animals

Animals have been identified as potential reservoirs and vectors of resistance genes, with studies showing that Gram-negative bacteria can acquire resistance through the horizontal transmission of resistance genes on plasmids. It is important to understand the distribution of antimicrobial-resistant bacteria and their drug-resistant genes in animals. Previous review articles mostly focused on a single bacterium or a single animal. Our objective is to compile all ESBL-producing bacteria isolated from various animals in recent years and provide a comprehensive viewpoint. Using a thorough PubMed literature search spanning from 1 January 2020 to 30 June 2022, studies exploring extended-spectrum beta-lactamase (ESBL) producing bacteria in animals were included. ESBL-producing bacteria are present in animals from various countries around the world. The most common sources of these bacteria were farm animals, and the most frequently isolated bacteria were Escherichia coli and Klebsiella pneumoniae. The most detected ESBL genes were blaTEM, blaSHV, and blaCTX-M. The presence of ESBL-producing bacteria in animals highlights the importance of the One Health approach to address the issue of antibiotic resistance. Further research is needed to better understand the epidemiology and mechanisms of the spread of ESBL-producing bacteria in animal populations and their potential impact on human and animal health.


Introduction
Antibiotics are important weapons for humans in fighting microbial infections and reducing overall mortality from infectious diseases. However, the increasing prevalence of antimicrobial-resistant bacteria (AMRs) in recent decades is a great challenge [1]. Studies suggest that animals are potential reservoirs and vectors of resistance genes [2]. Gramnegative bacteria (GNB), especially Enterobacterales strains, can acquire resistance through the plasmid-mediated horizontal transmission of resistance genes [3]. Increased use of antibiotics in livestock has been identified as a potential contributor to antimicrobial resistance in humans [4]. Therefore, it is also important to understand the distribution of antimicrobial-resistant bacteria and their drug-resistant genes in animals.
Bacteria that produce extended-spectrum β-lactamases (ESBLs) are considered one of the critical priority pathogens by the World Health Organization (WHO). ESBLs are a type of β-lactamase that can hydrolyze penicillins, first, second, and third-generation cephalosporins, and aztreonam but are unable to break down cephamycins or carbapenems [5]. The ESBLencoding genes can be grouped into several families: bla TEM , bla SHV , bla CTX-M , etc. In the past, TEM and SHV-type ESBLs were the mainstream of ESBLs. However, CTX-M-type enzymes are much more commonly found in the ESBL type in recent research [5].
The widespread use of antibiotics is a contributing factor in the rise of antimicrobial resistance, particularly in the case of ESBL-producing bacteria. Previous studies have shown that the use of antibiotics within the past three months and monotherapy with specific drug classes (cephalosporins, tetracycline, macrolide, and cotrimoxazole) are associated with the prevalence of these bacteria [6,7]. The plasmids responsible for ESBL production often carry genes encoding resistance to other drug classes, such as aminoglycosides, trimethoprim, and fluoroquinolones [8]. This makes the treatment of infections caused by ESBL-producing bacteria more challenging, as the presence of these plasmids exacerbates the problem of antibiotic resistance and limits therapeutic options. In short, the overuse of antibiotics creates a favorable environment for the spread of plasmids responsible for ESBL production, which in turn contributes to the rise of antibiotic resistance.
Animals have been identified as potential reservoirs and vectors of resistance genes. The widespread use of antibiotics in animals has been linked to the increasing prevalence of antimicrobial-resistant bacteria in humans, highlighting the importance of understanding the distribution of antimicrobial-resistant bacteria and their drug-resistant genes in both humans and animals.
There are many articles on the analysis of ESBL-producing bacteria and their drugresistance genes in particular animals. While some review articles have attempted to summarize these studies, most of them have focused on a specific type of bacteria (such as Escherichia coli or Klebsiella pneumoniae) or only reviewed one type of animal, lacking a comprehensive review. Our objective is to compile all ESBL-producing bacteria isolated from various animals in recent years, providing a comprehensive understanding of the distribution of ESBL-producing bacteria and genes in animals worldwide. This review attempts to underscore the role of animals in the rising incidence of ESBL-producing bacteria and the need for a coordinated effort to address this growing threat.

Results
The general findings of the reviewed articles are summarized in Table 1. Samples of ESBL-producing bacteria were mostly obtained from farms in Africa (Egypt, Kenya, Tunisia, Nigeria, and Algeria), Asia (Pakistan, India, Qatar, Iran, Malaysia, China, Saudi Arabia, Bangladesh, and Thailand), Europe (Finland, Portugal, Spain, Netherlands, Germany, France, and Switzerland), North American (USA), and South America (Brazil, Guadeloupe, and Peru). Other sampling locations included the airport, animal clinics, animal shelters, hunting grounds, petting zoos, slaughterhouses, research facilities, universities, and wild colonies. Most samples were obtained from rectal swabs and fresh feces of animals. However, other samples including raw milk, blood and visceral samples, cloacal swabs, uterine swabs, external surface and gut homogenates, urine, pus, and respiratory pathological specimens were also included. The most reported bacteria were Escherichia coli and Klebsiella pneumoniae. Other Enterobacterales were also in abundance while Pseudomonas aeruginosa was only found on the uterine swabs of farm cows, camels, and mares in one study from Saudi Arabia.
Our review included 23 articles on domestic animals, 6 articles on wild animals, and 1 article on both. Other than farm animals, pets, zoo animals, vampire bats, and cockroaches were sampled. Four studies emphasized that the specimens were sourced from diseased animals, including diseased companion animals, diseased horses, diseased cows, camels, mares, and diseased pigs. The compilation of animals screened across different countries is presented in Figure 1a. Table 2 summarizes the details of the ESBL genes. Most samples were grown using MacConkey agar. Fifteen articles included in our review utilized selective media supplemented with third-generation cephalosporins for initial ESBL screening. Most targeted bacteria were identified by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) and polymerase chain reaction (PCR). The prevalence of ESBL in the samples varied widely from 0 to 100%. Double-disc synergy test was mostly used for identifying ESBL-producing bacteria. The most detected ESBL genes were bla TEM , bla SHV , and bla CTX -M . Subtype distribution around the world can be found in Figure 1b. Primers used in the re-viewed articles are listed in Table 3. No standardized primer was used for each target gene. However, Woodford et al. (2006) was the most highly cited article for primers targeting specific groups of bla CTX-M .    (2) (17), bla SHV (1),     (17) 1 Including ampicillin (100 µg/mL), cefotaxime (1 µg/mL), meropenem (0.5 µg/mL), ciprofloxacin (1 µg/mL), or tetracycline (100 µg/mL). 2 Ciprofloxacin, cefotaxime, and ciprofloxacin + cefotaxime, at a final concentration of 8 mg/mL. 3 2 µg/mL cefotaxime, 2 µg/mL ertapenem, 9 µg/mL imipenem, and 3 µg/mL colistin, respectively. 4 Triple sugar iron agar, lysine iron agar, methyl red, Voges-Proskauer broth, the indole test, urea agar, Simmon's citrate agar, and a motility test. 5 Methyl red, Voges-Proskauer, indole, Simmon's citrate, urease, triple sugar iron agar, lysine decarboxylase, phenol red dulcitol, KCN, and malonate. 6 Triple sugar iron agar, indole production, Simmon's citrate, urease production, lysine decarboxylation, and sorbitol and cellobiose fermentation tests. 7

Discussion
The results of the literature review provide a comprehensive comparison with previous studies on ESBL-producing bacteria in animals. While most previous studies have focused on limited geographic regions and animal populations, the current literature review offers a broader perspective on the highly diverse nature of ESBL-producing bacteria. This review provides insights into the distribution and occurrence of ESBL-producing bacteria in different regions and animal populations, helping to fill gaps in our understanding of this important issue.
Although previous studies have established the spread of ESBL-producing Enterobacterales in food-producing animals and companion pets around the world [67,68], this review highlights the presence of ESBL-producing bacteria in wild vampire bats, wild ungulates, and cockroaches. These findings suggest that the spread of ESBL-producing bacteria is not limited to domesticated animals, but can also occur in wild animal populations. The presence of ESBL-producing bacteria in wild animals can have significant implications for their health, as well as for the health of other animal populations and humans that may encounter them. It is important to consider the potential sources of ESBL-producing bacteria in wild animals, including exposure to contaminated food and water sources, contact with domesticated animals and their environment, or exposure to antibiotics in the environment [69,70]. The spread of ESBL-producing bacteria in wild animal populations can also have ecological consequences, such as altering the balance of microbial communities and affecting the health of the animals and their habitat. It is crucial to continue monitoring the presence of ESBL-producing bacteria in wild animal populations and to implement strategies to reduce their spread. The One Health approach, which recognizes the interconnections between human, animal, and environmental health, is crucial in addressing the issue of ESBL-producing bacteria in wild animals. In the past, WHO had launched an integrated global surveillance on ESBL-producing E. coli. with the same approach [71]. Similar programs covering a wider range of ESBL-producing bacteria may be considered.
Samples in this review were mostly obtained from Enterobacterales-rich areas such as the rectum and fresh feces. However, ESBL-producing bacteria were also found in raw milk and blood and visceral samples of animals in this review [15,17,20]. Unlike most other samples where ESBL-producing genes were found in Enterobacterales, one study reviewed showed the presence of Pseudomonas aeruginosa in uterine swabs of farm animals [28]. The potential for animals to act as reservoirs and vectors of resistance genes is therefore not limited to Enterobacterales found in food-producing animals and pets in contact with humans and antibiotics. One study in Kenya was conducted to collect cloacal swabs from the chickens and fecal samples from the farms. Out of the 544 cloacal isolates of Enterobacterales, 30 were found to contain ESBL genes. Among these, 14 isolates had bla TEM , 5 had bla SHV , and 11 had bla CTX-M . In contrast, among the 47 human isolates, 3 were found to contain ESBL genes, including 2 with bla TEM and 1 with bla CTX-M [25].
There are many kinds of ESBL-encoding genes, including bla TEM , bla SHV , bla CTX -M , bla GES , bla VEB , bla IRT , bla CMT , bla BEL , bla TLA , and bla PER [5]. However, most studies reviewed that investigated bacteria from animals only screened for bla TEM , bla SHV , and bla CTX-M . Genes including bla GES , bla VEB , bla IRT , bla CMT , bla BEL , bla TLA , and bla PER were rarely described and did not occur as frequently as the former three. This is possibly due to the genes being encoded on the chromosomes and not plasmids [8,72]. Furthermore, researchers used various primers for the detection and sequencing of the target genes. The results of this review draw attention to the need for standardized and comprehensive surveillance of ESBL-producing bacteria in animal populations. The limited screening for only a few types of ESBL-encoding genes, such as bla TEM , bla SHV , and bla CTX-M , may not fully capture the diversity and distribution of ESBL-producing bacteria in animal populations. Standardized surveillance covering a wider range of animals and regions will be necessary to better understand the spread of ESBL-producing bacteria and the potential impact on human and animal health. In addition, the use of different primers for the detection and sequencing of ESBL-encoding genes can lead to variability in the results and limit the comparability of studies [73]. Standardized protocols for the detection and sequencing of ESBL-encoding genes are necessary to ensure consistent and accurate results, which is the cornerstone of a better understanding of the spread of these bacteria and their potential impact on human and animal health.
In recent years, there has been a growing concern over the emergence of bla ESBLharboring plasmids in animal isolates. These plasmids are capable of transmitting bla ESBL genes among different bacterial species and even among different hosts, including animals and humans [74,75]. Studies have shown that bla ESBL -harboring plasmids can be found in various animal isolates, including those from bovine, camels, dogs, cats, goats, and poultry [10,14,[76][77][78]. These plasmids can spread rapidly across and within bacterial populations, leading to the dissemination of antibiotic-resistance genes and the emergence of multidrug-resistant bacteria [79]. Overall, the occurrence of bla ESBL -harboring plasmids in animal isolates highlights the need for effective surveillance and control measures to limit the spread of antibiotic resistance in both animal and human populations.
PCR with oligonucleotide primers that are specific for a β-lactamase gene was the most common and simplest molecular method used to identify the presence of a β-lactamase enzyme belonging to a specific family. The chosen primers were designed to anneal to regions where no point mutations were known to occur [8,72]. However, in some cases, specific primers may have had special restrictions. For example, the bla CTX-M primer Forward (5 -CGCTTTGCGATGTGCAG-3 ) and Reverse (5 -ACCGCGATATCGTTGGT-3 ) should not be used to detect CTX-M enzymes in Klebsiella oxytoca as it would result in amplifying chromosomal bla oxy genes [80]. However, the direct comparison of the sensitivity and specificity of different primers in detecting specific ESBL genes is lacking. Therefore, exploring the impact of different primers on blaESBL epidemiology may be a potential research direction.
Though the presence of ESBL-producing bacteria could be seen around the world from this review, regions such as Australia, Canada, and Russia were not covered. This may be due to the fewer numbers of literature reviewed. The prevalence of the ESBL-producing bacteria ranged from 0-100% in this review. Results varied widely among different species and regions. Further research in different regions and animal populations is needed to gain a more comprehensive understanding of the distribution of ESBL-producing bacteria in animals. To limit the spread of ESBL-producing bacteria in animals, the development of new strategies, including the use of alternative treatments, improved animal husbandry practices, and increased public awareness are also vital.
Our review is subject to significant limitations. Specifically, our use of only one database (PubMed) and a limited set of keywords, as well as our exclusion of non-English literature. These may have resulted in some relevant publications being overlooked.

Literature Search Strategy
Using a thorough PubMed literature search from 1 January 2020 to 30 June 2022, studies that investigated bacteria from animals (whether wild or domestic) with details of ESBLs were included along with current contents and references from relevant articles. We combined the medical MeSH terminology with free-text terms to conduct a systematic literature search. These were the four keyword combinations we used to search: [(Animals) AND (extended-spectrum beta-lactamase)], [(Animals) AND ESBL], ["Animals" [Mesh] AND (extended-spectrum beta-lactamase)], and ["Animals" [Mesh] AND ESBL]. The bibliographic search was carried out by two researchers. The review protocol is provided as Supplementary Materials File S1.

Inclusion and Exclusion Criteria
The inclusion criteria include: (1) cultivation of bacteria from animal specimens, whether the animal is healthy or sick; (2) conducting ESBL testing on the bacteria, whether it is phenotype or genotype; and (3) the language of publication was English. The exclusion criteria were as follows: (1) specimens from humans or the surrounding environment of animals were excluded; (2) specimens from animals, humans, and the environment, with no clear distinction between them, were also excluded; and (3) specimens that may be contaminated by environmental or human factors were excluded, such as dairy products, supermarket-packaged raw meat, poultry litter, and pooled feces. However, raw milk and fresh feces are acceptable specimens, as we believe the probability of bacteria cultured from these two types of specimens being contaminated by environmental or human factors is low.

Study Selection
Our search uncovered 2430 bibliographic references to articles published between 1 January 2021 and 30 June 2022. Thereafter, 1187 duplicate records were removed. Finally, 1243 references remained for screening. The PRISMA 2020 flow diagram [81] for literature screening can be viewed in Figure 2. After the screening of the 1243 records, 1123 records did not match the type of articles we wanted to include. Hence, 120 pieces of full-text literature were assessed for eligibility. Sixty-five of them were excluded because the samples were from the surrounding environment of animals, not from animals themselves. Fortythree of them were excluded because the samples were not only from animals but also from humans or the environment, which could not be distinguished based on the content of the article. Twelve articles were excluded because no denominator (whether the number of animals, number of samples, or number of cultured bacteria) was provided. Finally, 30 documents were selected for further review and analysis.

Data Extraction
We extracted data from all selected literature using a standardized table. The data were grouped as follows: author, date of publication, countries, sampling date and location, sample type, animal species, targeted bacteria, selective media, methods for target

Data Extraction
We extracted data from all selected literature using a standardized table. The data were grouped as follows: author, date of publication, countries, sampling date and location, sample type, animal species, targeted bacteria, selective media, methods for target identification, the number of denominators, the number of ESBL target, methods for detecting ESBL, methods for detecting ESBL genes, and the number of particular ESBL genes. The collected data were entered into standardized data extraction sheets using Microsoft Excel 2019 (Microsoft Corp, Seattle, WA, USA) for data extraction.

Conclusion
The results of this systematic literature review show that ESBL-producing bacteria are present in animals from various countries around the world. We focused on articles where samples were obtained from animals, excluding data from the environment or humans. The most common sources of these bacteria were farm animals, and the most frequently isolated bacteria were E. coli and K. pneumoniae. The prevalence of ESBL in the samples varied widely and the most commonly detected ESBL genes were bla TEM , bla SHV , and bla CTX-M .
The presence of ESBL-producing bacteria in animals highlights the importance of the One Health approach to address the issue of antibiotic resistance. Further research is needed to better understand the epidemiology and mechanisms of the spread of ESBL-producing bacteria in animal populations and their potential impact on human and animal health.

Conflicts of Interest:
The authors declare no conflict of interest.