Whole-Genome Analysis of Two Copies of bla_NDM-1 Gene Carrying Acinetobacter johnsonii Strain Acsw19 Isolated from Sichuan, China

Introduction

Producing of carbapenemases, including the β-lactamases of Ambler classes A, B (metallo-β-lactamases) and D, are the most common mechanism of bacterial carbapenems resistance.^1–3 Especially, the New Delhi Metallo-β-lactamase (NDM), Klebsiella pneumoniae carbapenemase (KPC) and some Class D β-lactamases (CHDLs) have been identified worldwide in gram-negative bacterial isolates from clinical, environmental samples, and food animals, especially in Enterobacteriaceae,^1,4–9 and also in Pseudomonas aeruginosa and Acinetobacter species.^10–12 Carbapenem-resistant Acinetobacter species are mainly associated with the carbapenem-hydrolyzing NDM and CHDLs, such as bla_OXA-23-like, bla_OXA-24/40-like, bla_OXA-51-like, bla_OXA-134-like and bla_OXA-211-like gene.^13–18 Especially, co-carrying of the bla_NDM and bla_OXA in clinical, food, environmental derived isolates of Acinetobacter species were prevalent in the world.^11,19-21 In addition, the genes encoding bla_NDM and bla_OXA are known to be carried on some mobile genetic elements that inserted into the chromosome or plasmids, and it is suspected the mechanism of horizontal gene transfer (HGT) promotes the exchange of resistance genes among pathogenic microorganisms isolated from the clinical, environmental samples, and food animals.^11,22–25

In this study, we mainly characterized the genetic feature of a carbapenems resistant strain Acinetobacter johnsonii Acsw19 isolated from municipal sludge. This strain was found to carry two copies of bla_NDM-1, cmlB1-like gene, and bla_OXA-211-like gene along with other 8 antimicrobial resistance genes, three plasmids, 15 genomic islands and 8 prophages.

Materials and Methods

Bacterial Isolate, Identification and Antimicrobial Susceptibility Testing

The sample of municipal sewage was obtained from the influx of wastewater-related plant in Luzhou City (Sichuan Province, China) in March 2019. The sewage was 1:10 diluted and an aliquot (10 μL) was streaked onto a CHROM Agar Orientation (CHROMAgar, Paris, France) agar plate containing 2 mg/L meropenem (Solarbio, China) and then incubated at 37 °C overnight. Bacterial species identification was carried out by the Vitek2 system (BioMérieux, France), 16srRNA sequencing and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The minimal inhibitory concentrations (MICs) of 15 antimicrobial agents (Solarbio, China) including meropenem, imipenem, cefepime, cefotaxime, ceftazidime, piperacillin-tazobactam, amoxicillin-clavulanic acid, gentamicin, amikacin, aztreonam, erythromycin, chloramphenicol, sulfadiazine, colistin and ciprofloxacin were determined by broth microdilution method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI 2013, M100-S23). Escherichia coli strain ATCC 25,922 was used as quality control. Polymerase chain reaction (PCR) amplification and DNA sequencing were performed to identify the key carbapenemase-encoding genes (bla_NDM and bla_KPC) as previously reference.⁷

Whole-Genome Sequencing and Analysis

Genomic DNA of the strain A. johnsonii Acsw19 was extracted using the DNA Kit (QIAGEN, Germany). The 10kb sequencing library and a 300 bp paired-end library were constructed using the standard PacBio RS sample and Illumina DNA sample preparation instructions, and then sequenced on Pacific Biosciences RS II and MiSeq systems sequencing platforms (Novogene, China). The reads were de novo assembled using the software Celera Assembler (version 8.0). Gene prediction was performed for the whole genome with Glimmer 3.02 (http://www.cbcb.umd.edu/software/glimmer/).²⁶ And the annotation of the Acsw19 genome was achieved using the NCBI Prokaryotic Genome Annotation Pipeline. Pairwise alignment was performed by BLASTn search (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The average nucleotide identity (ANI) analysis was performed by the computer.²⁷ The resistome was identified using ResFinder 2.1 (https://cge.cbs.dtu.dk/services/ResFinder/)²⁸ (minimum threshold for identity, 85%; minimum coverage, 60%) and Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca/). The genomic island sequences were predicted based on three different genomic islands (GIs) prediction software (IslandPATH-DIMOB, IslandPick, and SIGI-HMM)^29–31 and the Prophage was predicted by using phiSpy.³²

Results and Discussion

Bacterial Isolate, Identification and Resistance Gene Detection

A gram-stain-negative, bla_NDM-1 and bla_OXA producing Acinetobacter johnsonii Acsw19 was isolated and identified by the Vitek2 system (BioMérieux, France), 16srRNA sequencing and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry in Luzhou City, Southwestern China. Acinetobacter johnsonii strain Acsw19 was resistant to meropenem, imipenem, cefepime, cefotaxime, ceftazidime, piperacillin-tazobactam, amoxicillin-clavulanic acid, gentamicin, amikacin, aztreonam, erythromycin, chloramphenicol and sulfadiazine and it was susceptible to colistin and ciprofloxacin according to CLSI breakpoints (M100-S23) (Supplementary Table 1). To the best of our knowledge, the occurrence of multiple antibiotic genes in the multi-drug resistant bacterial isolates from sewage has been evidenced in numerous studies, with the involvement of numerous species.^33,34

Characterization of the Whole Genome of Acinetobacter johnsonii Strain Acsw 19

We got 1,876,876,663 bp data of the whole genome by the WGS. The genome of Acinetobacter johnsonii Acsw19 consisted of a 3,433,749bp circular chromosome and three circular plasmids, pAcsw19-1, pAcsw19-2 and pAcsw19-3 in the size of 11,161bp, 351, 885bp and 38, 391 with the 35.66%, 38.79% and 33.76% G+C content, respectively. The chromosome has a 41.78% G+C content, 21 rRNA operons, 88 tRNAs, 44ncRNAs and 3413 predicted protein coding sequences (CDSs) (Table 1).

Table 1 Characteristic of the Whole Genome of Acinetobacter johnsonii Acsw19

Resistome Analysis

A total of 12 drug-resistance genes was detected in the whole genome of Acsw19. Specifically, 6 resistance genes [(bla_NDM-1, bla_OXA-211-like, aph(3")-Ib (strB), aph(6)-Id (strA), sul2, and floR)] were located in the chromosome genome and 6 [cmlB1-like, mph(E), msr(E), bla_OXA-58, bla_NDM-1 and aph(3ʹ)-VIa] in plasmid pAcsw19-2 (Table 2). None of the drug-resistance gene was detected in plasmid pAcsw19-1 and pAcsw19-3. The full lengthen of cmlB1-like gene consisted of 1266 nucleotides encoding a protein with 422 amino acids. Sequence analysis showed that the cmlB1-like gene shared 86.33%, 71.7% and 71.9% sequence similarities with the known cmlB1, cmlA4, and cmlA8 genes at nucleotide level and 67.8%, 44.8% and 44.5% at amino acid level, respectively. The full lengthen of bla_OXA-211-like gene consisted of 825 nucleotides encoding a protein with 275 amino acids. Sequence analysis showed that the bla_OXA-211-like gene shared 94.4%, 95.39%, 95.52%, 96% and 96.36% identity with the known bla_OXA-211, bla_OXA-643, bla_OXA-281, bla_OXA-645 and bla_OXA-652 gene at nucleotide level and 94.4%, 95.4%, 95.5%, 96% and 96.4% at amino acid level, respectively. Phylogenetic analysis showed that the bla_OXA-211-like gene and cmlB1-like gene had the closest evolutionary relationship with bla_OXA-643 (Figure 1) and cmlB1 (Figure 2). These results indicated that the bla_OXA-211-like and cmlB1-like genes maybe two new allelic variant of the gene bla_OXA and cmlB1. To our the knowledge, there are more new allelic variant of the resistance genes have been found from the sewage derived isolates.^33,35,36

Table 2 Distribution of the Resistance Genes in Acinetobacter johnsonii Strain Acsw19

Figure 1 Molecular phylogenetic analysis by maximum likelihood method with 1000 bootstraps of OXA-211 like (a novel variant of OXA-211 family) with the six Class D β-lactamases (CHDLs) OXA family (OXA-23, OXA-24/40, OXA-235/134, OXA-58, OXA-48, OXA-211) representative sequences retrieved from GenBank database. The black triangle was indicated the gene which was found in this study.

Figure 2 Molecular phylogenetic analysis by maximum likelihood method with 1000 bootstraps of CmlA/FloR family chloramphenicol efflux MFS (cmlB1-like) with the eight CmlA/FloR family chloramphenicol efflux MFS representative sequences retrieved from GenBank database. The black triangle was indicated the gene which was found in this study.

Characterization of Three Plasmids

Plasmid pAcsw19-1 contained 18 putative coding open reading frames (ORFs). Sequence analysis showed that pAcsw19-1 had 100%, 92%, 68% and 68% query cover and 96.55%, 91.5%, 87.8% and 87.8% sequence similarities with the plasmids p2_010062 (CP033122), p4_010055 (CP032283), p3_010030 (CP029391) and pALWEK1.4 (CP032107) at nucleotide level, and these reported plasmids were all harbored by the Acinetobacter species. None of antimicrobial resistance gene was determined in plasmid pAcsw19-1. Plasmid pAcsw19-1 carried two copies of plasmid replicons, two mobilization proteins (mobL-like), and several hypothetical ORFs. Complete sequence analysis showed that the 351,885 bp plasmid pAcsw19-2 contained 383 putative coding ORFs. Plasmid pAcsw19-2 had 87.34%, 14.76% and 7.06% query cover and 99.28%, 99.78% and 100% sequence similarities with the plasmids pXBB1-9 (CP010350), pACI-df08 (CP026426)³⁷ and pM131-2 (JX101647) at nucleotide level, respectively.¹¹

Plasmid pAcsw19-2 carried 6 resistance genes [cmlB1-like, mph(E), msr(E), bla_OXA-58, bla_NDM-1, and aph(3ʹ)-VIa] which were distributed in three regions. The first region carried the cmlB1-like gene. The genetic context of this region was ISL3-like transposase-cmlB1-like-LysR (Figure 2). This region was similar to the corresponding area of plasmid pOXA58_010030 (CP029396), pOXA58_010055 (CP032285), and pXBB1-9 (CP010351). The second region carried three resistance genes [mph(E), msr(E), and bla_OXA-58]. The first two resistance gene mph(E) and msr(E) were carried by the genetic context which was constituted by IS30 family transposase, helix-turn-helix-ORF, mph(E), msr(E), helix-turn-helix-ORF, plasmid stability associated protein-coding ORFs (addiction module antidote protein, RelE/ParE, brnA/T, and LysE family), ISAba1, and AraC transcriptional regulator gene. The AraC was linked to the third resistance gene bla_OXA-58 carrying area which was constituted by ISAba3-bla_OXA58-ISAba3 (Figure 3). The context of bla_OXA-58 carrying area (ISAba3-bla_OXA58-ISAba3) was same to the previous reports.^11,38-40

Figure 3 Schematic map of the genetic context of resistance gene regions in pAcSW19-2. The resistance genes are indicated by red arrows, the mobile genes are indicated by the green arrows and other function genes are indicated by the purple arrows. Gray areas between open reading frames (ORFs) denote nucleotide identities with the similarity context.

The third region carried 2 resistance genes [plasmid borne bla_NDM-1 and aph (3ʹ)-VIa]. Nucleotide sequence analysis revealed that the bla_NDM-1 gene was flanked in the upstream region of IS5 transposase-dmc-unknown ORF-umuD-Y-family DNA polymerase-unknown ORF-trpF-ble_MBL and downstream by the IS30 family transposase and 6 unknown ORFs carried region, which linked to the resistance gene aph(3ʹ)-Via carrying genetic context [ISAba125-aph(3ʹ)-Via -ISAba125]. This resistance area (ISAba125-aph(3ʹ)-Via-ISAba125) was similar to the corresponding region of the plasmid pAP43-OXA58-NDM1 (CP043053), which was harbored by Acinetobacter pittii.

The plasmid pAcsw19-3 contained 58 putative coding ORFs. Sequence analysis showed that pAcsw19-3 had 72%, 76%, and 83% query cover and 99.90%, 99.96% and 99.84% sequence similarities with the plasmids p3_010055 (CP032282.1), p2_010030 (CP029390), and p4_010060 (CP031712) at the nucleotide level, and these plasmids were all harbored by the Acinetobacter species strains. None of the antimicrobial resistance gene was determined in plasmid pAcsw19-3, either.

Characterization of the Genomic Islands (GI)

Fifteen genomic islands, named GI_Acsw19-1 to GI_Acsw19-15, were identified by the software IslandPATH-DIMOB, IslandPick and SIGI-HMM. Sequence analysis showed that the length of the 15 genomic islands were ranged from ~5.1 kb to ~94.86 kb with the average G+C context of 32.59% to 54.18%, respectively. Moreover, 13 genomic islands (GI_Acsw19-1 to GI_Acsw19-13) were located in the chromosome and 2 genomic islands (GI_Acsw19-14 and GI_Acsw19-15) in the plasmid pAcsw19-2. Among the 15 GIs, two (GI_Acsw19-11 and GI_Acsw19-12) were the resistant genomic islands. Genomic island GI_Acsw19-11 (26,133 bp) carried the aminoglycosides resistance genes strA and strB, sulphonamides resistance gene sul2 and phenicol resistance gene floR. Genomic island GI_Acsw19-12 (12,309 bp) carried the chromosome borne bla_NDM-1 (Table 3). Sequence analysis showed that GI_Acsw19-11 had 47%, 37%, and 34% query cover and 100% sequence similarities with the DNA sequence of A. baumannii MRSN15313 chromosome genome (CP033869), A. pittii WCHAP100004 plasmid pOXA58_100004 (CP027249), and uncultured bacterium HHV216 plasmid pHHV216(FJ012880). Based on the specific genetic content, GI_Acsw19-11 could be divided into two regions (regions A and regions B) (Figure 4). The region A was 15,713bp in length which served as the backbone of GI_Acsw19-11. It mainly carried the tyrosine-type recombinase/integrase gene (1137 bp) and an IS1 family tnpA, which might be responsible for encoding the site-specific resolvase and transposition. Additionally, the other genes of region A, including ferrous iron transporter gene feoAB, Cd(II)/Pb(II)-responsive transcriptional regulator encoding gene cadR, hydrolase encoding gene, some of hypothetical protein-encoding genes, were found to be located in the backbone. Sequence of region A was high similar to the corresponding region of other various Acinetobacter species.⁴¹ The 4 resistance genes (strA and strB, sul2 and floR) carried region B was 8980 bp in length (Figure 3). These four resistance genes genetic context is ISAba1-sul2-glmM-ISVsa3-LysR-floR-DUF3363-strB-strA-ISAba1. The sul2 and floR carrying area (6814 bp) was similar to the corresponding region of plasmid pOXA58_100004, while the strB and strA carrying area (2832 bp) was similar to corresponding region of plasmid pNDM-AP_882 harbored by the A. pittii AP_882 (CP014478.1).

Table 3 Overall Features of the Acinetobacter johnsonii Strain Acsw19 Genomic Islands

Figure 4 Schematic map of the genetic context of the GI_Acsw19-11. The resistance genes are indicated by red arrows, the mobile genes are indicated by the green arrows and other function genes are indicated by the purple arrows. Gray areas between open reading frames (ORFs) denote nucleotide identities with the similarity context.

Sequence analysis showed that GI_Acsw19-12 had 96%, 96%, and 96% query cover and 100%, 99.9%, and 99.9% sequence similarities with the corresponding region of sequences of A. baumannii AR_0083 genome (CP027528), A. pittii ST220 genome (CP029610), and A. baumannii AR_0083 genome (CP027528). GI_Acsw19-12 had 82%, 82%, and 84% query cover and 100%, 100%, and 99.9% sequence similarities with the plasmids including the pNDM1_060092 (CP035935), pNDM-GJ01 (KT965092)⁴² and pNDM-JVAP01(KM923969)⁴³ at nucleotide level. Moreover, the chromosome borne bla_NDM was located in the genetic element (10,023 bp) ISAba125-IS91-family tnpA-groL-groES-cutA-tat-trpF-ble-bla_NDM-1-ISAba125 (Figure 5). This bla_NDM carrying element was similar equal to the corresponding region of the pNDM1_010045.

Figure 5 Schematic map of the genetic context of the GI_Acsw19-2. The resistance genes are indicated by red arrows, the mobile genes are indicated by the green arrows and other function genes are indicated by the purple arrows. Gray areas between open reading frames (ORFs) denote nucleotide identities with the similarity context.

Characterization of Prophages

A total of eight prophages, named Pp_Acsw19-1 to Pp_Acsw19-8, was identified by phiSpy (Table 4). Sequence analysis showed that one prophage (Pp_Acsw19-1) was located in the plasmid pAcsw19-2 and 7 prophages (Pp_Acsw19-2 to Pp_Acsw19-8) were located in the chromosome genome. The lengthen of eight prophages was ranged from ~17.22 kb to ~159 kb with average G+C context of 40.03% to 47.12%, respectively. Interestingly, the bla_NDM-carrying genomic islands GI_ Acsw19-12 was located in the prophage Pp_Acsw19-7 and the cmlB1-like gene carrying prophage Pp_Acsw19-1 was located in the plasmid pAcsw19-2. These findings suggested that some mobile genetic elements can be excised from chromosome or mobile genetic elements and then integrated into the chromosome or other mobile genetic elements.^44–47 These situations may promote the exchange of resistance genes among pathogenic microorganisms.

Table 4 Overall Features of the Acinetobacter johnsonii Strain Acsw19 Prophage

Conclusions

The current study characterize the complete genome sequence of Acinetobacter Acsw19, which carried two copies of bla_NDM-1, cmlB1-like gene, bla_OXA-211-like gene along with other 8 antimicrobial resistance genes in 3 plasmids, 15 genomic islands and 8 prophages, which could provide great genetic plasticity for the host dissemination of antimicrobial resistance. The occurrence of multiple antibiotic genes in bacterial isolates from sewage has been evidenced in numerous studies, with the involvement of numerous species, it may be serving as an important reservoir of resistance genes and a hot spot for the transfer of resistance genes and mobile genetic elements. We should be vigilant these isolates or resistance genes transfer to the clinical bacterial.