First report of the chromosomal integration of carbapenemase gene blaIMP-19 in Acinetobacter baumannii AB322: the legacy of integron in phage-plasmid?

ABSTRACT Integration of carbapenemase gene blaIMP into the chromosome of carbapenem-resistant Acinetobacter baumannii (CRAB) has not been reported. The aim of this study was to explore the genomic characteristics of CRAB AB322 isolated from a Taiwanese patient diagnosed with bacteremia in 2011, whose chromosome harbors blaIMP-19. Disk diffusion and broth microdilution were employed to analyze the antimicrobial susceptibility of AB322 to 14 antimicrobials. Nanopore whole-genome sequencing platform was utilized for AB322 genome sequencing, and conjugation was further performed to investigate the transferability of blaIMP-19 to amikacin-resistant A. baumannii 218 (AB218) and Acinetobacter nosocomialis 254 (AN254). The results showed that AB322 was classified as multidrug-resistant A. baumannii but remained susceptible to ampicillin/sulbactam, colistin, and tigecycline. Whole-genome sequencing revealed the AB322 genome, consisting of a 4,098,985-bp chromosome, a 71,590-bp conjugative plasmid named pAB322-1, and an 8,726-bp plasmid named pAB322-2. Multilocus sequence typing analysis indicated that AB322 belonged to sequence type 1. AB322 chromosome harbored numerous acquired antimicrobial resistance genes, including aph(3′)-Ia, aadA1b, aadA1, aac(6′)-Ib3, aac (3)-Ia, blaADC-25, blaOXA-69, blaIMP-19, catA1, sul1, and tet(A), conferring resistance to β-lactams, aminoglycosides, chloramphenicol, sulfamethoxazole, and tetracyclines. Moreover, blaIMP-19 was identified to be situated within class 1 integron In240 and an incomplete PHAGE_Salmon_SJ46_NC_031129 on AB322 chromosome. However, conjugation experiments revealed that blaIMP-19 could not be transferred to AB218 and AN254 in our testing conditions. In conclusion, we first report the presence of chromosomal-integrated blaIMP-19 in CRAB, possibly mediated by integron. The future dissemination of blaIMP-19 among different species, leading to carbapenem resistance dissemination, requires close monitoring. IMPORTANCE The horizontal transfer of antimicrobial-resistant genes is crucial for the dissemination of resistance, especially as Acinetobacter baumannii has emerged as a clinically significant pathogen. However, in this study, we first report the integration of the blaIMP-19 gene into the chromosome of A. baumannii, and such horizontal transfer may be associated with integron-phage elements. Additionally, it is possible that these DNA fragments carrying antimicrobial-resistant genes could further spread to other pathogens by moving horizontally onto conjugative plasmids.

Carbapenems, commonly employed to combat life-threatening infections caused by multidrug-resistant (MDR) A. baumannii, face a growing challenge with increasing proportions of carbapenem-resistant A. baumannii (CRAB).Apart from porin dysfunction and efflux pump overexpression (1,2), carbapenemase genes present on the bacterial chromosome and plasmids are frequently linked to mobile genetic elements [including insertion sequences (ISs, part of transposon) and integrons], contributing to carbapenem hydrolysis and resistance (3,4).bla NDM-1 is the most prevalent carbapenem resistance gene identified in CRAB, followed by oxacillinase genes (OXAs, class D β-lactamases) and bla IMP-1 (4).Furthermore, these carbapenemase genes in CRAB are primarily dissemina ted through plasmid-mediation with prevalent genes being bla OXA-24 , bla OXA-58 , bla NDM , and bla GES .In contrast, bla NDM dominates in the occurrence of carbapenemase genes on CRAB chromosomes (4).
bla IMP-19 was initially identified in Enterobacter cloacae and Pseudomonas aeruginosa from Japan.Subsequently, it was found in an Aeromonas caviae isolate in France (9) and, more recently, in Achromobacter xylosoxidans and Acinetobacter spp.isolates from Japan (10,11).In a 2011 study by Yamamoto et al., it was noted that bla IMP-19 could be transmitted via plasmids between A. baumannii, Acinetobacter junii, and genospecies 3 (11).Subsequently, they reported the widespread distribution of Acinetobacter pittii ST119 harboring bla IMP-19 throughout the Kyoto-Shiga region in Japan (12).However, existing literature has primarily emphasized the plasmid-mediated dissemination of bla IMP-19 .In this study, we report the first occurrence of bla IMP-19 in A. baumannii and discover the integration of bla IMP-19 into the chromosome.
We further explored the integration of the bla IMP gene into the chromosomes of strains using the NCBI nucleotide database for BLAST analysis.The results revealed that, in addition to AB322, 55 other strains had the bla IMP integrated into their chromosomes (Tables S4 and S5).We found two strains of P. aeruginosa with duplicated integrons in their chromosomes: strain HPA0875 harbored In887, carrying aadA1, bla OXA-1 , and bla IMP-6 , while strain PA99 carried In994, containing bla IMP-1 (Table S5).On the chromo some of K. pneumoniae CPO109, there are two distinct integrons.In240 carried bla IMP-4 and catB3, while In498 harbors bla IMP-4 , catB3, and emrB.Meanwhile, three strains of P. aeruginosa, HPA0044, HPA0384, and HPA1406, each carry two bla IMP -integrons on their chromosomes, In498 and In887, and both integrons contain ARGs aadA1, bla OXA-1 , and bla IMP-6 .However, Morganella morganii N18-00103, Proteus mirabilis N18-00201, Providencia stuartii 2021CK-01196, and P. stuartii 2021CK-01296 all carried the bla IMP-27 gene on their chromosomes, but these genes were not located within integrons.In summary, In498 is the most common bla IMP -integron, not only in P. aeruginosa.Additionally, bla IMP-79 is the most common chromosomally encoded bla IMP gene (Table S4).Krahn et al. previously reported that the intraspecies transfer of the chromosomal A. baumannii bla NDM-1 gene might occur through phage-mediated transmission (15).Moreover, Pfeifer et al. showed that phage-plasmids obtained from clinical environments have substantiated their ability to infect susceptible strains, leading to the development of antimicrobial resistance (16).Our genome sequence analysis revealed five intact, one questionable, and three incomplete prophages in AB322.The bla IMP-19 gene was located in region 7 (incomplete Salmonella phage SJ46) (Table S6; Fig. S2).We analyzed the distribution of phage SJ46 (accession number NC_031129.1)among different bacteria using the NCBI database.Among the 90 identified sequences of SJ46, 88 were found to belong to plasmids.SJ46 was only detected in two chromosomes (Escherichia coli strains ST2747 and RM-096-CS), supporting the possibility of movement between chromosomes and plasmids of SJ46.
Conjugation is another pathway for genetic material transfer, and pAB322-1 possesses a type F conjugation system.Therefore, the liquid mating-out assay (5) was performed to determine the transferability of bla IMP-19 gene from AB322 isolates to amikacin-resistant A. baumannii 218 (AB218) and Acinetobacter nosocomialis 254 (AN254).However, we were unable to obtain transconjugants in our tested conditions.
We report for the first time the presence of bla IMP-19 in A. baumannii chromosome, and the bla IMP-19 gene may integrate into the chromosome via integron-plasmid-phage mediation, eventually evolving into an incomplete phage.The concern lies in dissemi nating ARGs facilitated by phage-plasmids, as it circumvents the need for cell-to-cell contact essential in traditional plasmid transfer by conjugation.However, the mechanism of integrated carbapenemase in pathogens still needs further investigation.Moreover, it is urgent to monitor the spread of A. baumannii ST1 isolates carrying bla IMP-19 on chromosomes in the environment and their evolution towards co-occurring with multiple ARGs.

FIG 1 (
FIG 1 (A) Comparative genomics of 10 chromosomes closely related to AB322.BRIG diagram illustrates homologous chromosome segments with the AB322 genome as a reference, highlighting the presence of bla IMP-19 .(B) Detailed depiction of the neighboring genomic region from 2,996,000 to 3,007,000 of bla IMP-19 within the AB322 chromosome was performed using the DNA features viewer in Python libraries.The bla IMP-19 gene (from 3,000,573 to 3,001,313) was annotated, and its representation was visually highlighted with a red coloration.