Infections due to carbapenemase-producing Enterobacteriaceae (CPE) remain pose a major threat to the public health (Nordmann et al., 2011; Tang et al., 2023). In particular, the co-production of two or three carbapenemases in a single bacterial isolate has become increasingly prevalent over the past 5 years, and resistance has shown an increase compared to the presence of a single gene. Such as in the study by Biez et al., the MICs of imipenem, meropenem and ertapenem in bla_NDM-1-E. coli J53 or bla_OXA-48-E. coli J53 transconjugants (Tc) or bla_VIM-1-E. coli TOP10 transformant (Tf) were significantly lower than the original strain NDM-1-, VIM-1- and OXA-48-producing C. freundii 255A1. In another report, the MIC of meropenem in the original strain 112,298 was the same as the highest MIC in the transformants (112298-KPC-TOP10 and 112,298-NDM-TOP10) (Feng et al., 2015; Biez et al., 2022). We should beware of the emergence of such strains. The bla_NDM-1 and bla_IMP-4 genes, both encoding metallo-beta-lactamases (MBLs) with high carbapenemase activity, enable them to hydrolyze nearly all β-lactams including carbapenems. In recent years, they have been frequently detected in a diverse array of gram-negative bacteria, leading to the occurrence of numerous serious outbreaks (Yong et al., 2009; Dolejska et al., 2016; Xiong et al., 2016; Matsumura et al., 2017; Guducuoglu et al., 2018; Roberts et al., 2020). The simultaneous presence of these two resistance genes in a single strain may result in the emergence of highly drug-resistant variants, presenting a significant challenge for the treatment of infections.

Citrobacter freundii, a member of Enterobacteriaceae family and widely existed in water, soil, and the intestines of both animals and humans, has been identified as an opportunistic pathogen responsible for various infections including urinary, gastrointestinal, respiratory, peritoneal and bloodstream infections (Bodey, 2005). Unfortunately, the indiscriminate use of carbapenems has led to an escalating acquired resistance to antibiotics in C. freundii in recent years. So far, carbapenemases such as KPC-2-, NDM-1-, IMP-4-, OXA-48- and VIM-1-type have been reported in C. freundii, with affected regions including China, India, Spain, France and Italy (Yong et al., 2009; Gaibani et al., 2013; Feng et al., 2015; Lalaoui et al., 2019; Biez et al., 2022). However, the coexistence of NDM-1 and IMP-4 in single C. freundii isolate, along with its characteristics of transmission and resistance, has been rarely documented.

In this study, we identified a ST22 isolate of C. freundii, named BC73, which is the first reported case of co-carrying bla_NDM-1, bla_IMP-4 and bla_SHV-12 from urine. Upon comprehensive investigation, we discovered that bla_NDM-1 and bla_IMP-4 were carried by a novel MDR plasmid and an IncN-IncU hybrid plasmid, respectively. Additionally, two extensive transposition units (MGE1 in pCFBC1, MGE2 in pCFBC2) harboring multiple resistance genes were identified, which were a potential contribution to the dissemination of multiple drug resistance.

Citrobacter freundii resistant to carbapenems has been gradually observed in patients with hospital-acquired infections (Hammerum et al., 2016; Zhang et al., 2023). However, studies on its transmission mechanisms and resistance characteristics, especially in case involving C. freundii carrying multiple carbapenemase genes, were scarce. In the routine collection of CRE strains, we obtained the C. freundii isolate BC73 co-carrying the carbapenemase genes bla_NDM-1, bla_IMP-4, and ESBLs gene bla_SHV-12. It was isolated from the urine of a 64-year-old patient with urinary tract infections resulting from total cystotomy and abdominal fistula drainage. Carbapenems resistant Citrobacter freundii isolates, especially multidrug-resistant strains, were gradually being found in urinary tract infections, which may cause the extension of infection and the increase of patients’ suffering (Prussing et al., 2020; Zhang et al., 2021; Ye et al., 2023). The AST results presented in Table 1 showed that C. freundii BC73 and transconjugant BC73-J53 were resistance to all β-lactams, chloramphenicol, minocycline, azithromycin, and gentamicin antibiotics tested. Conversely, they were susceptible to nitrofurantoin, tigecycline, polymyxin B, and amikacin. The resistant phenotype of C. freundii BC73 was consistent with resistant genotype of it, implying resistance determinants were likely responsible for multiple drug resistance.

Recently, IncX3 plasmids carrying bla_NDM have commonly been identified in different species of the Enterobacteriaceae (Ye et al., 2023), implying their significant role in bla_NDM transfer. Furthermore, a study from Zhang et al. showed a high transfer frequency were observed in pZY-NDM1, a IncX3 plasmid harboring bla_NDM-1 gene (Zhang et al., 2021). Interestingly, we identified a nonseparable plasmid, pCFBC-1, co-carrying bla_NDM-1, bla_SHV-12 and bla_DHA-1. In this plasmid, we found three noteworthy features: (1) pCFBC-1 (~131 kb) was a novel large plasmid carrying bla_NDM-1 gene. Firstly, compared to the most homologous plasmids in Figure 4A, we speculated that pCFBC1 underwent a genetic recombination and a conserved structure (groEL-∆groES-cutA-dsbD-trpF-ble_MBL-bla_NDM-1-∆ISAba125-IS3000) was recombined into pCFBC1. To our knowledge, this was the first time that the bla_NDM-1 gene was present in this type plasmid and the resistance of it may be increased. Second, the most homologous plasmids of pCFBC1 have not been systematically analyzed. Based on the above, we believed that pCFBC1 was a novel plasmid. (2) After comparison with the PlasmidFinder database, pCFBC1 was unable to obtain the replicon type of the plasmid and was defined as a nonseparable plasmid. According to the replicon type of the plasmid with higher homology to pCFBC1, we named it InFIB-like plasmid. (3) Lots of transposition units (ISs + resistant determinants) were discovered in MGEs of pCFBC-1 such as IS26-aac(3)-IId module (aminoglycosides resistance), IS6100-mph(A)-mrx-mphR module (macrolides resistance), chrA-IS5075-sul1 module (chromates and folate pathway antagonists resistance), ISCR1-sul1-qacE∆1-arr-3-catB3-IS1 module (folate pathway antagonists resistance, quaternary ammonium compounds, rifamycin and phenicol resistance) and IS26-based module (IS26-bla_SHV-12-IS26-tet(D)-IS26-catA2-IS26-ISVsa3-sul2-IS5075-∆Tn3-IS26-insB-IS26/β-lactams, tetracyclines, phenicol and folate pathway antagonists resistance) (Figure 4A). The presence of IS26 was highlighted as a potential contributor to the dissemination of resistance genes (Jia et al., 2022; Li et al., 2022). Wang et al. ever emphasized that genes encoding resistance could be recruited into a variable genetic locus flanked by IS elements and transposons, facilitating their common transfer in Enterobacteriaceae (Wang et al., 2017a). Additionally, three reports demonstrated (Toleman et al., 2006; Liu et al., 2015; Li et al., 2020) that ISCR1 may contribute to the mobilization of bla_NDM-1 through rolling-circle transposition, manifesting the potential of ISCR1 in transferring resistance genes. At present, while bla_IMP-4 has been found in various plasmid types (N, HI2, L/M and A/C), the IncN type, known for its broad-host-range and self-conjugative properties, remains predominant for the spread of bla_IMP-4 in China (Lai et al., 2017; Wang et al., 2017b; Liu et al., 2021). In our study, we identified a IncN-IncU hybrid plasmid, pCFBC-2, carrying bla_IMP-4 and a class 1 integron In823. The elements related to conjugation transfer such as traKN, kikA, oriT, relaxase, the type IV coupling proteins (T4CP) and the type IV secretion system (T4SS) (virB1-11) were revealed in pCFBC2. Furthermore, an extensive transposition unit (MGE2 in pCFBC2) were described. Compared to the most homologous IncN plasmids in Figure 4B, pCFBC2 not only possessed both IncN and IncU replicon types, but also added Tn3 family, aac(6′)-Ib3 and arr-3 on it, making its host range wider and mobility more flexible.

Horizontal gene transfer (HGT) plays a crucial role in the dissemination of bacterial resistance. The primary vehicle of HGT included plasmids, transposons (Tn), insertion sequences (IS) and integrons (In), which possessed the capability to capture and recombine genes associated with antibiotic resistance, heavy metal resistance and virulence, disseminating them with mobile characteristics (Qiao et al., 2023). Our investigation revealed that C. freundii BC73 successfully transferred bla_NDM-1 and bla_IMP-4, along with a carbapenem non-susceptible phenotype, to the recipient E. coli J53. This confirmed the natural horizontal gene transfer characteristic across species for bla_NDM-1 and bla_IMP-4. From the gene point of view, pCFBC2 had the complete conjugation transfer system, which further verified its autonomous conjugation transfer ability. According to our experimental validation in Figure 3, although pCFBC1 lacked elements related to conjugation transfer except part of relaxase, bla_NDM-1 gene in it can be transferred to the recipient E. coli J53, suggesting that pCFBC1 may have been transferred to the recipient E. coli J53 with the help of pCFBC2. In pCFBC-1, bla_NDM-1 was located in a conserved structure: ISCR27-groEL-∆groES-cutA-dsbD-trpF-ble_MBL-bla_NDM-1-∆ISAba125-IS3000, resembling the structures found in pZY-NDM1 and pNDM-Cf7308. This structure was a combination of Tn3000 and Tn125 remnants. Previous studies have described the prototype structures of Tn3000 and Tn125 associated with bla_NDM-1, as vital vehicles for its dissemination, namely IS3000-groEL-groES-cutA-dsbD-trpF-ble_MBL-bla_NDM-1-ISAba125-IS3000 and ISAba125-ISCR27-groEL-∆groES-cutA-dsbD-trpF-ble_MBL-bla_NDM-1-ISAba125, respectively (Poirel et al., 2012; Campos et al., 2015). Compared with traditional Tn3000 and Tn125, one copy of IS3000 and ISAba125 was absent and another copy of ISAba125 was incomplete in pCFBC-1 (Figure 5A). The genetic background of bla_IMP-4 was intI1-bla_IMP-4-Kl.pn.13-mobC-IS6100 (MGE1 of pCFBC2). Compared with the genetic context of bla_IMP-4 in other plasmids (VR1 of Figure 5B), we observed its subtle changes in pCFBC-2, suggesting the occurrence of gene recombination. Class 1 integrons should be responsible for the transfer of bla_IMP gene. Thus far, bla_IMP-4-associated class 1 integrons, including In809, 823, 823b, 1,377, 1,456, 1,460, and 1,589, has been reported in Enterobacteriaceae (Lee et al., 2017; Matsumura et al., 2017; Dolejska et al., 2018; Liang et al., 2018; Liu et al., 2021; Zhao et al., 2021). The distinction between In823 and In823b depends on the integrality of the intI1 gene. Furthermore, we identified a large MGE2 situated between two fragments (fipA∆1 and fipA∆2) in pCFBC2. In addition to containing the common Tn6292 (IS2-IS26-qnrS1, ISKpn19 and tnp genes) (VR2 of Figure 5B), one repB gene, two toxin-antitoxin proteins (higA and yafQ), mobile elements (IS5075-∆Tn3-IS26) and a class 1 integron carrying gene cassettes aac(6′)-Ib3 and arr-3 were assembled on it. The interruption of the fipA gene has been reported could promote the accumulation of plasmids in diverse hosts and facilitate the aggregation of mobile elements (Yang et al., 2018), which was a beneficial explanation for the formation of this MGE2.

Phylogenetic analysis (Figure 6) was conducted to unveil evolutionary characteristics and homology of C. freundii. The results revealed that C. freundii BC73 clustered with C. freundii MEI002, C. freundii CAV1321, C. freundii 064C1, C. freundii CF8_ST22, C. freundii IDR1800045912-01-00, C. freundii P7699, C. freundii MH17-012 N and C. freundii DY2007. Interestingly, these isolates all belonged to the ST22 C. freundii strain and were distributed in different countries over the span of a decade, which suggested that the ST22 C. freundii strains have disseminated globally and they may be highly clonal. Notably, C. freundii BC73 co-carrying bla_NDM-1 and bla_IMP-4 and C. freundii DY2007 harboring bla_NDM-5, isolated from dongyang, China in 2020 (Ye et al., 2023), were found to be the most closely related isolates. Previous report has described that the differences between bla_NDM-1 and bla_NDM-5 were represented by mutations at only two specific sites (88, 154) (Sun et al., 2019). Based on above findings, we proposed a bold hypothesis that C. freundii BC73 likely evolved from DY2007 through vertical propagation.

Since the initial discovery of NDM-1 and IMP-4 in Enterobacteriaceae, these carbapenemases have rapidly disseminated worldwide (Chu et al., 2001; Yong et al., 2009). In recent years, NDM-1 or IMP-4 producing C. freundii has frequently identified in the clinical setting, which further aggravated the concerns for public health (Wu et al., 2016; Liu et al., 2021). However, C. freundii with the coexistence of NDM-1 and IMP-4 has been rarely reported. To our knowledge, only one such isolate, named wang9, has been reported in China. The C. freundii wang9 isolate belonged to ST415, and the genes bla_NDM-1 and bla_IMP-4 were located on a conjugative IncHI1B plasmid, pwang9-1. The bla_NDM-1 gene was located on the transposon TnAS3 (IS91-sul-ISAba14-aph (3′)-VI-IS30-bla_NDM-1-ble_MBL-trpF-dsbD-IS91) while the bla_IMP-4 gene was carried by integron In1337 (intI1-bla_IMP-4-∆Kl.pn.13-qacG2-aac(6′)-Ib4-∆catB3) (Qiao et al., 2023). However, the above characteristics were markedly distinct in our isolate. The more attention should be paid on further monitoring and genetic analysis of NDM-1 and IMP-4-producing C. freundii isolates and flexible transposition units for better understanding of multiple drug resistance transfer.

In this study, we identified and characterized the genome of C. freundii BC73 co-carrying bla_NDM-1, bla_IMP-4 and bla_SHV-12 from an inpatient with urinary tract infection after bladder cancer surgery. The bla_NDM-1 and bla_IMP-4 genes were located in a novel MDR plasmid and an IncN-IncU hybrid plasmid (pCFBC1, pCFBC2), respectively. In addition, multiple transposition units (ISs + resistant determinants), especially including two extensive transposition units (MGE1 in pCFBC1, MGE2 in pCFBC2), were found on it. The dissemination of NDM-1 and IMP-4-producing C. freundii isolates and ISs + resistant determinants should be of close concern in future clinical surveillance.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The ethical protocol was approved by the Ethics Committee of The Fifth Clinical Medical College of Henan University of Chinese Medicine (Zhengzhou People’s Hospital).

NL: Data curation, Funding acquisition, Resources, Software, Visualization, Writing – original draft, Writing – review & editing. BT: Data curation, Funding acquisition, Software, Supervision, Writing – review & editing. HW: Software, Writing – review & editing. XiC: Software, Writing – review & editing. PW: Data curation, Resources, Writing – review & editing. ZW: Data curation, Writing – review & editing. XuC: Data curation, Writing – review & editing. XG: Supervision, Writing – review & editing. JG: Supervision, Writing – review & editing. YS: Funding acquisition, Supervision, Writing – review & editing.