Aeromonas spp. belongs to Aeromonadaceae, a kind of Gram-negative, rod-shaped, facultative anaerobic bacteria, which are ubiquitous in the aquatic environment, soil, fish, animals and foodstuffs (Janda & Abbott, 2010; Fernandez-Bravo & Figueras, 2020). More than 30 different Aeromonas species that threaten various environmental niches and human health have been identified (Hoel et al., 2019; Fernandez-Bravo & Figueras, 2020). Aeromonas veronii bv.sobria, Aeromonas hydrophila, and Aeromonas caviae (A. caviae) are pathogenic to humans and are responsible for a broad spectrum of human intestinal and extra-intestinal infections (Janda & Abbott, 2010). Gastroenteritis associated with Aeromonas infection is primarily caused by the consumption of contaminated water or foods. Extra-intestinal infections in humans include biliary tract infection, urinary tract infection, soft-tissue infection, peritonitis, pneumonia, and bacteremia. Previous studies have indicated that Aeromonas causes infections in both immunosuppressed and immunocompetent persons (Dwivedi et al., 2008; Chuang et al., 2011). Individuals with underlying diseases, such as liver cirrhosis, malignancies, and biliary tract diseases, are at a higher risk of infection when exposed to the pathogens (Janda & Abbott, 2010; Chao et al., 2013).

A. caviae has been proved to reside in the human gastrointestinal tract and is considered a significant etiological agent of gastrointestinal infections. Although intestinal infections caused by A. caviae may be self-limited, invasive extra-intestinal infections in immunocompromised individuals or patients with severe underlying diseases may be life-threatening or even fatal. The increasing number of extra-intestinal infections worldwide poses severe threats to public health (Luo et al., 2022; Xu et al., 2022). A study in Japan found A. caviae to be the most frequent pathogen causing Aeromonas bacteremia (Kimura et al., 2013). According to Lamy et al., A. caviae and A.veronii were the most common Aeromonas species causing bacteremia and gastroenteritis in France (Lamy et al., 2009). Chen et al. reported that A. caviae predominated in primary bacteremia and biliary tract infections in southern Taiwan (Chen et al., 2021). However, epidemiological characteristics, antimicrobial resistance profiles, virulence genes and phyloevolution of A. caviae causing extra-intestinal infections remain unclear.

The present study aims to investigate the epidemiological characteristics of extra-intestinal infections caused by A. caviae and to assess the antimicrobial resistance profiles and virulence genes to provide relevant microbiological data as the basis for effective strategies in the prevention and control of drug resistance. Phyloevolution analysis was subsequently conducted to understand the evolutionary path of A. caviae and the relationship between species. 47 clinical A. caviae causing extra-intestinal infections within a 4-year period were collected at the First Medical Center of Chinese PLA General Hospital. Aeromonas spp. were identified, and virulence and resistance genes were detected through whole-genome sequencing (WGS). Phylogenetic characteristics of 568 strains, including 521 strains obtained from the National Center for Biotechnology Information (NCBI) database and 47 clinical strains in our study, were analyzed.

In this study, it was found that more than 60% of extra-intestinal infections caused by A. caviae developed in elderly patients aged ≥ 65 years, most having immunocompromised conditions or underlying diseases, including hepatobiliary diseases, malignancies, diabetes mellitus, and renal disease. Furthermore, over 50% of patients with A. caviae infection had coexisting hepatobiliary diseases, including biliary stones, obstructive biliary disease, gallbladder or biliary tract tumor, and liver cancer, of which 8 patients suffered from bloodstream infections, 16 patients had biliary tract infections, and 1 patient had biliary tract infection followed by bloodstream infection. The high occurrence of infection with A. caviae may be attributable to biliary tract obstruction or stasis with increased intraductal pressure during hepatobiliary diseases. Moreover, translocation of A. caviae present in the gastrointestinal tract into intrahepatic and extrahepatic bile ducts, as well as hepatic veins and lymphatics, remain a possible cause of biliary tract infection and bacteremia (An et al., 2021). Polymicrobial infections occurred in 14 of 19 patients with A. caviae biliary tract infection, demonstrating that patients with biliary tract infection have a higher risk of developing polymicrobial infections. Malignant tumor patients who have received chemotherapy are more vulnerable to foodborne infections due to the disruption of their intestinal mucosal barrier, and the ensuing neutropenia makes the host susceptible to opportunistic infections (Baden et al., 2016). Consistent with previous studies, malignancy was found to be a common underlying disease in our study (Wu et al., 2015; Chen et al., 2021). Furthermore, liver cirrhosis has been recognized as a predisposing condition associated with Aeromonas bacteremia in regions with a high prevalence of chronic hepatitis (Tang et al., 2014). However, none of the 12 patients with A. caviae bacteremia had liver cirrhosis, and only one patient with liver cirrhosis developed urinary tract infection. Although A. caviae has been considered the primary culprit of bacteremia and biliary tract infections, A. caviae has also been reported in a few cases of urinary tract infections (Chao et al., 2012). In our study, more than 15% (7/46) of patients suffered from urinary tract infection, 5 of which are immunocompromised or undergoing invasive therapeutical procedures. Therefore, A. caviae should be considered a possible pathogen in immunocompromised patients, individuals suffering from underlying diseases, elderly patients, and patients undergoing invasive medical procedures.

CLSI has recommended third and fourth generation cephalosporins, fluroquinolones, and trimethoprim-sulfamethoxazole for the treatment of infections with Aeromonas spp. To date, there are only a few studies on the antimicrobial resistance of extra-intestinal A. caviae isolates, with one study reporting 15%-30% resistance rates for ceftazidime, cefepime, and ceftriaxone from 2012 to 2017 (Yang et al., 2019). Results of our study demonstrated that the resistance rate for cefotaxime was over 30%, and the resistance rates for ceftazidime, cefepime and trimethoprim-sulfamethoxazole were over 20%. Although the incidence of MDR reached to 40%, resistance rates to several antibiotics remained below 10%, including piperacillin-tazobactam, levofloxacin, gentamicin, amikacin, imipenem, and meropenem. These findings provide guidance for selecting appropriate empirical treatment before obtaining AST results.

In the present study, bla _MOX was detected in more than 90% of isolates. This result concurs with previous findings that AmpC β-lactamases genes are species-specific to Aeromonas spp. and that all A. caviae isolates carry bla _MOX (Fosse et al., 2003; Wu et al., 2015). However, only 50% (22/44) of the strains carrying bla _MOX exhibited the cephalosporins or cephamycins non-susceptibility. Thus, the presence of the bla _MOX gene was not associated with corresponding antibiotics non-susceptibility as reported previously (Walsh et al., 1997).

The class A extended-spectrum β-lactamases (ESBLs) gene bla _PER-3 shared 99% identity with bla _PER-1 and was initially identified within an isolate of Aeromonas punctata in France adjacent to a copy of ISCR1 (insertion sequence common regions, ISCRs), and then detected in two A.caviae isolates from a medical center in Taiwan and A.veronii isolated from chicken cloaca (Toleman et al., 2006; Wu et al., 2011; Wang et al., 2020). We found 7 bla _PER-3-producing A. caviae isolates, all of which were MDR strains. Previous research revealed that the horizontal transfer of genetic elements, such as plasmids and integrons, could lead to an increased incidence of MDR among environmental Aeromonas isolates (Jacobs & Chenia, 2007). The spread of Enterobacteriaceae carrying the bla _PER-1 gene as a chromosomal insert has been reported in Europe (Perilli et al., 2007). Therefore, caution should be exercised to prevent transmission of drug resistance genes between Aeromonas spp. by genetic elements.

Carbapenem resistance has been detected in A.hydrophila and A.veronii isolates, but is rarely found in A. caviae strains (Chen et al., 2012). However, recent studies have found that plasmid-encoded or non-plasmid encoded bla _NDM-1, bla _OXA-181, bla _VIM, and bla _KPC-2 genes contribute to carbapenem resistance in clinical A. caviae isolates, indicating the possibility of evolution and transmission of resistance genes in clinical strains (Adler et al., 2014; Anandan et al., 2017; Tang et al., 2020; Luo et al., 2022; Xu et al., 2022). In this study, two strains isolated from urine harbored bla _NDM-1 and exhibited carbapenem resistance, one of which co-harbored of bla _CTX-M-3 and class D β-lactamases gene bla _OXA-1, which conferred resistance to third or fourth generation cephalosporins. The emergence of MDR strains poses a threat that challenges the diagnosis, clinical treatment, and control of infectious diseases. Studies on the genetic characteristics of these two strains are being conducted to elucidate the role of mobile genetic elements, such as plasmids and integrons, in the transmission of resistance.

According to one study, the AmpC β-lactamase gene bla _AQU-2 was found only in A. hydrophila and A. jandaei strains isolated from chicken rinse (Wang et al., 2021). The MBL gene bla _CphA, which is involved in the intrinsic resistance of Aeromonas to carbapenems, was found in the majority of A.hydrophila isolates but rarely detected in A. caviae strains (Wu et al., 2015; Wu et al., 2019). In our study, one bla _CphA-carrying A. caviae, which co-harbored both bla _AQU-2 and the class D β-lactamases gene bla _OXA-724, conferred resistance to third generation cephalosporins, but did not exhibit carbapenem resistance in vitro, possibly attributed to the difficulty in detecting carbapenemase activity of carbapenemase hydrolyzing Aeromonas (CphA) through conventional AST, or genetic modifications that alter the expression of CphA (Wu et al., 2012).

To date, the virulence and pathogenic mechanism of Aeromonas remain obscure. Many virulence factors, including cytotoxins, enterotoxins, hemolysins, cell surface structures, lipases, proteases, aerolysins, and secretory systems, contributed to survival, environmental adaptation, and disease pathogenesis (Li et al., 2015; Wu et al., 2019; Zhou et al., 2019; Chen et al., 2021; Sun et al., 2021). Previous studies observed lower virulence and lower fatality rates in A. caviae compared to other Aeromonas spp. (Wu et al., 2015; Wu et al., 2019). However, findings from several studies on virulence genes and virulence phenotype of A. caviae remain controversial. In the present study, we revealed 9 virulence patterns composed of one to eight genes. Additionally, we found that the majority of the A. caviae strains carried at least one of the virulence genes, and three or more virulence genes were detected in more than 20% of these isolates. Pablos et al. reported that A. caviae infrequently carried aerA, hlyA, and ast genes (Pablos et al., 2010). Wu et al. found that the major genotype in clinical A. caviae isolates was lip, col, and ela (Wu et al., 2019). However, our finding contradicts the results of previous studies. More than 90% of isolates carried hlyA, and none carried lip, col, or ela. In accordance with previous research, our study showed that ast was absent in clinical A. caviae isolates (Aguilera-Arreola et al., 2007). Research has demonstrated that flagella glycosylation in A.hydrophila plays a vital role in Hep-2 cell adhesion and biofilm formation (Merino et al., 2014). Previous study showed that fla and laf were highly prevalent in A. caviae isolated from human faeces (Santos et al., 2011). In a recent study, 92.3% of A. caviae isolated from clinical specimens harbored fla (Miyagi et al., 2021). This study deteced fla and laf in only 26% and 23% of extra-intestinal A. caviae isolates, respectively. The type III secretion system (TTSS) of Aeromonas has been proved to play an essential role in pathogenicity (Yu et al., 2004). Studies found ascV and ascF-G in only a few extra-intestinal A. caviae isolates (14.3%, 2/14) (Chacon et al., 2004). Chen et al. demonstrated that infection with ascF-G Aeromonas was associated with mortality (Chen et al., 2021). We found that more than 20% of extra-intestinal A. caviae isolates (21%, 10/47) possesed these TTSS encoding genes, but only one patient, who was infected with A. caviae harboring ascV and ascF-G, died in the hospital.

One patient (case 46) infected with A. caviae carrying 8 virulence genes was cured after 14 days of hospitalization. However, another patient (case 18) infected with A. caviae possessing only hemolysin encoding gene hlyA died 45 days after admission. The significant difference between the two patients was that the latter had liver cancer. Our work showed that the overall mortality of extra-intestinal A. caviae infection was 15% (7/46). Additionally, we found that most of the strains (86%, 6/7) isolated from the 7 patients who died harbored only one virulence coding gene hlyA, and 5 of the 7 patients suffered from cancer. It is possible that host factors, such as underlying diseases, prolonged hospitalization, and not the virulence of A. caviae itself, are the main causes of death. We did not find an association between virulence genes and the pathogenicity of extra-intestinal A. caviae stains, suggesting the need for further research to identify specific virulence gene and the mechanism of pathogen-host interaction in extra-intestinal A. caviae infection.

In this retrospective study, we analyzed the genomic evolutionary characteristics of A. caviae based on 47 clinical isolates and 521 public strains available in the NCBI database. The genetic relationships of the 47 strains were identified, and cluster analysis indicated differences in SNPs among the 47 strains. These isolates had low genetic relationships, which might be due to the long separation interval of these strains. Genomic evolutionary analysis showed that stains in L3 and L4 contained more drug resistance genes than those in L2, presumably because changes in some key SNPs determined the subsequent evolution, and some drug resistance genes were inserted during the evolution. A high degree of homology between S108 and S109, but low homology between other stains indicated that there existed a risk of nosocomial transmission, but no clustering of hospital-onset infections was noted.

The epidemiological characteristics, antimicrobial resistance profiles, virulence genes, and phylogenetic traits described in this study strengthen our understanding of A.caviae strains that cause extra-intestinal infections. It may contribute to the management of extra-intestinal infections as well as the prevention and control of drug resistance.

The data presented in the study are deposited in the National Library of Medicine repository (https://www.ncbi.nlm.nih.gov/sra/PRJNA902936), accession number PRJNA902936.

This study involving human participants was reviewed and approved by the Ethics Committee of First Medical Center of Chinese PLA General Hospital.

YS, L-FW, D-XS, and YC designed the study. KZ, SL, LG, L-YY, and JG performed the experiments and interpreted the data. YS, YC, and L-FW wrote the first draft of the paper. YS, YC, L-FW and D-XS reviewed and approved the final report. All authors contributed to the article and approved the submitted version.