Complete genetic characterization of carbapenem-resistant Acinetobacter johnsonii, co-producing NDM-1, OXA-58, and PER-1 in a patient source

The emergence of carbapenemase-producing Acinetobacter spp. has been widely reported and become a global threat. However, carbapenem-resistant A. johnsonii strains are relatively rare and without comprehensive genetic structure analysis, especially for isolates collected from human specimen. Here, one A. johnsonii AYTCM strain, co-producing NDM-1, OXA-58, and PER-1 enzymes, was isolated from sputum in China in 2018. Antimicrobial susceptibility testing showed that it was resistant to meropenem, imipenem, ceftazidime, ciprofloxacin, and cefoperazone/sulbactam. Whole-genome sequencing and bioinformatic analysis revealed that it possessed 11 plasmids. bla OXA-58 and bla PER-1 genes were located in the pAYTCM-1 plasmid. Especially, a complex class 1 integron consisted of a 5′ conserved segment (5′ CS) and 3′ CS, which was found to carry sul1, arr-3, qnrVC6, and bla PER-1 cassettes. Moreover, the bla NDM-1 gene was located in 41,087 conjugative plasmids and was quite stable even after 70 passages under antibiotics-free conditions. In addition, six prophage regions were identified. Tracking of closely related plasmids in the public database showed that pAYTCM-1 was similar to pXBB1-9, pOXA23_010062, pOXA58_010030, and pAcsw19-2 plasmids, which were collected from the strains of sewage in China. Concerning the pAYTCM-3 plasmids, results showed that strains were collected from different sources and their hosts were isolated from various countries, such as China, USA, Japan, Brazil, and Mexico, suggesting that a wide spread occurred all over the world. In conclusion, early surveillance is warranted to avoid the extensive spread of this high-risk clone in the healthcare setting.


Introduction
Acinetobacter spp.are ubiquitous in nature and are usually identified in the hospital environment, and some of these species have been reported in a variety of nosocomial infections (Wong et al., 2017).The most common species to cause infections is A. baumannii, followed by A. calcoaceticus and A. lwoffii.However, A. johnsonii, a kind of potentially opportunistic pathogen in Acinetobacter spp., generally distributed in natural or nosocomial environments, such as agricultural soil (Wang et al., 2019;Jia et al., 2021).
Carbapenems are the main antimicrobial agents for the treatment of infections with multidrug-resistant Acinetobacter spp., including A. johnsonii (Tang et al., 2020).However, the problem of carbapenem resistance is being increasingly reported, which has contributed to a huge challenge for clinicians (Bonnin et al., 2014).The carbapenem resistance mechanism was usually mediated via enzymatic inactivation (such as carbapenemases), efflux pump overexpression, and target site modification (i.e., altered penicillin-binding proteins) (Mohd Rani et al., 2017;Castanheira et al., 2023).Upon previous studies, more than 210 b-lactamases have been identified in Acinetobacter spp. with class D b-lactamases being the most widespread carbapenemase (Mohd Rani et al., 2017), including OXA-23, OXA-24, and OXA-58 (Liu et al., 2021).Moreover, several insertion sequence (IS) elements such as ISAba1 and ISAba3 could increase the expression of class D b-lactamase genes (including bla OXA-58 -like and bla OXA-23 -like genes) when they were found upstream of these IS elements (Mohd Rani et al., 2017).
Considering the increasing resistance to carbapenems and almost all other antimicrobial agents, Acinetobacter spp.are important resistant microorganisms with a global public health threat, which are associated with severe nosocomial infections including pneumonia, urinary tract, bloodstream, and wound infections (Gonzalez-Villoria and Valverde-Garduno, 2016).However, limited knowledge concerning the carbapenem resistance was known in A. johnsonii strains.Until now, researchers only reported some genome sequences and described the features of A. johnsonii strains which are isolated from the environment, especially in hospital sewage (Feng et al., 2016;Zong et al., 2020).However, little is known about this species which was collected from a patient source in the hospital.Here, we investigated the genetic characteristics of one carbapenem-resistant A. johnsonii, co-producing NDM-1, OXA-58, and PER-1 in a patient's sputum in 2018 in China.To the best of our knowledge, this is the first comprehensive description of one carbapenem-resistant A. johnsonii from a patient source.

Mating experiments
To determine whether the plasmids carrying bla NDM-1 , bla OXA- 58 , and bla PER-1 were transferable, conjugation experiments using E. coli J53 (sodium azide resistant) as the recipient strain were carried out using the filter mating method (Yang et al., 2021).Transconjugants were screened on Mueller-Hinton (MH) agar plates containing sodium azide (100 mg/L) and meropenem (2 mg/L).The identity of putative transconjugants was confirmed via PCR and MALDI-TOF MS.
Stability experiments of plasmids carrying bla NDM-1 , bla OXA-58 , or bla PER-1 genes A. johnsonii AYTCM strain was grown overnight at 37°C in 2 mL of Luria broth (LB) without antibiotics, followed by serial passage of 2-µL overnight culture into the 2-mL LB (1:1,000) each day, with a yield 10 generations, lasting for 7 days (Tian et al., 2022).On the last day, samples were collected and streaked onto antibiotic-free MHA plates.Colonies were selected randomly, and the presence of bla NDM-1 , bla OXA-58 , or bla PER-1 genes was confirmed by PCR with specific primers.

Genome annotations and subsystem categories
Genome was annotated using PGAP and RAST.Based on PGAP annotation, there are 3,980 genes in total, of which 3,731 are proteincoding genes, 136 are pseudo genes, and the remaining 113 are predicted RNA-coding genes.Compared with the PGAP server, 4,182 genes, including 109 RNA-coding genes, belonged to 293 subsystems when annotated using RAST.The statistics of the subsystem is shown (Figure 1).Most of them belonged to metabolism (427), amino acids and derivatives (252), and carbohydrates (130).Additionally, 14 CDS were sorted into "Phages, transposable elements, plasmids" and only 2 and 1 CDS belonged to "cell division and cell cycle" and "dormancy and sporulation," respectively.Functional comparison showed that most subsystems were metabolism among three A. johnsonii strains.However, a huge difference was found in "Phages, Prophages, Transposable elements, Plasmids".There are two CDS that belonged to "Phages, Prophages, Transposable elements, Plasmids" in A. johnsonii C6 and MB44 strains.However, 14 subsystems of this function were identified in A. johnsonii AYTCM strain.
ANI, core-genome phylogeny, lipooligosaccharide outer core, and capsular polysaccharide (KL) According to the ANI analysis, the result showed that 95.82% two-way ANI between A. johnsonii AYTCM and A. johnsonii C6 and 95.86% ANI were found between A. johnsonii AYTCM and A. johnsonii MB44 and only 79.89% two-way ANI between A. johnsonii AYTCM and A. baumannii ATCC 17978.Core-genome phylogeny analysis showed a close genetic relationship among A. johnsonii AYTCM, C6, and MB44 strains.However, a huge diversity was observed among A. baumannii, A. pittii, and other A. seifertii strains based on the phylogenetic tree (Figure S2A).Similar results of SNP difference are shown in Figure S2B.
Kaptive revealed that AYTCM strain contains OC locus 1c (OCL-1c), matching the 92.01%nucleotide identity.The K locus in A. johnsonii AYTCM strain is KL19, to which it matches with an overall nucleotide identity of 72.75%.
Transfer ability and stability of plasmids in the A. johnsonii AYTCM strain Mating assays were performed to explore the transfer ability of bla NDM-1 , bla OXA-58 , and bla PER-1 genes; results showed that only bla NDM-1 could transfer to the recipient strain.The stability assays revealed that all three resistance genes were quite stable even after 70 passages under antibiotics-free conditions.

Genome characterization of the chromosome and 11 plasmids
Hybrid assembly of the short and long reads generated a 3,567,832-bp size circular chromosome with a GC content of 41.60% (Table 1).One intrinsic resistance gene, bla OXA-652 , was identified in the chromosome.Of note, A. johnsonii AYTCM strain carries 11 plasmids, namely, pAYTCM-1 to pAYTCM-11, with sizes between 2,356 bp and 378,197 bp and GC contents ranging from 34.38% to 42.44% (Table 1).Apart from pAYTCM-2, pAYTCM-5, pAYTCM-9, pAYTCM-10, and pAYTCM-11, various kinds of resistance genes were found in other plasmids.Analysis of rep genes showed that only pAYTCM-7 possessed one identified name with Aci1.

Genetic context characterization of pAYTCM-1 multidrug-resistant plasmid
pAYTCM-1 is a huge 378,197-bp multidrug-resistant plasmid with an average GC content of 39.93%.It comprises different regions, including type IV secretion system (T4SS) region, class 1 integron region, and mercury resistance region (Figure 2A).bla OXA- 58 and bla PER-1 genes were located in the pAYTCM-1 plasmid.Concerning the genetic context of bla OXA-58 , three intact and one truncated ISAjo2 were located upstream or downstream.9-bp TSD sequences were observed in the upstream and downstream of ISAjo2 genetic elements.Nevertheless, the TSD sequences were all different (Figure 2B).Importantly, a complex class 1 integron complex consisted of a 5′ conserved segment (5′ CS) and 3′ CS, which was found to carry sul1, arr-3, qnrVC6, and bla PER-1 cassettes (Figure 2C).Of note, 10 XerC and XerD-like binding sites (pdif sites) were found in the pAYTCM-1 plasmid (Table 2).In addition, no oriT was identified in the pAYTCM-1 plasmid and no transconjugants were obtained via conjugation.Moreover, results of the Circoletto tool showed that there were many similar segments  Tian et al. 10.3389/fcimb.2023.1227063Frontiers in Cellular and Infection Microbiology frontiersin.orgbetween pAYTCM-1 and pXBB1-9 (GenBank accession number: CP010351) plasmids (Figure 3).Genetic structure comparison revealed that 98% coverage and 99.91% identity were identified between pAYTCM-1 and pXBB1-9 plasmids, which was found in the A. johnsonii XBB1 isolate from a hospital sewage in 2010 in Chengdu, western China.

Prophage regions in the chromosome
Prophage regions were predicted by the PHASTER tool; results showed two intact, two questionable, and two incomplete regions in the chromosome (Figure 5A).Based on the PHASTER tool, regions 4 and 5 were predicted to be intact due to the score of >90.In addition, regions 1 and 6 were classified as questionable due to the scores of 70-90.However, regions 2 and 3 were shown as incomplete due to the low scores.Gene functions of the two intact and two questionable prophage regions are shown, including attachment, phage integration, and cell lysis (Figure 5B).

Track and characteristics of closely related plasmids in the public database
To track the closely related plasmids from different countries, a wide search was performed via the BacWGSTdb server.Data s h o w e d t h a t p A Y T C M -1 w a s s i m i l a r t o p X B B 1 -9 , pOXA23_010062, pOXA58_010030, and pAcsw19-2 plasmids (Table 3).Their sizes are all >300 kb, and they were collected from the strains of sewage in China.However, the species were various, including A. johnsonii, A. wuhouensis, and A. defluvii.
Concerning the closely related plasmids of pAYTCM-3, results showed that hosts, also carrying the bla NDM-1 -related plasmid, were collected from several different sources, including feces, blood, sputum, pus, sewage, and hospital environment, from 2005 to 2023.These bla NDM-1 -harboring plasmids were all collected in Acinetobacter spp.and not in P. rettgeri and K. pneumoniae.Their hosts were isolated from various countries, such as China, USA, Japan, Brazil, and Mexico.

Discussion
Emergence of carbapenemase-producing Acinetobacter spp.has become dominant in several countries, and it is being increasingly considered a quite important nosocomial pathogen and poses a huge challenge to the healthcare setting (Mohd Rani et al., 2017).Class D b-lactamases (mainly OXA-23), commonly named as OXA, are responsible for carbapenem resistance in Acinetobacter spp.species (Zong et al., 2020).However, the reports of other b- lactamases (e.g., NDM-1) are relatively rare, especially for those with a high resistance level.In this work, NDM-1 and OXA-58 were found in our strain, which leads to a high-level carbapenem resistance.To promote better understanding regarding the genomic features of our A. johnsonii strain, whole-genome sequencing and further RAST software were used to classify the different CDS into subsystems based on their function.Consistent with other A. pittii strains, the majority of the CDS belong to the function of "Metabolism" (Chapartegui-Gonzalez et al., 2022).
Mobile genetic elements (MGEs), including ISs, integrons, and transposons, play a particularly important role in the movement and dissemination of resistance genes (Gorbunova et al., 2021).Concerning the acquisition of the bla OXA-58 gene, many copies of ISAjo2 were identified in the pAYTCM-1 plasmid and located upstream and downstream of bla OXA-58 .Nevertheless, considering the various 9-bp TSD sequences of ISAjo2, we failed to find direct evidence to conclude that bla OXA-58 was embedded into the plasmid via different ISAjo2.Interestingly, XerC/XerD-like recombinase sites (pdif sites) were considered as a new approach for the transfer of carbapenem resistance genes, such as bla OXA-24 , bla OXA-72 , and bla OXA-58 (Merino et al., 2010;Kuo et al., 2016;Liu et al., 2021).Here, 10 pdif sites were identified in the pAYTCM-1 plasmid.Furthermore, we observed that the bla OXA-58 gene was flanked by two pdif sites.Consequently, the bla OXA-58 gene might have been introduced by pdif site-mediated specific recombination.This is consistent with previous research (Feng et al., 2016).Moreover, considering the high coverage and identity with pXBB1-9 (Feng et al., 2016), we deduced that the pAYTCM-1 plasmid may come from a hospital environment-related A. johnsonii isolate XBB1 strain and underwent slight evolution.Another finding in this study is that bla PER-1 was also located in the pAYTCM-1 plasmid.Liu et al. reported that the production of PER-1 in A. baumannii is the key mechanism of cefiderocol resistance (Liu et al., 2022b).However, the MIC of cefiderocol was low and considered as susceptible in our A. johnsonii AYTCM strain.We inferred that the resistance in A. baumannii was caused by species specificity.
In our previous study, we reported that bla NDM-1 was located in the chromosome, which was mediated by two ISAba125-based Tn125 composite transposons, highlighting the importance of Tn125-mediated transfer of bla NDM-1 resistance determinants (Tian et al., 2022).However, we could not find the composite Tn125 transposon in the A. johnsonii AYTCM strain due to that only one copy of ISAba125 was identified.In addition, two studies from Krahn et al. and Abouelfetouh et al. showed that prophages may play a key role in the carbapenem resistance genes, such as bla NDM-1 and bla OXA-23 (Krahn et al., 2016;Abouelfetouh et al., 2022).In addition, a study demonstrated the presence of resistance genes (including mcr-1 and vanA) in the phage fraction and its role on the acquisition and transfer of these resistance genes (Pires et al., 2023).However, the bla NDM-1 gene is not part of any of the prophages.Hence, the relationship of these prophages and the bla NDM-1 gene should be further confirmed through induced experiments.Concerning the bla NDM-1 -harboring plasmids, we discovered that they were located in diverse sources and hosts and in various countries.These data indicated that a wide spread of bla NDM-1 -bearing plasmids has occurred all over the world.However, these plasmids usually transferred among different Acinetobacter species.Concerning the various resistance plasmids in A. johnsonii AYTCM strain, it is revealed that our strain has great potential to capture plasmids that contribute to its resistance.Since our strain is of patient origin, there may be a great possibility that this strain will emerge and further spread between patients and the environment in the hospital.More importantly, Lam et al. reported that the Aci1 plasmid usually was found in extensively and panresistant A. baumannii isolates which belong to global clones GC1 and GC2 (Lam et al., 2023).Here, the Aci1 plasmid has been identified in A. johnsonii strain, further suggesting that the Aci1 plasmid has transferred among various Acinetobacter species.
Apart from resistance determinants, virulence factors should also be paid attention in bacteria.However, the low content of virulence factors in A. johnsonii AYTCM strain is in clear contrast to the high number of resistance genes.Thus, in the surveillance of A. johnsonii, researchers should probably pay more attention to the antimicrobial resistance when compared with virulence.This is a different aspect from the hypervirulent carbapenem-resistant K. pneumoniae (Pu et al., 2023).

Conclusion
This study is the first comprehensive description for the complete genome characteristics of a carbapenem-resistant A. johnsonii, co-producing NDM-1, OXA-58, and PER-1 from a patient source.The A. johnsonii isolate AYTCM carried 11 plasmids, which revealed great genome plasticity for this species, which possesses huge potential to capture resistance plasmids.Moreover, the Aci1 plasmid has been identified in A. johnsonii strain using the current plasmid typing system.However, other eight plasmids failed to type.Therefore, the rep genes for the plasmid typing system need to be further explored.Early surveillance of this kind of carbapenem-resistant isolate is warranted to avoid the extensive spread of this high-risk clone in the healthcare setting.

FIGURE 1
FIGURE 1RAST annotation of A. johnsonii AYTCM strain.The number of each subsystem category is shown on the right of column.
FIGURE 2 Circular map and genetic environment of the pAYTCM-1 plasmid.(A) Circular map of the pAYTCM-1 plasmid.Different filled boxes indicate various open reading frames (ORFs).The GC content and GC skew are shown in the inner rings.Resistance genes (red filled boxes), mobile genetic elements (yellow filled boxes), T4SS region (green filled boxes), and mercury resistance region (purple filled boxes).Light blue represents other ORFs.(B) Genetic environment of the bla OXA-58 gene.The red filled arrow indicates the position of the bla OXA-58 gene.Blue filled arrows indicate ISAjo2 and ISAjo2D.Purple filled arrow indicates ISAlw15.Arrows' directions indicate the ORF directions.9-bp target site duplications (TSD) are shown upstream and downstream of ISAjo2 and ISAjo2D using white or red filled circles, respectively.(C) Structure of the class 1 integron containing bla PER-1 .intl1 is shown as a black filled box.attl is shown as a white filled box.The 5′ conserved segment (5′ CS) and 3′ CS of class 1 integron are labeled.The various kinds of resistance genes were shown as different colors with the names labeled above with the orientation indicated by thin black arrows.

FIGURE 4
FIGURE 4 Circular map of pAYTCM-3.Different filled boxes indicate various open reading frames (ORFs).The GC content and GC skew are shown in the inner rings.Resistance genes are shown as a red filled box.Mobile genetic elements are shown as a yellow filled box.The T4SS region and T4CP are indicated as a green filled box.The relaxase-encoding gene is shown in the purple filled box.Light blue represents other ORFs.Moreover, the position of oriT was also labeled.
FIGURE 5 Predicted prophage regions within the A johnsonii AYTCM chromosome.(A) Six prophage regions positions in the chromosome.Green filled boxes mean the intact prophage regions (score > 90), filled boxes mean the questionable prophage regions (score 70-90), filled boxes mean the incomplete prophage regions (score < 70).(B) Structure of two intact and two questionable prophage regions.Genes are colored based on predicted functions.

TABLE 1
Molecular characterization of the genome of A. johnsonii AYTCM strain.
ND, not detected.