Introduction
Poyang Lake, located in northern Jiangxi Province, is the largest freshwater lake in China and the largest wetland reserve in Asia. Due to its rich aquaculture resources, it has become an important aquaculture area in Jiangxi Province and even in China (Wang et al., 2020). The aquaculture in this area mainly produces grass carp (Ctenopharyngodon idella), silver carp (Hypophthalmichthys molitrix), bighead carp (Aristichthys nobilis) and Pengze crucian carp (Carassius auratus var. Pengze). The main cultivation method applied in the Poyang Lake area is intensive pond culture. However, the growth of the aquaculture industry in this region has resulted in some health problems. Common pathogens include bacteria, viruses and parasites. Among the various pathogens, bacteria are the main pathogens and are closely related to human health. Some aquatic bacteria can cause zoonoses that directly endanger human health, and more importantly, some can harm human health through the horizontal transfer of virulence genes and antimicrobial resistance (AMR) genes (Carnelli et al., 2017; Rahman et al., 2020).
The major bacterial infections of freshwater fish generally involve one of several species of the genus Aeromonas, such as A. veronii and A. hydrophila (Fernández-Bravo and Figureueras, 2020). A. veronii is the primary pathogen isolated from freshwater fish in South China, including Jiangxi Province (Ran et al., 2018; Xu et al., 2018, 2019, 2021; Gao et al., 2020; Rao et al., 2020; Wang et al., 2021). The cultured fishes in the Poyang Lake area are also affected by A. veronii, which is the most frequently detected bacterium in freshwater (Ran et al., 2018; Pessoa et al., 2020). A. veronii is widely distributed and has been isolated from diseased aquatic animals and aquatic systems in multiple countries. It has a variety of hosts and can live in humans, aquatic animals, human food and the environment, posing risk to humans (Fernández-Bravo and Figureueras, 2020). Thus, it is necessary to carry out genetic and epidemiological studies of A. veronii to prevent diseases caused by this pathogen.
A. veronii is recognized as an opportunistic pathogen (Fernández-Bravo and Figureueras, 2020). Identifying the epidemiological relationships of A. veronii strains is important for understanding the behaviour of this microorganism in human infections (Martínez-Murcia et al., 2000). A number of typing methods are applied to Aeromonas strains for epidemiological purposes (Łaganowska and Kaznowski, 2004; Naviner et al., 2011; Fernández-Bravo and Figureueras, 2020). Compared with other typing methods, enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC-PCR), a DNA-based typing method is more rapid and economical, is technically simpler and has a higher discriminatory power for determining the distribution of a single strain among individuals (Xu et al., 2017).
The virulence of Aeromonas results in potential damage (Gonçalves Pessoa et al., 2019), and it is impossible to establish a hierarchical classification of virulence factors according to their role in the disease process (Aguilera-Arreola et al., 2007). Therefore, it is necessary to continuously survey the presence of several accepted virulence factors in A. veronii isolates, this monitoring is also important for understanding both A. veronii pathogenesis and the epidemiology of A. veronii infections (Aguilera-Arreola et al., 2007).
Antimicrobials are occasionally used in feed or directly in water in aquaculture systems for disease prevention and treatment in China (Liu et al., 2017). Antibiotics boost the risk of emerging AMR in aquatic bacteria, which is commonly caused by AMR genes. Antimicrobial-resistant bacteria and AMR genes result in biological contamination of the environment (Carnelli et al., 2017). In aquatic environments, antibiotic resistance genes may spread horizontally and be transferred to bacteria colonizing humans and other animals through food and drinking water (San, 2018; Sabbagh et al., 2021).
To our knowledge, there have been no reports characterizing the genetic diversity, virulence and AMR of A. veronii in the Poyang Lake area. In this study, the genetic diversity and virulence and AMR genes prevalence in A. veronii strains isolated from aquaculture systems in the Poyang Lake area were examined. The results of this study are of great significance for improving the food safety assessment of freshwater aquatic products and understanding its relevance to animal and public health.
Discussion
ERIC-PCR is one of the most popular methods for genotyping Aeromonas because it is easy to carry out, does not require any expensive equipment and is highly reproducible (Fernández-Bravo and Figureueras, 2020). Genetic analysis of A. veronii performed via ERIC-PCR has revealed the epidemiological relationships of various strains and the genetic diversity of this species (Ye et al., 2012). In this study, the population of A. veronii was genetically heterogeneous, with the 46 strains producing 36 ERIC-PCR types and lacking dominant clones. Some strains from the same source produced different ERIC types: for example, FZQPS, FZ2N, FZQP, and FZSY were isolated from the same aquatic farm but had different ERIC types. A similar result was observed for strains DC2-2-1 and DC2S-2. In contrast, some strains from different sources sampled in different years exhibited similar genotypes, such as the type vii strains DC2-2-1 and LJLC and the type xix strains LTFS4 and BL16103. The high diversity of A. veronii poses a problem for disease control. Different types of bacteria have different properties and environmental adaptabilities and correspondingly can infect a wide range of hosts, which is in line with the prevalence of A. veronii infection in a variety of aquatic animals in southern China in recent years (Ran et al., 2018; Gao et al., 2020; Wang et al., 2021).
We examined the numbers and types of virulence genes that are essential for Aeromonas infection. Aravena-Román et al. (2014) expounded, in general, that pathogens should possess the virulence genes necessary to gain entry, adhere, and colonize host tissue, causing damage, while evading host defence mechanisms and, in some cases, spreading, leading to systemic infection. In this study, virulence genes related to entry (gcaT, exu, ser, lip, ascV, TraJ), adherence (fla), colonization (ahy, act, alt, ast, hly, aer) and other processes were selected for survey.
The genes exu, fla and aer were the most frequently observed genes, with frequencies of 93, 91 and 89%, respectively. These results are consistent with those of Khor et al. (2015), who found that the frequencies of the genes exu, fla and aer were 95, 86 and 93%, respectively, in 44 strains of A. veronii isolated from freshwater lakes in Malaysia. In addition, the results are consistent with those of Nawaz et al. (2010), who found that among 81 strains of A. veronii isolated from farm-raised catfish, 80 and 96% contained the genes fla and aer, respectively. The gene aerA codes for aerolysin, and had been identified as the key virulence factor of A. veronii in southern China (Cirauqui Diaz et al., 2017; Ran et al., 2018). The high percentages of isolates positive for these genes highlight the relevance of these genes for the maintenance of Aeromonas spp. in the host.
Haemolysin, which is encoded by haemolysin genes (such as hlyA), associated with aerolysin (aerA) and participates in virulence by inducing incomplete erythrocyte lysis (a-haemolysins) or the complete destruction of erythrocytes (β-haemolysins; Tomás, 2012). In this study, the frequency of hlyA was 65%, consistent with the frequency of 65% reported by Dahanayake et al. (2020), among A. veronii strains isolated from Tegillarca granosa in Korea.
Three enterotoxin genes (act, ast and alt) related to public health were detected in this study. Any one of these three enterotoxins can lead to severe diarrhoea. The presence of all three enterotoxin genes in an Aeromonas isolate could be devastating to patients (Chopra et al., 2009). In this study, 2 out of the 46 strains contained all three of these genes. These three enterotoxin genes are related to human diseases and cause diarrhoea, enteritis and other conditions. Among them, ast and alt are generally abundant in A. hydrophila. For example, Aravena-Román et al. (2014) reported that among A. hydrophila strains, 87 and 91% of clinical strains carried alt and ast, respectively, and 83 and 100% of environmental strains carried alt and ast, respectively. Similarly, Zhou et al. (2019) reported that 66% of A. hydrophila strains carried the gene alt and that 100% carried the gene ast. In this study, ast and alt were detected in A. veronii, which does not exclude the possibility that virulence genes may be transferred between A. hydrophila and A. veronii through mobile genetic elements (MGEs), such as plasmids, insertion sequence (IS) elements, transposons, genomic/pathogenicity islands, and integron-associated gene cassettes, between A. hydrophila and A. veronii (Piotrowska and Popowska, 2015).
The TTSS is an important virulence factor in Aeromonas strains (Reyes-Rodríguez et al., 2019). In this study, the frequency of the gene ascV, which encodes a component of the TTSS, was found to be 50%, which is slightly lower than the 66% reported by Reyes-Rodríguez et al. (2019) and higher than the 31% reported by Aravena-Román et al. (2014). The frequency of ascV was higher in clinical strains (53%) than that in environmental strains (45%) in this study, which is consistent with the findings of Vilches et al. (2004) and Reyes-Rodríguez et al. (2019). However, Aravena-Román et al. (2014) observed the opposite pattern, reporting that 50% (4 of 8) of environmental strains carried ascV, while only 23% (6 to 26) of clinical strains had this gene. Based on these results, it appears that the TTSS might not be a key virulence factor of A. veronii and that the distribution patterns of the TTSS genes are strain- and source-dependent (Aravena-Román et al., 2014).
TFSSs are deployed by many bacterial species to deliver DNA, protein, or other macromolecules to bacterial or eukaryotic cell targets or even to the environment (Wallden et al., 2010; Li et al., 2019; Costa et al., 2021). TFSSs have not been extensively studied in A. veronii (Matys et al., 2020). Rangrez et al. (2010) reported a case of a TFSS encoded by a plasmid. In this study, only 2 clinical strains were found to carry the investigated TFSS gene. However, because TFSSs can mediate horizontal gene transfer (Wallden et al., 2010), which greatly facilitates adaptation to environmental changes and is the basis for the spread of virulence and even drug resistance among bacteria, the distribution of TFSSs in A. veronii warrants continued attention.
The frequencies of lip, ahyB, ser and gcaT, genes that encode extracellular enzymes, were 72, 76, 63 and 17%, respectively, in this study. These findings differ to some extent from those of other reports. For example, Dahanayake et al. (2019) reported that the frequencies of lip, ahyB, and gcaT were all 20%, and that the frequency of ser was 0. Nawaz et al. (2010) reported frequencies of ser, gcaT and lip of 82, 78 and 85%, respectively. Zhou et al. (2019) found 8.3% positivity for the gene lip among 36 A. veronii strains.
The A. veronii strains isolated from the Poyang Lake area contained multiple virulence factors, and the main types were alt + act + aer + fla + exu + lip (9/46) and act + aer + fla + exu + lip + ahyB + hly (24/46). These results are similar to those of Khor et al. (2015) and Roges et al. (2020), which indicates that the virulence of A. veronii is a synergistic effect of multiple virulence factors. Notably, 78% (36/46) of the strains contained both act and aer. According to Nawaz et al. (2010), bacteria containing the genes act and aer are virulent strains, which indicates that the virulent strains of A. veronii are dominant in the Poyang Lake area.
The results of AMR gene detection by PCR amplification showed that AMR genes were widely distributed among A. veronii strains isolated from the Poyang Lake area. The percentage of strains containing two or more AMR genes was 98% (45/46), and 76% of the strains contained five or more AMR genes, which to some extent reflected the law of “survival of the fittest” for bacteria (Munita and Arias, 2016). That is, widespread antibiotic contamination in the natural environment has placed selective pressure on A. veronii such that populations with few or no AMR genes have been unable to persist in the natural environment. Only individuals with multidrug resistance genes have been able to survive in the presence of the antibiotic components in the environment that are derived from human activities. The results also showed that there has been antibiotic abuse in the aquaculture system in the Poyang Lake area: the majority of individuals in the group carried five or more drug resistance genes, indicating the existence of selection for antibiotic resistance.
In this study, an abundance of PMQR genes was observed among the tested A. veronii strains. Among these genes, qnrB (85%) was the most prevalent among the isolates, followed by qnrA (83%), and their abundances were higher than those in previous reports (Verner-Jeffreys et al., 2009; Hossain et al., 2018). The lowest abundance of the qnr genes was 57% in this study. There may be geographical differences associated with the abundance of these genes. Quinone drugs, such as enrofloxacin, have been authorized for use in Chinese aquaculture, and are used in large quantities in China (Liu et al., 2017). The increased use of quinolones has led to a rise in bacterial resistance (Kolár et al., 2001).
The tetracycline resistance genes tetE and tetA were detected in 24 and 11% of the isolates, respectively, whereas tetB was not detected in any isolate. The genes tetA and tetE are the most common tetracycline resistance determinants of Aeromonas spp. (Hossain et al., 2018). In this study, two tet genes were detected in the A. veronii strains isolated from the Poyang Lake area, occasionally in the same strain (strains JX3-2-2 and XG1-2-1). This result indicated that resistance to tetracycline was high among strains in the Poyang Lake area, which was also suggested by the drug susceptibility test results of this study. For example, a total of 22% of the strains analysed in this study were resistant to tetracycline. The reason may be related to the fact that tetracycline has been approved for and is frequently used in veterinary medicine in China. A large number of tetracycline drugs have been used for the prevention and treatment of aquatic diseases, resulting in A. veronii developing resistance to tetracycline. Some strains in which tet genes were not detected also presented resistance phenotypes, indicating that there might be other genes conferring resistance to tetracycline that were not detected, possibilities include the tet (34) gene, which is widely distributed in A. veronii (Tekedar et al., 2020).
In this study, two aminoglycoside resistance genes, strA-strB and aac(6β)-Ib, were detected in 39 and 9%, respectively, of A. veronii strains. This result is consistent with a previous report in which 5% of A. veronii strains contained the gene aac(6β)-Ib (Deng et al., 2014). In contrast, Dahanayake et al. (2019) and Hossain et al. (2018) reported that the frequency of aac(6β)-Ib in A. veronii reached 67 and 56%, respectively. These differences among studies show that there are regional differences in the distribution of aminoglycoside resistance genes, as drug use policies vary among countries, differences in selective pressure exist, leading to differences in the distribution of resistance genes. In China, only one aminoglycoside drug, neomycin sulfate, has been approved for use in aquaculture, which might explain the lower resistance to aminoglycosides than to other antibiotic drugs among the genotypes and phenotypes of A. veronii in the Poyang Lake area (Committee of Chinese Veterinary Pharmacopoeia, 2021). Among the surveyed ESBL genes, only blaSHV was observed, detected in 7% of the isolates. BlaTEM, BlaCTX-M and BlaOXA were not detected in this study. These results slightly differ from those of previous studies. For example, Dahanayake et al. (2019) detected the blaSHV and blaCTX in 33 and 20%, respectively, of A. veronii strains isolated from marketed cockles in Korea. Fauzi et al. (2021) reported that 47% of A. veronii strains isolated from fish carried the gene BlaTEM, and that 11% of strains carried BlaSHV. These differing results show that there are also geographic differences in the distribution of ESBL genes among A. veronii strains.
Integrons, transposons and mobile plasmids are the most frequent genetic elements involved in the horizontal spread of resistance genes and have facilitated the global antibiotic resistance crisis. They are a potential force in the adaptation and rapid evolution of bacteria (Alvarado et al., 2012; Gillings, 2014; Khademi et al., 2021). Some researchers have analysed the genomes of A. veronii strains and, shown that strains containing integrons, transposons and plasmids have more putative AMR genes than other A. veronii strains (Tekedar et al., 2019). In this study, 20% of the A. veronii strains isolated from the Poyang Lake area carried class 1 integrons, this finding is consistent with previous reports, showing that 21% ~ 31% of Aeromonas spp. strains isolated from aquatic environments were positive for class 1 integrons (Carnelli et al., 2017; Dhanapala et al., 2021). It seems that the frequency of class 1 integrons in Aeromonas spp., including A. veronii strains isolated from polluted environments, is high. Therefore, class 1 integrons of A. veronii can be used as indicators of pollution originating from human activity, and the antibiotic resistance phenotypes can be comprehensively evaluated by detecting the distribution frequency of class 1 integrons in A. veronii strains. In addition, slass 2 integrons that confer antibiotic resistance in clinical contexts have been described. The frequency of class 2 integrons in environmental microorganisms is low. This study confirms the existence of class 2 integrons in A. veronii. Similar to class 1 integrons, class 2 integrons evolve under selective pressure imposed by human activity and continue to accumulate new gene cassettes relevant to resistance, pathogenicity and virulence (Gillings, 2014). Thus, the monitoring of class 2 integrons should be increased to reveal the mechanisms of bacterial resistance, pathogenicity and virulence.
Plasmids can mediate horizontal gene transfer of antibiotic resistance, virulence genes, and other adaptive factors across bacterial populations (Lopatkin et al., 2017; Redondo-Salvo et al., 2020). Aeromonas spp. carry a variety of plasmids. Some plasmids showed template or host genes acquired from Pseudomonas monteilii, Salmonella enterica, Escherichia coli or other species (Nwaiwu and Aduba, 2020). In this study, plasmid gene fragments of Escherichia, Salmonella and Yersinia were detected in A. veronii, indicating that plasmids of these bacteria could be transferred to A. veronii. Among the plasmids detected in A. veronii, the Escherichia coli resistance plasmid R64 was the most common, with a transfer frequency reaching 100%. Previous studies have reported that both Escherichia and Aeromonas are abundant in water, with similar ecological niches (Araujo et al., 1989; Carnelli et al., 2017; Solaiman and Micallef, 2021). By employing metagenomics technology, researchers have revealed the existence of “habitat-specific gene pools,” suggesting that transmission is more frequent among species occupying the same ecological niche (Redondo-Salvo et al., 2020). The results of this study are consistent with these findings. Of course, the presence of AMR genes in plasmids may not necessarily translate to strong phenotypic expression (Nwaiwu and Aduba, 2020). However, plasmids are a transferable AMR gene pool, and the threat they pose to public health should be noted. Continuous monitoring can help us better understand the impact of plasmids on the evolution and persistence of bacterial species (Redondo-Salvo et al., 2020).
Transposons are important for bacterial evolution and adaptation because they play a central role in disseminating resistance to antibiotics in gram-negative bacteria. They are often associated with multidrug resistance plasmids in Aeromonas (Del Castillo et al., 2013). In this study, transposons were found in 35% of the A. veronii strains; this frequency is higher than the 14% reported in Aeromonas spp. isolated from aquatic environments of Switzerland (Carnelli et al., 2017). A possible reason for this differences is that the strains studied in this paper originated from aquaculture systems, while the strains in the study of Carnelli et al. (2017) originated from four water bodies, including unpolluted waters, this differences suggests a positive correlation between the frequency of transposons in A. veronii and the human use of antibiotics. Previous reports have shown that transposons are important for bacterial adaptation and evolution in marine ecosystems (Dahlberg and Hermansson, 1995). This study indicated that transposons may also play important roles in evolution and adaptability in freshwater ecosystems.
There are four varieties of natural antibiotics and two classes of synthetic antibiotics approved by the Ministry of Agriculture and Rural Affairs of the People’s Republic of China for production and use in aquaculture: neomycin, doxycycline, florfenicol, thiamphenicol, sulfonamides, and quinolones (Committee of Chinese Veterinary Pharmacopoeia, 2021). In this study, these approved antibiotics and other common antibiotics were selected for drug sensitivity testing, and the results showed that all A. veronii isolates were highly ampicillin resistant. This finding is likely due to high intrinsic β-lactam resistance (Tayler et al., 2010). Furthermore, high resistance to quinolones, tetracyclines and aminoglycosides was detected, which might be due to the extensive consumption of such antimicrobials in aquaculture systems in the Poyang Lake area. Additionally, although the phenotypic resistance of most strains was consistent with the AMR genotype in our study, some strains with drug resistance genes, such as strains BL16106 and CCG1, did not show phenotypic resistance. This result is consistent with previous studies identifying discrepancies between phenotypic and genotypic traits; these studies indicate that these discrepancies might be the result of resistance gene silencing or mutation (Carnelli et al., 2017; Dahanayake et al., 2019; Baunoch et al., 2021). These observations indicate that one cannot predict the bacterial antibiotic response only by detecting AMR genes in A. veronii.