2.1 A. johnsonii M19 has high carbapenem resistance
Strain M19 was isolated from hospital sewage and identified as A. johnsonii based on the 16S rDNA sequence (Fig. 1). MICs of imipenem, meropenem and ertapenem for A. johnsonii M19 were 128 mg/L, 48 mg/L and 24 mg/L, respectively, which were higher than those reported for most A. johnsonii strains (Table 1) [4,27-32], indicating that strain M19 had striking resistance to carbapenems.
The whole genome of M19 was sequenced, and the assembled genome contained one 3.75 Mb circular chromosome with 41.4% GC content, and one 55 kb circular plasmid, here named pFM-M19, with 35.8% GC content. The general features of the complete genome sequence are included in Table S1. Overall, 197 genes (5.24% of the total genes) could be assigned to a VFDB number, and 228 genes (6.07% of the total genes) to a PHI number, indicating that M19 has a high pathogenic potential for humans or other hosts.
Dozens of ARGs were identified in the genome of M19 (Table S2). Three classes of β-lactamase-encoding genes (class B, class C and class D) were identified, including genes encoding six metallo-β-lactamases (MBLs), two AmpCs and two OXAs (OXA-23 and OXA-211). Furthermore, other antibiotic resistance genes, including efflux pumps, a porin and an aminoglycoside-modifying enzyme gene, were also identified.
Insert Fig. 1
Insert Table 1
2.2 M19 harbours two oxacillinases genes, blaOXA-23 and blaOXA-211
Genome annotation of A. johnsonii M19 revealed the presence of two OXA-encoding genes, which are responsible for carbapenem resistance, blaOXA-211 in the chromosome and blaOXA-23 in plasmid pFM-M19. In addition, blaOXA-211 in M19 has the same genetic context conserved in other A. johnsonii strains (Fig. S1) and which appears to be ubiquitous in this species .
OXA-23 encoded by plasmid pFM-M19 exhibited extremely high amino acid identity with OXA-23 found in A. baumannii (CAB69042.1), A. pittii (AUF80820.1), A. wuhouensis (AYO52469.1), A. indicus (ANG65640.1), A. nosocomialis (AKL90363.1), E. coli 521 (AIE13834.1), A. baylyi (AER61544.1), A. radioresistens (ABX00637.1) and Klebsiella pneumoniae (WP_063864531.1) (Fig. S2). M19 OXA-23 also has the conserved active-sites (Fig. S2) (for example Ser-79, Ser-126, Lys-216, Phe-110 and Met-221) that essential to carbapenemase activity [33,34].
The tertiary structure of M19 OXA-23 was modelled based on the crystal structure of 4JF4, which is an OXA-23 from A. baumannii (GenBank accession number CAB69042.1) and which had the highest amino acid similarity with M19 OXA-23 in the Protein Data Bank (Fig. 2). In this model, the hydrophobic tunnel was formed by Phe-110 and Met-221, and had an elongated shape. Meropenem, imipenem and ertapenem were able to traverse the hydrophobic tunnel and bound to similar positions in the tunnel (Fig. 2). Additionally, Phe-110, Lys-216, Thr-217, Met-221 and Arg-259 were the conserved reactive amino acids (Fig. S3).
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2.3 blaOXA-23 is located in the novel transposon Tn6681 in pFM-M19
To evaluate the potential for horizontal transfer of blaOXA-23, the genetic context of blaOXA-23 was investigated. Notably, sequence analysis found that the region containing blaOXA-23 formed a composite transposon with the components ISAba14-HP-ATPase-blaOXA-23-ΔISAba1-ISAba14 (Fig. 3); this novel transposon has been named Tn6681 in the Transposon Registry and GenBank (Accession number: MN081614). Further alignment analysis showed that Tn6681 was highly similar to a chromosome fragment of A. baumannii CBA7, which was isolated in Korea (Accession number: CP020586.1) . However, the blaOXA-23 context region of CBA7 is ISAba10-HP-ATPase-blaOXA-23-ISAba1-ISAba15, which differs somewhat from Tn6681. In addition, two ISAba14 genes, marked as ISAba14L and ISAba14R, were found upstream (3,63,408 bp) of the ISAba10 gene and downstream (78,188 bp) of the ISAba15 gene in the CBA7 chromosome and share 99.91% identity with ISAba14 in Tn6681 (Fig. 3). Given their overall similarity, we propose that Tn6681 and this region of the CBA7 chromosome have the same ancestor.
ISAba14 genes belong to the IS3 family and have been previously identified as part of the active composite transposon Tn2114 in A. baumannii RAB . Analysis of the inverted repeats (IRs) of ISAba1 showed that the right inverted repeat (IRR) of ISAba1 remained only 9 bp and the direct repeat and inverted repeat sequences vanished, but the left inverted repeat (IRL) of ISAba14L shared sequence similarity with IRR of ISAba14R (17/26) in particular the motif TATTT(TG/AT)GCG in their extremities (Fig. 4a). The direct repeat sequences (ATCACTT) of 7 bp were also identified (Fig. 3). This overall structure formed a composite transposon Tn6681, which is a novel blaOXA-23 containing transposon.
Additionally, the arrangement ATPase-blaOXA-23-ISAba1 constitutes a classic genomic organisation found in Tn2008 of A. pittii (GenBank accession number MF078634) and Tn2008B from A. baumannii (GenBank accession number LN877214.1) . In Tn2008, the promoter of blaOXA-23 was overlapped by ISAba1 upstream of the start codon of OXA-23, and both the -10 and -35 regions of this promoter are within the sequence of the ISAba1 gene [37-39]. In Tn6681, the insertion of ISAba14 into ISAba1 generated two ΔISAba1, but the complete -10 and -35 regions of the blaOXA-23 promoter were fully maintained (Fig. 4b), indicating that blaOXA-23 should be expressed normally in M19.
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2.4 Conjugative plasmid pFM-M19 disseminates blaOXA-23 and carbapenem resistance
To evaluate the ability to transfer blaOXA-23 and carbapenem resistance, the conjugation systems of pFM-M19 were analyzed. Components of conjugative machinery were identified in pFM-M19, such as a relaxase; the type IV coupling protein (T4CP) gene (traG) for initiation of conjugation; type IV secretion system (T4SS)-related genes, including the translocation channel protein genes (trbD, trbL, trbF, trbG and trbI); the pilus protein genes (trbC and trbJ) and the ATPase genes (trbE, trbB and traG), indicating that pFM-M19 was a conjugative plasmid (Fig. 5a).
Mating experiments between M19 and E. coli 25DN were carried out. The results revealed that the conjugal transfer efficiency of pFM-M19 from M19 to E. coli 25DN was approximately 1.6 ×10-4 CFU/donor when 8 mg/L meropenem was used as the selective pressure. The MICs of carbapenems in six transconjugants were 20 mg/L (imipenem), 16 mg/L (meropenem) and 4 mg/L(ertapenem), which were weaker than that of the donor strain M19 but much higher than that of strain 25DN (Table 2). PCR analysis was performed to confirm the dissemination of carbapenem resistance via plasmid pFM-M19 and blaOXA-23. The results showed that both pFM-M19 and blaOXA-23 were detected in all transconjugants (Fig. 5b and 5c) and suggested that E. coli 25DN obtained carbapenem resistance due to the acquisition of blaOXA-23 along with pFM-M19.
Insert Table 2