Emergence of blaIMI-2- and blaIMI-16-Producing Enterobacter asburiae in the Aquaculture Environment of Jiangsu, China

IMI carbapenemases have been detected in clinical isolates of many bacterial species with systemic infection and cause a further burden on clinical treatment in China, but their source and distribution are still unclear. The study systematically investigated the distribution and transmission of the blaIMI gene in aquaculture-related water bodies and aquatic products in Jiangsu Province, China, which is famous for its rich water resources and developed aquaculture industry. ABSTRACT Carbapenem-resistant Enterobacteriaceae strains have emerged as a serious threat to global public health. In recent years, blaIMI, a carbapenemase gene that drew less attention before, has been increasingly detected in both clinical and environmental settings. However, the environmental distribution and transmission of blaIMI, especially in aquaculture, require systematic investigation. In this study, the blaIMI gene was detected in fish (n = 1), sewage (n = 1), river water (n = 1), and aquaculture pond water samples (n = 17) collected from Jiangsu, China, demonstrating a relatively high sample-positive ratio of 12.4% (20/161). Thirteen blaIMI-2- or blaIMI-16-carrying Enterobacter asburiae strains were isolated from blaIMI-positive samples of aquatic products and aquaculture ponds. We also identified a novel transposon (Tn7441) carrying blaIMI-16 and a conserved region containing several truncated insertion sequence (IS) elements harboring blaIMI-2, all of which may play important roles in blaIMI mobilization. The occurrence of blaIMI-carrying Enterobacter asburiae in aquaculture-related water samples and fish samples highlights the risk of transmission of blaIMI-carrying strains through the food chain and the need for effective measures to prevent further dissemination. IMPORTANCE IMI carbapenemases have been detected in clinical isolates of many bacterial species with systemic infection and cause a further burden on clinical treatment in China, but their source and distribution are still unclear. The study systematically investigated the distribution and transmission of the blaIMI gene in aquaculture-related water bodies and aquatic products in Jiangsu Province, China, which is famous for its rich water resources and developed aquaculture industry. The relatively high prevalence of blaIMI in aquaculture samples and the identification of novel mobile elements harboring blaIMI enhance our knowledge of blaIMI gene distribution and highlight the public health risk and urgency of surveillance of aquaculture water systems in China.

their living environments. The abundance of antibiotic-resistant isolates or genes found in farm animals and their environment has been investigated extensively. In contrast, less attention has been paid to aquatic food products and the associated water environment, although both are important sources of farm-to-table antimicrobial resistance gene transmission.
IMIs are class A carbapenemases, with IMI-1 first described in a clinical isolate of Enterobacter cloacae in 1984 (3). Since then, IMI carbapenemases have been detected in clinical isolates of many bacterial species, such as Enterobacter asburiae, Enterobacter cloacae, Escherichia coli, Raoultella ornithinolytica, and Klebsiella variicola (4)(5)(6). Furthermore, the gene encoding IMIs, bla IMI , has been detected in environmental samples, especially those from the aquatic environment. In 2005, bla IMI-2 was identified in a self-transferable plasmid of an Enterobacter asburiae strain isolated from a river in the United States (7). That study was followed by several others in which bla IMI -producing Enterobacteriaceae were detected in rivers (8,9), estuaries (10), market foods (11), and seafood (12). Thus far, 22 assigned variants of IMI enzymes have been reported (IMI-1 to -22; http://bldb.eu). However, the studies mentioned above mainly reported the emergence of strains carrying the bla IMI gene. Only two of them demonstrated the bla IMI -positive ratios of 10% and 4% in the water ecosystems of the United States (8) and Spain (9), respectively. The epidemiological analysis for the environmental distribution and transmission of bla IMI , especially in China, has not been investigated systematically, and the mobile element(s) and specific genetic environment(s) responsible for the spread of bla IMI remain unclear (7,13).
In this study, we surveyed the prevalence of bla IMI genes and their host bacteria in aquatic food products and the associated water environments in Wuxi, Jiangsu Province, China, which is famous for its aquaculture industry. A relatively high sample-positive ratio of 12.4% for bla IMI genes was detected, and bla IMI -carrying Enterobacter asburiae strains were isolated from bla IMI -positive samples obtained from aquatic products and aquaculture ponds. A novel transposon, Tn7441, and a conserved region containing several insertion sequence (IS) elements were also identified, which may play important roles in the mobilization of bla IMI . Moreover, our study revealed that bla IMI -carrying Enterobacter asburiae has spread extensively in the waterbodies and aquatic products of Wuxi, highlighting the risk of bla IMI -carrying strain transmission to humans through farm-to-table processes and the need to promote effective measures to prevent further dissemination.

RESULTS AND DISCUSSION
Prevalence of bla IMI in aquatic ecosystems. Jiangsu Province is developed in the aquaculture industry. Its total fishery economy ranks second in China, and the output value of its freshwater farming ranks first. Wuxi, a city in Jiangsu Province, is surrounded by lakes of all sizes, giving rise to a large aquaculture industry. From 10 to 11 June 2019, 161 samples from aquaculture ponds, rivers, sewage, and aquatic products were collected in Wuxi and subjected to PCR analysis to detect bla IMI . Twenty (12.4%) samples were positive for bla IMI genes, comprising 1 each from fish, sewage, and river water and 17 from aquaculture pond water (see Table S1 in the supplemental material). In mainland China, IMI-2 was reported in an Enterobacter cloacae isolate in Zhejiang in 2006 (5). Then, in 2017, IMI-3-producing Raoultella ornithinolytica RJ46C and IMI-2-producing E. coli RJ18 were identified in a hospital in Shanghai (6). Thus far, no other bla IMI -positive isolates had been reported from local healthy or clinical people. Therefore, this was a relatively high bla IMI positivity rate reported in China, even higher than those determined in the water ecosystems of Spain (4%) (9) and the United States (10%) (8).
The bla IMI -positive samples were inoculated into culture medium to screen for bla IMI -producing bacteria, resulting in the isolation of 13 carbapenem-resistant strains from 5 samples, of which 4 were water samples collected from the fish ponds, and 1 was a fish sample obtained from another fish pond. The presence of the bla IMI gene in the 13 strains was confirmed by PCR. Six (46%) isolates harbored bla IMI-16 , and seven (54%) harbored bla IMI-2 ; the latter included the isolate from the fish sample. The colonies were identified using the Vitek 2 compact assay (bioMérieux, Inc.) and confirmed by 16S rRNA sequencing and whole-genome sequencing (WGS) taxonomy classification through Kraken2. All 13 strains were Enterobacter asburiae, indicating the importance of the bacterium as a carrier of bla IMI in aquaculture. Other studies from different countries have reported bla IMI -carrying Enterobacter asburiae in clinical settings (14)(15)(16). Our results thus expand the known habitat niches of bla IMI -carrying Enterobacter asburiae.
The bla IMI -carrying isolates were resistant to most b-lactam antibiotics. Antimicrobial susceptibility testing showed that all 13 isolates were resistant to carbapenems (imipenem, meropenem, and ertapenem). Testing of the isolates against expanded-spectrum cephalosporins showed that all were resistant to first-generation cephalosporins (cefazolin). Some strains exhibited intermediate resistance to second-generation cephalosporins (30.8% for cefuroxime, 69.2% for cefoxitin), and all except strain CW35-4 (92.3%) were susceptible to third-and fourth-generation cephalosporins (ceftriaxone and cefepime). Additionally, in contrast to the other strains, strain CW35-4 also showed intermediate resistance to nitrofurantoin. The 13 strains exhibited full or intermediate resistance to amoxicillin-clavulanic acid and ampicillin-sulbactam. Thus, in summary, the tested strains were resistant to most b-lactam antibiotics and susceptible to several non-b-lactam antibiotics (trimethoprim-sulfamethoxazole, chloramphenicol, colistin, quinolones, aminoglycosides, and tetracyclines).
In order to further investigate the antibiotic resistance mechanism of these 13 strains, their whole-genome sequences were sequenced using Illumina HiSeq 2000. Then, the antibiotic-resistant genes (ARGs) were annotated by ResFinder. Besides bla IMI , another b-lactam antibiotic resistance gene, bla ACT, was also detected in all the isolates. Additionally, oqxA/B, qnrS1 (quinolone resistance), mdf(A) (multidrug efflux pump), and fosA (fosfomycin resistance) were also found to be carried by 2 to 6 isolates (Fig. S1). Furthermore, the antibiotic residues were detected for the water samples, aiming to explore whether the prevalence of antibiotic-resistant genes was related to antibiotic usage. Several commonly used antibiotics, such as chloramphenicol, sulfonamides, quinolones, macrolides, and tetracyclines, could be detected in about 2.6% to 61.5% of samples. The presence of ARGs like oqxA/B, qnrS1, mdf(A), and fosA in aquatic settings might be mainly under selective pressure of the detected antibiotics. However, no b-lactams were detected in any samples (Table S2). The negative detection results might be related to the rapid degradation and low dosage of lactam drugs. Nevertheless, the heavy metal residues and/or other chemicals caused by use of disinfectants in the aquaculture industry may exert coselection pressure on ARGs (17). Thus, it is difficult to suggest the selection pressure and origin of the b-lactam resistance, especially bla IMI , of the aquatic settings. Notably, it was reported that IMI had the ability to hydrolyze carbapenems but not expanded-spectrum cephalosporins. In our result, the strain CW35-4 was resistant to third-and fourth-generation cephalosporins (ceftriaxone and cefepime), which is not consistent with previous understanding and needs to be explored in future studies.
Transfer of the bla IMI-2 gene among plasmids mediated by a conserved region. Based on the genome sequences, multilocus sequence typing (MLST) analysis showed that the seven bla IMI-2 -carrying Enterobacter asburiae strains belonged to two new sequence types (STs). The strain from the fish, CFB52, was ST1336, and the other six (85.7%) strains, isolated from the water of two fish ponds, were ST1337 (Table 1). Further pairwise homologous alignment of the bla IMI-2 -carrying scaffolds of the bla IMI-2 -positive strains resulted in their classification into two types, with lengths of 94 kb (strain of ST1336) and 46 kb (strain of ST1337). Strains representative of these two types of bla IMI-2 -positive scaffolds, CFB52 (ST1336) and CW35-3 (ST1337), were selected for sequencing by the PacBio platform to obtain the complete genomes. According to the sequencing results, the bla IMI-2 genes were located on two plasmids, pCW353_IMI and pCFB52_IMI, carried by two Enterobacter asburiae clones. No bla IMI -carrying strain was isolated from the corresponding pond water of CFB52, which was derived from a fish sample, or, conversely, from the corresponding aquatic products from the pond waters hosting the other six isolates. Thus, whether direct transmission between the aquatic products and the aquaculture environment occurred could not be determined. Nonetheless, the high prevalence of bla IMI -carrying strains in the aquaculture environment and the contamination risk to aquatic products raise concern.   ERT Emergence of bla IMI in the Aquaculture Environment Microbiology Spectrum pCW353_IMI, a 46,684-bp plasmid carrying bla IMI-2 , has an average GC content of 51% and belongs to the IncFII (Yp) group (Fig. S2). pCFB52_IMI, a 95,423-bp plasmid carrying bla IMI-2 , has an average GC content of 51% and encodes the RepA protein, which has 84.48% identity to IncFII (Yp) RepA (Fig. S3). Except for bla IMI , no other resistant gene is encoded by these two plasmids. Sequence comparisons of pCW353_IMI and pCFB52_IMI showed that they mainly shared three regions (Fig. 1a). The first (region 1) is 8,918 bp in length and contains both the bla IMI-2 and bla IMI-R genes and many ISs flanking the two genes, including an ISSen7-like element, ISEae2, two IS1H-like elements, an ISSba14-like element, and ISEae1. The second region contains mainly genes encoding the UmuC subunit of DNA polymerase V, toxin, and antitoxin, while the third contains genes encoding the outer membrane usher protein FimD, chaperone protein FimC, type 1 fimbrial protein, and transposase. Alignment of pCW353_IMI and pCFB52_IMI against sequences in the NCBI nonredundant nucleotide (nr/nt) database identified three plasmids (p3442-IMI-2, pJF-787, and pN151247-1) as the best hits (identity $ 99%; 42% and 18% average coverage of pCW353_IMI and pCFB52_IMI with the three plasmids, respectively), as shown in Fig. 1b. Plasmid p3442-IMI-2 (GenBank accession no. CP033468) originated from Enterobacter cloacae and was isolated from Penaeus vannamei in the Netherlands (12). Plasmid pN151247-1 (GenBank accession no. KY680213) originated from Klebsiella aerogenes and was isolated in Canada from shrimp imported from Bangladesh. Plasmid pJF-787 (GenBank accession no. KX868552) originated from clinical isolates of K. variicola in the United Kingdom (4). All three plasmids were present in Enterobacteriaceae despite being collected from three different countries and sampled from patients and aquatic organisms, which suggests a worldwide prevalence of the bla IMI-2 gene in animals and humans. Sequence comparisons of these five bla IMI-2 -carrying plasmids revealed a 5,153bp conserved region containing bla IMI-2 and several truncated ISs (Fig. 1b). In this conserved region, the resistance gene bla IMI-2 and the LysR-type regulator gene bla IMI-R are flanked by two truncated IS1H-like elements. In addition, an ISSba14-like element and ISEae1 are found upstream of the truncated IS1H-like elements (Fig. 1b). Outside of this Emergence of bla IMI in the Aquaculture Environment Microbiology Spectrum conserved region, the genetic environment of these plasmids varies widely, suggesting that pCW353_IMI and pCFB52_IMI are novel bla IMI-2 -carrying plasmids. Of note, the two bla IMI-2 -encoding plasmids are nonconjugative, in contrast to previously reported bla IMI-2carrying plasmids (4,5,7,(12)(13)(14). The high density of IS elements flanking bla IMI-2 in the conserved region indicated that these sequences might be responsible for the mobilization of bla IMI-2 between conjugative and nonconjugative plasmids in Enterobacteriaceae.
The bla IMI-16 gene is located on a novel transposon, Tn7441. The bla IMI-16 genetic environment was the same among the six bla IMI-16 -harboring strains of Enterobacter asburiae, and MLST analysis showed that all were ST1585. We therefore selected one strain (CW1-2.1) for sequencing by PacBio platform to obtain the complete genome. The results showed that the bla IMI-16 gene of CW1-2.1 was located on pCW1_IMI, an IncFII (Yp) group plasmid with an average GC content of 51%. pCW1_IMI contains 79 predicted open reading frames, but no resistance gene besides bla IMI-16 was found on the plasmid (Fig. S4).
A BLAST similarity analysis against the nr database revealed one bla IMI-6 -harboring plasmid and two bla IMI-3 -harboring plasmids as the best hits against pCW1_IMI, but with low coverage (42% on average and .96% identity) (Fig. 2a), suggesting that pCW1_IMI is a novel bla IMI -carrying plasmid. Plasmid pRJ46C (GenBank accession no. KT225520) was isolated from clinical strain Raoultella ornithinolytica in China, pGA45 (GenBank accession no. KT780723) was isolated from an uncultured bacterium from Chinese river sediment, and pIMI-6 (GenBank accession no. KX786187) was isolated from a clinical strain of Enterobacter cloacae in Canada. In the two bla IMI-3 plasmids (pRJ46C and pGA45), bla IMI-3 was embedded in the same Tn6306 element, and the gene structures were similar, whereas the genetic structure of bla IMI-6 within plasmid pIMI-6 was similar to that of Tn6306, carrying extra genes encoding two partitioning proteins (ParA and ParB), a protease, and a resolvase (Fig. 2b). The presence of ISEcl1-like elements on both ends is a feature of Tn6306 (6). In addition, ISEae1 was located on one side of the Tn6306 or Tn6306-like element (Fig. 2b). In our pCW1_IMI, there was only a homologous gene of the ISEcl1-like element, ISEcl3, located downstream of bla IMI-R -bla IMI-16 . However, there were two ISEae1-like genes, located ;7 kb upstream and ;6.5 kb downstream of the bla IMI-16 gene. Sequence analysis showed that these two ISEae1-like genes had the same orientation, and both contained two 13-bp complete inverted repeats. Furthermore, the whole 16.8-kb region was flanked by 2-bp direct duplications immediately adjacent to each of the ISEae1-like genes (Fig. 2b). This is a typical transposon structure, which is another Tn6306-like element. Thus, we named it Tn7441, according to the nomenclature of transposons (https://transposon.lstmed.ac.uk/). In Tn7441, several other IS elements and function genes located between the bla IMI-16 and ISEae1-like genes were identified. The novel transposon Tn7441 and other inserted sequences may play important roles in bla IMI-16 mobilization.
Conclusions. Compared to previous studies of the prevalence of the bla IMI gene, the relatively high rate of bla IMI gene detection in our samples indicates that IMI carbapenemases are dispersed in the water bodies and aquatic products of Jiangsu, China. Isolation of bla IMI-2 -and bla IMI-16 -producing Enterobacter asburiae strains, especially the bla IMI-2 -carrying strain from fish, suggests that antibiotic resistance can be transferred from aquaculture ponds to aquatic products. A novel transposon, Tn7441, harboring bla IMI-16 and a conserved region in bla IMI-2 -carrying plasmids were identified in this study, and they may play important roles in bla IMI mobilization. Our study is just cross-sectional research. There are limitations in the time span and regional representation of sample collection. However, considering the risk of transferring bla IMI -carrying strains from the environment to humans through the food chain, our findings highlight the need for surveillance of resistance genes in aquatic products and in the aquaculture environment, as well as effective measures to prevent further dissemination of carbapenem resistance.

MATERIALS AND METHODS
Sample collection. The sampling area is located in Ehu town of Wuxi, Jiangsu Province of China. Wuxi has Taihu Lake, the third-largest freshwater lake in China. Ehu town is near Taihu Lake, and its aquaculture industry is developed. It has a total area of about 9 square kilometers and a fishing pond area of about 2 km 2 , and it is rich in black carp, with an annual output of about 1 million kg. Meanwhile, aquatic products such as silver carp, crab, Chinese turtle, and aquatic plant Semen Euryales are all main Emergence of bla IMI in the Aquaculture Environment Microbiology Spectrum local aquaculture species. From 10 to 11 June 2019 (during the production period), a total of 161 samples comprising 141 water, 11 fish, 7 shrimp. and 2 crabs samples were collected from aquaculture ponds (n = 141) in two villages (A, 31.520°N, 120.585°E; B, 31.575°N, 120.582°E) of Ehu town (see Fig. S5 in the supplemental material). About 100 mL of water samples was collected by submerging a sterile water sample collector approximately 30 cm below the water surface at three randomly selected locations in each pond. Then, the water samples from the same pond were merged into one sterile sample transport bag, resulting in a total of 141 water samples (300 mL for each). Eleven fish samples, 7 shrimp samples, and 2 crab samples were collected from 20 randomly selected ponds of the 141 ponds (one for each) and placed in sterile sample transport bags. All samples were delivered to the laboratory on ice.
We investigated the antimicrobial agent usage and water management measurements by questionnaire when sampling. Enrofloxacin, florfenicol, and sulfonamides were commonly used antibiotics during the aquatic production period, and no carbapenems were used in aquaculture and animal husbandry in China. A water purification area was set up for each fish pond. The tailwater at the end of aquaculture is first discharged into the water purification area and then discharged after ecological and physical purification by the fish, plant, and filter device in the water purification area.
Extraction of total DNA and detection of bla IMI genes. For the water samples, after being fully mixed, 50 mL was taken to centrifuge at 8,000 rpm/minute for 30 min at 4°C and gentle separation of the supernatants and the sediments. The supernatants were collected in new clean tubes for antibiotic detection, while the sediments were resuspended in 1 mL sterile phosphate-buffered saline (PBS) for DNA extraction. Two-hundred-microliter sediment suspensions were used for DNA extraction by using QIAamp Fast DNA stool minikit (Qiagen, Germany). For sampled fish, crab, and shrimp, the intestines were cut open with a sterile surgical blade and rinsed in 10 mL sterile PBS. After fully mixing and discarding the tissues, the mixtures were centrifuged at 8,000 rpm/min for 30 min at 4°C, we gently discarded the supernatants, and we resuspended the sediments in 1 mL sterile PBS. We used 200 mL resuspensions for DNA extraction by using QIAamp Fast DNA stool minikit. Next, the DNA was used to detect the carbapenemase gene bla IMI following a new high-throughput real-time PCR assay (18). In brief, gene bla IMI detection was performed through Roche LightCycler 480 (LC 480) system. The primers and probe used in reverse transcriptase PCR (RT-PCR) are listed as follows: blaIMI-F, 59-GGTGTCTACGCTTTAGACACTGGC-39; blaIMI-R, 59-CTGTGTTTAGATCT AACTCCCAACGA-39; and blaIMI-P, FA M-TGGTCCTGAGGGTATG-MGB. The amplification conditions were 2 min at 50°C and 1 min at 95°C, followed by 40 cycles of two-step amplification of 15 s at 95°C and 1 min at 60°C.
Determination of antibiotics in water samples. The collected supernatants of water samples were loaded into the QTRAP 5500 liquid chromatography-tandem mass spectrometry system (AB Sciex, USA) after 0.45-mm film filtration to determine the antibiotic contents. The chromatographic condition and mass spectrum condition are outlined as follows. The determination was performed on an HSS T3 column (column length was 100 mm, inner diameter was 2.1 mm, and particle size was 1.8 mm). Phase A was 0.1% formic acid aqueous solution, and phase B was methanol. The flow rate was 0.3 mL/min. The column temperature was 40°C. The automatic injector temperature was 5°C. The injection volume was 10.0 mL. The ionization mode was positive ion electrospray ion source (ESI 1 ). The ion source temperature was 550°C. The spray voltage was 5.5 kV. Curtain gas was 35 lb/in 2 . Atomizing gas was 55 lb/in 2 . Auxiliary gas was 65 lb/in 2 . The detection mode was multireactive ion monitoring mode.
Isolation and identification of bla IMI -carrying bacteria and antimicrobial susceptibility testing. For the bla IMI -positive water samples, the water samples were fully mixed, and a 20-mL aliquot was taken from each bla IMI -positive sample. The aliquots were centrifuged at .10,000 rpm for 2 min, and the pellet was suspended in 1 mL nutrient broth (NB). An overnight culture was prepared by inoculating 0.1 mL of the suspension into plates of Enterobacteria enrichment (EE) agar medium containing 0.5 mg ertapenem/L and incubating the plates at 35 6 2°C for 18 h. As for the bla IMI -positive aquatic product samples, the intestinal content resuspensions obtained in "Extraction of total DNA and detection of bla IMI genes" were used to isolate bla IMI -resistant strains. We plated 0.1 mL of the resuspensions on EE agar medium containing 0.5 mg/L ertapenem and incubated the plates at 35 6 2°C overnight. All the carbapenemnonsusceptible single colonies were selected, and colony PCR was performed to detect the bla IMI gene. The primers and probe and the amplification conditions used in colony PCR were the same as those of RT-PCR for bla IMI gene detection described above. Then the bla IMI -positive colonies were identified using the Vitek 2 compact assay (bioMérieux, Inc.) and confirmed by 16S rRNA sequencing and WGS taxonomy classification through Kraken2 (19). The MICs of several antibiotics against the isolated strains (Table 1) were determined using the broth microdilution method through Phoenix automated system (BD Phoenix 100, USA); with the results interpreted according to the Clinical and Laboratory Standards Institute guidelines (CLSI M100-S28) (20).
Data availability. The genomic sequences of the bla IMI -carrying strains used in this study are deposited in China National Microbiology Data Center (NMDC; https://nmdc.cn/en) under BioProject accession number NMDC10018005.