AST of target strains

AN70, NFYY023, E202, and NCTC10498 strains were identified by BD Mérieux, and the strains were A. faecalis, C. testosteroni, B. trematum, S. maltophilia, respectively. AST showed that these strains were multi-drug resistant bacteria. All of these strains are resistant to carbapenems. Detailed AST results of strains are presented in Table 1.

Table 1 Antibiotics susceptibility testing of target strainsPhylogenetic relationship and secondary structure analysis of AFM

Before the occurrence variants of blaAFM-1, by comparison with several B1 carbapenemases genes, blaAFM-1 showed the highest similarity to blaNDM, with 93% query coverage and 86% identity at the nucleotide level, followed by blaANA-1 and blaSPN79-1 (both 51% homology with blaAFM-1 gene at the nucleotide level) (data do not show). Phylogenetic tree analysis (MEGA 7.0) also revealed that the blaAFM-1 gene was close to blaNDM gene family (Fig. 1), which is in line with previous studies [12]. The phylogenetic tree of AFM-1 amino acids and other common B1 metalloenzyme amino acids can be seen in the Additional file 1: Fig. S1. Over time, three subtypes of blaAFM genes occurred. Compared with the blaAFM-1 gene, the blaAFM-2, blaAFM-3, and blaAFM-4 genes have 99% homology with the blaAFM-1 gene at the nucleotide level, and only a single base mutation (C → T), and the mutation sites are located at the 44th, 37th, 40th position, respectively, and the mutated amino acid corresponding to A14V, P12S, P12L, respectively.

Fig. 1

The phylogenetic tree of blaAFM gene and common class B1 carbapenemases gene. Note: blaAFM-1 gene is highlighted by a black triangle symbol.

The blaAFM-1 gene contained 804 bp and encoded a protein of 267 amino acids with a molecular mass of approximately 28 KDa. Compared with NDM-1 and NDM-6, AFM differs from NDM by 43 amino acids at the amino acid level (Fig. 2).

Fig. 2

Amino acid alignment among NDM-1 with NDM-6, AFM-1 ~ AFM-4. Note: NDM-1 is at the top of the sequence listed

Spatial prediction model of AFM enzymes

AFM-1 was composed of an αβ/βα sandwich structure, with two zinc atoms at its active site (see in Additional file 1: Fig. S2), similar to common B carbapenemases. For NDM-1, the active site around the zinc ions was surrounded by amino acids, including His120, His122, His189, His250, Cys208, and Asp124 [23, 24]. While for AFM-1, the key zinc ions coordinating residues were His117, His119, His186, Asp121, Cys205, and His247.

WGS of A. faecalis AN70

A. Faecalis AN70 contained a 3922717 bp chromosome with 50.7% G + C content and a 61,915 bp plasmid (pAN70-1) with 58.1% G + C content. The chromosome of AN70 contained 3,640 coding genes (it also harbored six resistance genes, including strA, strB, sul1, catB3, blaOXA-21, and AAC (6′)-IIa), including a new class 1 integron consisting of aacA3-blaOXA-21-catB3-dfrA1b, termed In1675 (http://integrall.bio.ua.pt/?acc=CP036294), which promoter is “PcWTGN-10 + P2”. pAN70-1 contained 81 coding genes, including 10 ARGs (blaAFM-1, msrE, mphE, dfra14, aac(6’)-Ib, blaOXA-10, emrE, sul1, dhfrX, bleMBL) (Fig. 3). Of note, the blaAFM-1 gene was first identified by us and was submitted to the NCBI database under accession number MK143105.1, and pAN70-1 belongs to the IncW plasmid.

Fig. 3

Circle Diagram of pAN70-1

WGS of C. testosteroni NFYY023, B. trematum E202, S. maltophilia NCTC10498

C. testosteroni NFYY023 contained a 4136480 bp chromosome with 59.43% G + C content, and a 89553 bp truncate plasmid (pNFYY023-1) with 57.66% G + C content, a 17,394 bp plasmid (pNFYY023-2) with 52.34% G + C content, and a 6656 bp plasmid (pNFYY023-3) with 52.52% G + C content. ARGs located on C. testosteroni NFYY023 chromosome were cmxA, tetG, dfrV, sul1, aadA5, aadA3, floR, aadA1, aac(6')-Ib9, while blaAFM-1, bleMBL, sul1, strA, strB etc. ARGs carried on pNFYY023-1.

B. trematum E202 contained a 4457823 bp chromosome with 65.47% G + C content. ARGs carried on chromosomes were aadA2, sul1, floR, bleMBL, blaAFM-1, mphA, aadA, cmlA5, AAC(6′)-IIa.

S. maltophilia NCTC10498 contained a 4928653 bp chromosome with 66.25% G + C content. ARGs carried on chromosomes were blaAFM-1, bleMBL, smeR, AAC(6')-Iz, facT, smeF, smeE, smeD, smeC, smeB, smeA, smeS, etc.

Cloning expression

pET-28a( +)-AFM-1-Top10 clone and pET-28a( +)-NDM-1-Top10 clone were successfully constructed, and the PCR/sequencing of these clones using common primers showed that pET-28a( +)-AFM-1-Top10 clone harbored blaAFM-1 gene, while pET-28a( +)-NDM-1-Top10 clone carried blaNDM-1 gene. Etest exhibited that the pET-28a( +)-AFM-1-Top10 clone non-susceptible to carbapenems, which demonstrated AFM-1 has the ability to hydrolyze carbapenem agents (Fig. 4).

Fig. 4

MBL-producing test results. Note: The results of MBL of A. faecalis AN70 strain was shown in (a), the results of MBL of E.coli J53 was indicated in (b), the results of MBL of pET-28a( +)-AFM-1-Top10 clone was highlighted in (c)

Phenotype of AFM-1

Carba NP test showed that AFM-1-producing strains were positive, which was consistent with the positive control (NDM-1-producing strain) (Additional file 1: Table S3), which indicates AFM-1 has the activity of carbapenemase.

Assessment of horizontal transmission of bla AFM-1 gene

Transconjugants were grown on MH agar plates containing 4 µg/ml meropenem and 150 µg/ml sodium azide. PCR analysis and sequencing using AFM-1 primers confirmed that the transconjugants contained blaAFM-1 gene. The pAN70-1 plasmid was successfully transferred from isolate AN70 into E.coli J53 by conjugation, at a frequency of 1.3 × 10−5. The MIC of meropenem for transconjugant (E.coli J53-AN70) was higher than E.coli J53 (Table 2). Conjugation assay was carried out on pNFYY023-1 according to the same procedure. Unfortunately, the pNFYY023-1 plasmid failed to complete the conjugation assay.

Table 2 MICs (mg/L) of AN70, E.coli J53, E.coli J53 -AN70Genetic environment of bla AFM gene

By retrieving from the NCBI database, we found several sequences carrying the blaAFM genes as follows: the pAN70-1 plasmid of A. faecalis AN70 strain (this study), a pNFYY023-1 plasmid from C. testosteroni NFYY023 strain (this study), two chromosomes of B. trematum E202 strain and S. maltophilia NCTC10498 strain (this study), pSS332-218 K plasmid from A. hydrophila SS332 strain [11], a pHS17-127 plasmid from carbapenem-resistant P. aeruginosa HS17-127 strain [12], PA13SY16 of P. aeruginosa strain (Accession NO. MKEM01000335), SWCO2 of C. testosteroni strain (Accession NO. QURR01000056). Plasmids pNDTH10366-KPC and pNDTH9845 harbored blaAFM-2 from the P. aeruginosa strain [13]. Plasmid pWTJH17 that carried blaAFM-3 isolated from P. aeruginosa strain [13]. Plasmid pAR19438 that carried blaAFM-4 recovered from the P. aeruginosa strain [14]. Blastn analysis among these sequences and genetic context among blaAFM gene were displayed in Fig. 5. All of these sequences contain the component of “floR-blaAFM-bleMBL-trpF”. Insertion sequences analysis revealed that all sequences contained an ISCR27-like module, which was named ISCR27n1, ISCR27n2, ISCR27n3, and ISCR27n4. Compared with ISCR27 sequence, ISCR27n1-4 had 98.67%, 94.42%, 95.33%, 97.17% identity with ISCR27, respectively.

Fig. 5

A schematic presentation of the genetic context of blaAFM gene. blaAFM gene was highlighted in red arrow, other antibiotic resistance genes were indicated by gray arrow, mobile genetic elements were showed by green arrow

Comparative genome analysis of plasmid pAN70-1

Plasmid pAN70-1 (Sequence ID: MK089784.1), which plasmid was the first to report to harbor the blaAFM-1 gene. Homology comparison by Blastn revealed that seven plasmids share > 50% coverage with the pAN70-1 sequence, which was pPROV002-IMP (Sequence ID: MH882484.1), pMAK3 (Sequence ID: AB366442.1), p538_S (Sequence ID: AP025181.1), R388 (Sequence ID: BR000038.1), pMTY10660_IncW (Sequence ID: P018350.1), pHH2-227 (Sequence ID: JN581942.1), and IncW pIE321 (Sequence ID: EF633507.1). Above all these seven sequences, pPROV002-IMP had the highest coverage (59%) with pAN70-1.

The common feature of the pAN70-1 sequence and the pPROV002-IMP sequence is that they both harbored the B1 subclass metalloenzyme gene, that is, the pAN70-1 sequence carries the blaAFM-1 gene, while the pPROV002-IMP sequence carries the blaNDM-1 and blaIMP-1 gene. Meanwhile, these two plasmids have the same replication repA, and they all contain the type IV secretion system complex VirB11, VirB10, VirB9, VirB8, VirB6, VirB4, VirB3, and VirB1 in plasmid conjugal transfer region.

The difference between the pAN70-1 sequence and the pPROV002-IMP sequence is that the pPROV002-IMP sequence without mercury resistance-related genes (merR, merT, merP, merD, merA and merE, etc.), and pPROV002-IMP didn’t form a “floR-blaAFM-1-bleMBL-trpF” composite-like module.

Using pAN70-1 plasmid (harboring blaAFM-1 gene) as a reference, the homologous identity of pNDTH9845 plasmid (carrying blaAFM-2 gene), pWTJH17 plasmid (carrying blaAFM-3 gene) and AR19438 plasmid (carrying blaAFM-4 gene) were 44.18%, 50.8%, and 53.32%, respectively.