Genomic analysis of CTX-M-115 and OXA-23/-72 co-producing Acinetobacter baumannii, and their potential to spread resistance genes by natural transformation

Abstract

Objectives

To characterize Acinetobacter baumannii strains co-producing the ESBL CTX-M-115 and carbapenem-hydrolysing class D β-lactamases (CHDLs), and to assess the potential diffusion of their resistance genes by horizontal transfer.

Methods

Nineteen CTX-M-115/CHDL-positive A. baumannii were collected between 2015 and 2019 from patients hospitalized in France. Their whole-genome sequences were determined on Illumina and Oxford Nanopore platforms and were compared through core-genome MLST (cgMLST) and SNP analyses. Transferability of resistance genes was investigated by natural transformation assays.

Results

Eighteen strains were found to harbour CHDL OXA-72, and another one CHDL OXA-23, in addition to CTX-M-115, narrow-spectrum β-lactamases and aminoglycoside resistance determinants including ArmA. cgMLST typing, as well as Oxford Scheme ST and K locus typing, confirmed that 17 out of the 18 CTX-M-115/OXA-72 isolates belonged to new subclades within clonal complex 78 (CC78). The chromosomal region carrying the bla_CTX-M-115 gene appeared to vary greatly both in gene content and in length (from 20 to 79 kb) among the strains, likely because of IS26-mediated DNA rearrangements. The bla_OXA-72 gene was localized on closely related plasmids showing structural variations that occurred between pdif sites. Transfer of all the β-lactamase genes, as well as aminoglycoside resistance determinants to a drug-susceptible A. baumannii recipient, was easily obtained in vitro by natural transformation.

Conclusions

This work highlights the propensity of CC78 isolates to collect multiple antibiotic resistance genes, to rearrange and to pass them to other A. baumannii strains via natural transformation. This process, along with mobile genetic elements, likely contributes to the considerable genomic plasticity of clinical strains, and to the diversity of molecular mechanisms sustaining their multidrug resistance.

Introduction

The opportunistic pathogen Acinetobacter baumannii is an emerging cause of nosocomial infections worldwide. Because of the diffusion of MDR strains at the global level, this member of the ESKAPE group of hard-to-treat bacterial species is considered by the WHO as a priority for the development of novel antimicrobial strategies.^1–3 For instance, high rates of MDR A. baumannii are consistently reported in some European countries.⁴

The resistance of A. baumannii strains to carbapenems most often results from acquisition of Ambler’s class D (CHDLs, carbapenem-hydrolysing class D β-lactamases) and class B (MBL) β-lactamases, while the resistance to broad-spectrum cephalosporins is essentially due to IS-driven overexpression of intrinsic class C β-lactamase ADC.^5,6 Transferable ESBLs seem to be less common in A. baumannii; those belonging to PER-, GES- and VEB-types being more prevalent than those of the TEM-, SHV- and CTX-M-types.^7–14 In contrast to Enterobacterales, very few strains have been found to produce enzymes such as CTX-M-2 (Japan, USA), CTX-M-15 (India, Haiti) and CTX-M-43 (Bolivia), respectively.¹⁵ Conversely, the CTX-M-115 variant has been detected almost exclusively in A. baumannii.^16–20 This ESBL of the CTX-M-2 group differs from the prototype enzyme by three amino acid changes (V251I, I279V and G290S) and from the progenitor KluA-1 (accession number AJ272538) harboured by Kluyvera ascorbata by a single substitution (G290S). Its encoding gene, bla_CTX-M-115, was detected in A. baumannii isolates from Russia, the USA, Germany and Brazil.^16–19 All the reported strains were found to belong to the clonal complex 78 (CC78), known to be part of the ‘Italian’ international clone 6 that caused outbreaks in Mediterranean hospitals.²¹ However, limited information was provided on the genetic environment and support of bla_CTX-M-115.

Conjugation-mediated exchanges of plasmids are considered as key drivers of resistance gene diffusion among bacteria.²² However, recent studies have pointed to the somewhat neglected role of natural transformation in these molecular cross-talks.^23,24A. baumannii is able to capture exogeneous DNA fragments under certain conditions, thanks to specific genes required for DNA uptake.^25,26 Some of these encode type IV pili while others like comEC are thought to determine a DNA uptake channel.²³ Recently, it was demonstrated that A. baumannii can acquire foreign DNA during a specific time frame that correlates with the production of external appendages involved in the DNA acquisition process, especially type IV pili.²⁷

The present work reports on the characterization of 19 CTX-M-115/CHDL-positive A. baumannii clinical strains referred to the French National Reference Center for Antibiotic Resistance (NRC-AR) between 2015 and 2019. The genotypic relatedness of the isolates, the genetic environment and support of β-lactam resistance genes, as well as the mechanisms by which these latter can be horizontally transferred were investigated.

Materials and methods

Bacterial strains and antimicrobial susceptibility testing

The 19 CTX-M-115-producing A. baumannii isolates were collected between February 2015 and November 2019 by French medical laboratories and were referred to the NRC-AR for a detailed analysis of their resistance mechanisms to β-lactams. Strains used for cloning and transformation experiments are detailed in Table S1 (available as Supplementary data at JAC Online). The bacterial strains were grown aerobically on Mueller–Hinton agar (MHA; Bio-Rad, Marnes-la-Coquette, France) or in Mueller–Hinton broth (MHB; Becton Dickinson, Heidelberg, Germany) at 35 ± 1°C.

Bacterial susceptibility to antibiotics was determined by the disc diffusion method with MHA plates, and was interpreted according to the CLSI guidelines.²⁸ MICs of amoxicillin/clavulanic acid, ampicillin/sulbactam, ceftriaxone and cefotaxime were determined by using gradient tests (Etest; BioMérieux, Marcy-l’Etoile, France), and those of other antibiotic molecules were determined by broth microdilution with customized 96-well microplates (Sensititre; ThermoFisher Scientific, Villebon-sur-Yvette, France).

WGS analyses

Total bacterial DNA was purified from overnight cultures by using the PureLink^TM Genomic DNA Mini Kit (ThermoFisher Scientific) and was then quantified on a NanoDrop spectrophotometer (Ozyme, Saint-Quentin-en-Yvelines, France). WGS was carried out on an Illumina NextSeq platform with v2 chemistry using 150 bp paired-end reads (Microsynth, Balgach, Switzerland). The Fastp v0.19.10 was used for quality filtering of Illumina reads and SPAdes for short-reads assembly.^29,30 Long reads were obtained from four selected strains (AbCTX1, AbCTX5, AbCTX9 and AbCTX13) using the ligation sequencing kit 1D SQK-LSK109 with the barcoding extension kit EXP-NBD 104 according to the Oxford Nanopore Technologies protocols (Oxford, UK). A flowcell FLO-MIN106 connected to the MinION sequencing device was used to sequence the library for 48 h. Real-time base calling of MinION reads was performed with the MinIT and integrated into Guppy software to produce fastQ files. The long reads were de novo assembled with the Illumina short reads using Unicycler v.0.4.7.³¹ Genome annotation was performed by the National Center for Biotechnology Information prokaryotic genome annotation pipeline (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/). The total raw sequence data of A. baumannii isolates were uploaded to the ResFinder server for resistance gene identification, with a sequence identity threshold set at 90% (https://cge.cbs.dtu.dk/services/ResFinder/). Bioinformatic analyses were conducted with CLC Genomic Workbench v10.1.1 (Qiagen, Courtaboeuf, France). The ST of A. baumannii strains (https://pubmlst.org/abaumannii/) were determined according to both the Oxford (ST^Ox) and Pasteur (ST^Pas) schemes.^32–34 K locus (KL) typing was assessed using the Kaptive’s web interface (https://kaptive-web.erc.monash.edu/). Comparison of the strains by core-genome MLST (cgMLST) was performed according to the scheme developed by Higgins et al.,³⁵ while SNPs were identified with Snippy (v4.4.5) set as follows: minimum depth at SNP position = 10, minimum distance between SNPs = 11 bp. Snippy was also used to compare the chromosomal regions flanking the transferred genetic element in the transformants to those of the recipient strain. The pdif sites in plasmids were screened by BLAST software by identifying the XerD binding site (5′-ATTTAACATAA-3′) distant by 6 bp from the XerC binding site (5′-TTATGCGAAAT-3′). Only sites that were at least 75% identical to the reference were taken into account.

Cloning experiments, plasmid DNA analysis and horizontal gene transfer assays

In order the compare the hydrolysis spectrum of CTX-M-2 and CTX-M-115, the corresponding genes were cloned into the pABEC plasmid vector and subsequently expressed in Escherichia coli and A. baumannii (see Supplementary Appendix 1). Plasmid DNA extraction and transformation assays are detailed in the supplementary material (Appendix 1).

GenBank accession numbers

This Whole Genome Shotgun project was deposited at GenBank under accession number PRJNA644570.

Results and discussion

Case characteristics and clonal population structure

From 2015 to 2019, 19 CTX-M-115-positive A. baumannii strains were recovered from 19 patients admitted to 12 distant French hospitals (Table 1), of which three are located in French overseas territories, namely Martinique island in the Caribbean Sea, and French Guiana in South America. A travel history was reported by five patients mentioning Georgia twice and Ukraine once. All the isolates were considered by clinicians as colonization, except one (AbCTX13), which was responsible for a digestive infection in a paediatric patient (Table 1).

Table 1.

Clinical features of patients harbouring the CTX-M-115-positive A. baumannii isolates

Patient	Hospital	Travel history	Ward	Date of isolation (month/year)	Site of isolation	Infection/colonization	Isolate	Genome Accession
1	A	none	ICU	02/2015	Throat	Colonization	AbCTX1	CP060029
2	B	Guadeloupe (OT)	Surgery	10/2015	Abdominal wound	Colonization	AbCTX2	JACGEH000000000
3	B	NA	Surgery	10/2015	Sputum	Colonization	AbCTX3	JACBOM000000000
4	C (D before)	none	NA	11/2015	Skin	Colonization	AbCTX4	JACBOL000000000
5	D	none	ICU	11/2015	Skin	Colonization	AbCTX5	CP060505 (chromosome), CP092029 (plasmid)
6	E (OT)	none	Surgery	06/2016	Urine	Colonization	AbCTX6	JACBOK000000000
7	E (OT)	none	ICU	06/2016	Tracheal aspiration	Colonization	AbCTX7	JACBOJ000000000
8	B	none	Endocrinology	10/2016	Bone biopsy	Colonization	AbCTX8	JACBOI000000000
9	F (OT)	NA	ICU	04/2017	Bronchial aspiration	Colonization	AbCTX9	CP060504 (chromosome), CP092030 (plasmid)
10	E (OT)	NA	Urology	08/2017	Skin	Colonization	AbCTX10	JACBOH000000000
11	G (OT)	NA	Physical medicine and rehabilitation	05/2017	Urine	Colonization	AbCTX11	JACGEI000000000
12	G (OT)	NA	Surgery	06/2017	Skin	Colonization	AbCTX12	JACBOG000000000
13	H	Georgia	Paediatrics	08/2017	Peritoneal fluid	Infection	AbCTX13	CP059729 (chromosome), CP059730 (plasmid), CP059731 (plasmid)
14	E (OT)	NA	ICU	11/2017	Tracheal aspiration	Colonization	AbCTX14	JACBOF000000000
15	G (OT)	NA	Medicine	02/2018	Stool	Colonization	AbCTX15	JACBOE000000000
16	I	Ukraine	Surgery	06/2018	Stool	Colonization	AbCTX16	JACBOD000000000
17	J	Georgia	Oncology	03/2019	Throat	Colonization	AbCTX17	JACGEK000000000
18	K	Israel and Germany	ICU	04/2019	Stool	Colonization	AbCTX18	JACBOC000000000
19	L	NA	Infectious diseases	11/2019	Stool	Colonization	AbCTX19	JACGEJ000000000

NA, not available; ICU, Intensive Care Unit; OT, French overseas territories.

MLST analysis confirmed that AbCTX19 was genotypically distinct from the rest of the collection, as it belonged to ST^Pas1 and ST^Ox231. Like the CTX-M-115 A. baumannii previously isolated in Russia, the USA, Germany and Brazil, the other 18 isolates belonged to CC78 (Figure 1).^16–19

Figure 1.

Phylogenetic tree of CTX-M-115-producing strains based on 2242 core gene products, resistome and STs of all isolates. The scale shows the number of SNPs in the core genes. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Antibiotic susceptibility and resistome of A. baumannii isolates

The 19 CTX-M-115-positive strains were all resistant to penicillin/inhibitor combinations, broad-spectrum cephalosporins, carbapenems, fluoroquinolones and cotrimoxazole (data not shown). Eighteen of them remained susceptible to minocycline and colistin, thus exhibiting an XDR phenotype,³⁶ while the remaining strain (AbCTX13) showed an additional susceptibility to aminoglycosides.

Analysis of the resistome of isolates identified genes encoding a CHDL in all of them, either the OXA-23 enzyme (n = 1), or the OXA-24/40 variant OXA-72 (n = 18 strains). One strain (AbCTX17) turned out to harbour an additional gene coding for a new single point variant of OXA-72, named OXA-897 (accession number MN920418.1) (Figure 1). Since their first description in China in 2007, OXA-72-producing A. baumannii strains have been spotted in many countries, especially in South America, Southern Asia and Europe, from human, animal and environmental samples. The wide dissemination of the bla_OXA-72 gene in A. baumannii populations seems to have been promoted by both self-transferable plasmids and epidemic strains, such as the international clone 2.^19,37–43 Based on our sequence data, most of the isolates of the collection also appeared to produce a narrow-spectrum β-lactamase (either TEM-1 or CARB-16), various aminoglycoside-modifying enzymes and the 16S rRNA methylase ArmA that confers panaminoglycoside resistance (Figure 1). In all strains, the insertion sequence ISAba1 was mapped upstream of the gene encoding variants of intrinsic class C β-lactamase ADC, namely ADC-152 in 16 isolates, ADC-170 (a V236A variant of ADC-152) in two isolates, and ADC-30 in one isolate (Figure 1). In contrast, none of the bacteria contained a mobile element ahead of the gene of OXA-51-like intrinsic carbapenemase (OXA-90 and OXA-69 variants in 18 and 1 strains, respectively). The high-level resistance of all strains to fluoroquinolones was associated with a S81L substitution in the QRDR of DNA gyrase subunit GyrA, and to a S84L substitution in QRDR of the topoisomerase IV subunit ParC.^44,45

Resistance profile conferred by β-lactamase CTX-M-115

As mentioned above, CTX-M-115 differs from CTX-M-2 by three amino acids. The I279V substitution is present in most of CTX-M enzymes, including CTX-M-15, while V251I and G290S have only been identified in few CTX-M variants (CTX-M-77, CTX-M-124 and CTX-M-216). Expression of CTX-M-2 and CTX-M-115 enzymes in the same genetic E. coli DH5α background yielded identical resistance profiles to ticarcillin, broad-spectrum cephalosporins and aztreonam (Table S2). However, CTX-M-115 provided A. baumannii strain CIP70.10 with a significantly higher resistance to cefepime (8-fold) and ticarcillin/clavulanic acid (≥4-fold), as compared with CTX-M-2 (Table S2).

Phylogenetic analyses

Assessment of the refined phylogeny of our CC78 isolates by cgMLST revealed three clusters (Figure 1). A first cluster (cluster CI in Figure 1) was composed of 11 closely related strains belonging to ST^Pas78/ST^Ox944, which differed from each other by a maximum of 12 SNPs. Of note, eight of those isolates came from three hospitals located in French overseas territories (Table 1). Three other strains (AbCTX2, AbCTX3, AbCTX8) were isolated in a same hospital in mainland France. However, sanitary repatriation of the AbCTX2-positive patient from the Caribbean zone was followed by a small outbreak in this hospital. Analysis of our collection by cgMLST also documented a second cluster consisting of two ST^Pas78/ST^Ox1757 strains (AbCTX4 and AbCTX5) from two distant hospitals (named C and D in Table 1; Figure 1). Supporting the notion of cross-contamination, we found that both patients had stayed in hospital D at the same time before the transfer of one of them to hospital C. Finally, the third cluster was composed of two ST^Pas78/ST^Ox1104 strains (AbCTX13 and AbCTX17), exhibiting a difference of only eight SNPs (cluster CIII in Figure 1). These strains were collected from patients who had travelled in Georgia before their admission to different French hospitals, thus illustrating probable cases of contamination abroad. The remaining three strains of the collection could not be phylogenetically grouped (Figure 1). AbCTX16 and AbCTX18 belonged to new Oxford scheme STs within CC78 (ST^Ox2018 and ST^Ox2019, respectively). AbCTX1 was found to belong to ST^Pas1077, which differs from ST^Pas78 by a single allele (rpoB).

The KL cluster, harbouring genes involved in capsular polysaccharide synthesis, has been previously proved to be an interesting epidemiological tool to trace genetic lineages of A. baumannii.⁴⁶ In the present study and as already reported, multiple KL types were identified within a single Pasteur scheme ST (five KL types within ST^Pas78; Figure 1).⁴⁶ However, the perfect correlation found between the subclades (obtained by cgMLST), Oxford scheme ST and KL types (Figure 1) emphasizes the accuracy of those latter two in separating distinct phylogenetic subclades.

Five representative ST^Pas78 isolates (AbCTX2-4-13-16-18) were then compared with nine genomes of the same ST downloaded from public data repositories. At a global phylogenic level, our ST^Pas78 isolates defined a novel monophyletic group besides the subclades ST78A and ST78B described in 2019 (Figure S1).⁴⁷

Genetic context of bla_CTX-M-115 gene

The genetic environment of this ESBL-encoding determinant was reminiscent of but not strictly identical to the one depicted in CTX-M-115-positive Brazilian strains.¹⁹ In these bacteria, a truncated ISEcp1 element is inserted 43 bp upstream of bla_CTX-M-115, and a 156-bp-long chromosomal sequence from K. ascorbata lies downstream. However, whereas ISEcp1 is disrupted by IS26-v5 (former IS15-ΔIV) in the Brazilian strains, this inactivation involved IS26-v1 (a two-nucleotide variant of IS26-v5) in 12 of our strains (cluster CI and AbCTX13),⁴⁸ and involved ISKpn26 in the other seven strains.

Despite the clonal relatedness of French CTX-M-115-positive isolates, the rest of the genetic environment of bla_CTX-M-115 was highly variable. Indeed, analysis of nanopore genomic data showed that the chromosomal region bearing this gene ranged from 20 to 79 kb in size and contained different combinations of resistance determinants to β-lactams (bla_TEM-1, bla_CARB-16), aminoglycosides (armA, aadB, aphA7, aadA5, aac(6′)-Ian), sulphonamides (sul1, sul2), macrolides (msrE, mphE), chloramphenicol (catA1, floR), disinfectants (qacEΔ1) and heavy metals (merRTPCADE operon) (Figure 2 and Figure S2). In addition, multiple copies of IS26-v1 and other ISs (up to 12) forming putative transposons and/or promoting possible sequence rearrangements were detected in these dynamic resistance islands (Figure 2 and Figure S2). It is worth noting that IS26-v1 differs by only three mutations from IS26; a member of IS6 family whose importance in disseminating antibiotic-resistant genes clusters is now clearly established.^49–52 The global architecture of the region surrounding bla_CTX-M-115 was somewhat more conserved in very closely related strains of cluster CI, with the exception of small-sized insertions/deletions (Figure S2). In spite of their high variability, those structures shared genes identified on various plasmids identified in Enterobacterales. Therefore, a plausible hypothesis would be that CTX-M-115-positive A. baumannii strains emerged following the chromosomal insertion of one of these plasmids, and subsequent remodelling of the resistance region through IS26-like mobile genetic elements.

Figure 2.

Genetic configurations of bla_CTX-M-115-carrying structures. The syntenic regions with near 100% identity are highlighted with grey shading. Antibiotics and heavy metal resistance genes are shown in red and pink, respectively. IS26-v1 and other IS are displayed as black and yellow arrows, respectively. Corresponding genes in the AYE chromosome are shown in blue. White arrows represent putative ORFs. The schematic representations are drawn to scale.

Chromosomal insertion region of the bla_CTX-M-115-carrying element

In most strains (AbCTX2-3-4-5-6-7-10-12-14-15-16-19), the resistance islands described above replaced an 8-kb-long sequence located downstream the glmS gene, between the ABAYE0080 and ABAYE0075 (hutU) genes, according to the annotation of AYE genome (accession number CU459141.1). Small variations of the insertion site were observed for AbCTX1 (ABAYE3313), AbCTX8 (hutC gene), AbCTX11 (murI gene) and AbCTX18 (ABAYE0081) (Figure 2 and Figure S2). In two strains, a Tn6171 (accession number CP01252.1),⁵³ inserted 25 bp downstream of the glmS gene, was truncated by the bla_CTX-M-115-carrying DNA region (in fbsE for AbCTX13 and in fbsJ for AbCTX17). Contrasting with other Cluster I strains, we found that the 37 kb resistance island of AbCTX9 interrupts the dsbD gene that codes for a disulfide interchange protein precursor (ABAYE0138), with the presence of two 8 bp direct repeats (DRs, GTTTAGTT) bracketing the IS26-v1 copies (green arrows in Figure S2).

It is worth noting that all the bla_CTX-M-115-carrying fragments were inserted at different but close positions not only into the genomes of CC78 strains but also into the single CC1 isolate AbCXT19 (Figure 2). Interestingly, in ST^Pas1 AbCTX19, the adjacent chromosomal region of the bla_CTX-M-115 element had more sequence similarities with the chromosome of ST^Pas78 than that of native ST^Pas1 (Figure 3), suggesting that large recombination events occurred between clonally unrelated isolates, as already reported.^54,55

Figure 3.

Chromosomal location of bla_CTX-M-115-carrying element in ST^Pas1 AbCTX19 isolate compared with ST^Pas78 AbCTX6 and ST^Pas1 AYEΔAbaR1 isolates, respectively. Hashed and black lines show chromosome sequences from ST^Pas78 and ST^Pas1 isolates, respectively. Black arrows indicate the genes truncated by insertion of acquired genetic structures. A white line shows the bla_CTX-M-115-carrying element. The syntenic regions with near 100% identity are highlighted with grey shading and lower identities are specified.

Additional genomic features of CTX-M-115-positive strains

Chromosomal gene comM is a hotspot for insertion of resistance islands in strains belonging to clonal complex CC1 or CC2.^56,57 These islands, named AbaR, can carry a great diversity of antibiotic and heavy metal resistance genes. Not surprisingly, we found that in the CC1 isolate AbCTX19, comM was truncated by a copy of transposon Tn6019, whose structure was itself disrupted by another transposon, Tn6018.⁵⁶ The situation was different for 17 out of the 18 ST^Pas78 strains since the disruptive element was a Tn6177-like transposon, sharing 98% homology with the native Tn6177 (accession number MG954377.1) and carrying an operon encoding resistance to heavy metals (arsenic and mercury). Finally, in the remaining CC78 strain AbCTX1, comM was interrupted by the resistance island AbaR4 bearing the bla_OXA-23 gene embedded in a Tn2006 element. This latter strain was the only example of the series, harbouring an antimicrobial resistance determinant located within the comM gene.

Genetic structure of bla_OXA-72-like-positive plasmids

Analysis of the plasmid content of CTX-M-115 A. baumannii revealed the presence of 1–3 plasmids per strain, whose size ranged from ∼8 to ∼140 kb (data not shown). Mining of whole DNA sequence data enabled us to localize the bla_OXA-24-like gene on several of these replicons (from 7055 to 17003 bp in size). All ST^Pas78 strains (AbCTX2 to AbCTX18) appeared to possess a ca. 15–20 kb plasmid, which contained an unvariable part sharing 99% sequence identity with plasmid pIBAC_OXA58_20C15 (accession number KY202458) and carrying a DNA replication protein identical to that of plasmid pMMA2 (GQ377752).⁵⁸ Those replicons also carried 3 or 4 putative pdif sites, which likely mediated several intra-molecular inversion events (pAbCTX2, pAbCTX11, pABCTX13a, pAbCTX17a, pAbCTX16 in Figure 4). Another bla_OXA-72-carrying plasmid of 7 kb (pAbCTX13 in Figure 4), detected in the two ST^Ox1104 strains AbCTX13 and AbCTX17, possessed a replicase-encoding gene sharing 99% homology with repGR12 of plasmid p2ABSDF from A. baumannii SDF strain (NC_010396). On the other hand, the plasmid pAbCTX19 from ST^Pas1 isolate AbCTX19 exhibited a 98.7% sequence identity with a replicon previously identified in an OXA-72-positive Acinetobacter nosocomialis strain from Taiwan (accession number CP033553) and carried a replicase-encoding gene identical to repAci1 of pACICU1 (NC_010605). As already reported, the dynamic and variable plasmid structures described here provide further support for the exchange of the bla_OXA-72 genes between different replicons through co-integrate structures.⁵⁹ Other investigators have stressed the important role of Xer recombinases in mobilization and evolution of plasmids in Acinetobacter sp., by site-specific recombination mechanisms.⁵⁹

Figure 4.

Schematic representation of gene bla_OXA-72-like-carrying plasmids. Homologous sequences with near 100% identity are highlighted in grey. Genes of toxins/antitoxins systems are shown with grey arrows, whereas antimicrobial resistance genes are with black arrows and replicase-encoding genes with hashed arrows. XerC/XerD putative binding sites (pdif sites) are represented by vertical black bars; the direction of each site is shown above. The schematic representations are drawn to scale.

Horizontal transfer of antimicrobial resistance genes

Three representative clinical strains (AbCTX1, AbCTX6, AbCTX9) were selected to decipher the possible ways of resistance genes horizontal transfer. Transformation assays were performed using whole DNA extracts from those three strains and A. baumannii AYEΔAbaR1 as recipient. Those experiments yielded clones exhibiting various resistance phenotypes depending upon the antibiotic used for selection. Repeated attempts with non-transformable mutant AYEΔAbaR1ΔcomEC systematically failed, which indirectly confirmed the transfer of resistance genes to the parent strain through transformation. Transformation experiments with the DNA of cluster CI strains AbCTX6 and AbCTX9 resulted in selection by ticarcillin of a first type of resistant clones (illustrated by AYEΔAbaR1 FT6.1, FT6.2 and FT9.1 in Table 2 and Figure 5), with an average transformation efficiency of 2.2 ± 1.2×10² transformants per μg of DNA. These clones were more resistant to penicillins, cephalosporins, aminoglycosides and cotrimoxazole than the recipient strain (Table 2). Genome sequencing of AYEΔAbaR1 FT6.1 and FT6.2 (using AbCTX6 as donor) and FT9.1 (AbCTX9 as donor) revealed the insertion of various DNA fragments carrying bla_CTX-M-115 as well as other resistance determinants, in the AYEΔAbaR1 chromosome (Figure 5a and b). None of these sequenced transformants contained DRs flanking the inserted fragments; an observation supporting the notion that homologous recombination rather than transposition events occurred. For instance, despite the presence of a seemingly functional bla_CTX-M-115-carrying transposon bracketed by two 8 bp DRs in strain AbCTX9, a homologous recombination involving the chromosomal regions flanking this element (9 kb and 6.2 kb at its 5′- and 3′-ends, respectively), was evident in the transformed strains (Figure 5b). Further sequence analyses showed the replacement of small (100 bp) to large (13 kb) genomic fragments of the recipient AYEΔAbaR1 strain by the corresponding sequences of ST^Pas78 donor strains (Figure 5).

Figure 5.

Chromosomal location of transferred genetic structures in AbCTX6 (a), AbCTX9 (b) and AbCTX1 (c), compared with their corresponding sequenced transformants. Green and orange lines show chromosome sequences from AYEΔAbaR1 (recipient strain) and CTX-M-115-producing clinical A. baumannii strains, respectively. Green arrows indicate the genes truncated by insertion of acquired genetic structures. A blue line shows the acquired genetic structures with antibiotic resistance genes represented by red boxes and IS26-v1 by grey boxes (a and b). The syntenic regions with near 100% identity are highlighted with grey shading and lower identities are specified.

Table 2.

Antimicrobial susceptibility of strain A. baumannii AYEΔAbaR1 and six selected transformants

Isolate	Originating strain	Selective antibiotic	Transferred resistance genes	TIMa	SAMb,c	TZPd	ATM	CAZ	FEP	IPM	MEM	AMK	GENb	TOB	SXT
AYEΔAbaR1			none	32	8	64	64	24	12	≤1	≤1	≤4	4	2	≤0.5/9.5
AYEΔAbaR1 TF6.1	AbCTX6	TIC	blaTEM-1, blaCTX-M-115, aadA5, sul1, armA, msrE, mphE, sul2, aadB, aph(3′)-I	128	64	>256	>256	48	>256	≤1	≤1	>32	>256	>8	>4/76
AYEΔAbaR1 TF6.2	AbCTX6	TIC	blaTEM-1, blaCTX-M-115, aadA5, sul1, armA, msrE, mphE, sul2, aadB, aph(3′)-I	128	64	>256	>256	48	>256	≤1	≤1	>32	>256	>8	>4/76
AYEΔAbaR1 TF6.3	AbCTX6	GEN	aadB, aph(3′)-I	32	8	64	64	24	12	≤1	≤1	≤4	>256	>8	≤0.5/9.5
AYEΔAbaR1 TF6.4	AbCTX6	IPM	blaOXA-72	>128	8	>256	48	32	16	>32	>32	≤4	4	2	≤0.5/9.5
AYEΔAbaR1 TF1.1	AbCTX1	IPM	blaOXA-23	>128	8	>256	48	32	16	>32	>32	≤4	4	2	≤0.5/9,5
AYEΔAbaR1 TF9.1	AbCTX9	TIC	blaCTX-M-115, aadA5, sul1, armA, msrE, mphE, sul2, aph(3′)-I	128	64	>256	>256	64	>256	≤1	≤1	>32	>256	>8	>4/76

TIC, ticarcillin; TIM, ticarcillin/clavulanic acid; SAM, ampicillin/sulbactam; TZP, piperacillin/tazobactam; ATM, aztreonam; CAZ, ceftazidime; FEP, cefepime; IPM, imipenem; MEM, meropenem; AMK, amikacin; GEN, gentamicin; TOB, tobramycin; SXT, trimethoprim/sulfamethoxazole.

MIC values (mg/L) in bold indicate a 4-fold increase compared with the reference strain.

a

MIC values of ticarcillin with a fixed concentration of clavulanate equal to 2 mg/L.

b

MIC values were determined with Etests.

c

MIC values of ampicillin with a fixed concentration of sulbactam equal to 4 mg/L.

d

MIC values of piperacillin with a fixed concentration of tazobactam equal to 4 mg/L.

Transformation assays with isolate AbCTX6 as a donor of genetic material, and gentamicin as selective agent, yielded a transformant only resistant to aminoglycosides (AYEΔAbaR1 FT6.3) that harboured a short piece of extra DNA carrying the genes of two aminoglycoside-modifying enzymes (Table 2 and Figure 5a).

Transformants of two other types, both displaying resistance to penicillins and carbapenems, were selected on MHA plates supplemented with imipenem. A first one was documented when the unique ST^Pas1077/ST^Ox1631 isolate of our collection, AbCTX1, was used as source of DNA. Occurring at relatively high efficiency (4.5 ± 0.3 × 10⁵ transformants per μg of DNA), these transformants (illustrated by AYEΔAbaR1 TF1.1 in Table 2 and Figure 5c) acquired the bla_OXA-23-carrying AbaR4 genomic island. In addition, the use of DNA from AbCTX6 or AbCTX9 allowed the easy selection (4.4 ± 1 × 10⁴ transformants per μg of DNA) of the second type of transformants (illustrated by AYEΔAbaR1 FT6.4 in Table 2) that contained the 15 kb bla_OXA-72-carrying plasmid present in the donors.

Conclusions

This report highlights the high variability of the bla_CTX-M-115 region detected in closely related A. baumannii strains belonging to international lineage CC78. Lines of evidence suggest that this region of likely plasmid origin is shaped by complex recombination and deletions events, mostly dependent on IS26-like insertion sequences. In addition, our results support the notion that CC78 strains are able to transfer to isolates belonging to other lineages both plasmid-borne and chromosomal genes conferring resistance to carbapenems (OXA-23, OXA-72-like β-lactamases), broad-spectrum cephalosporins (CTX-M-115) and aminoglycosides, through natural transformation. This straightforward mode of gene exchange does not require cell-to-cell contacts and does not depend on the intervention of mobile genetic elements. According to our observations, integration of pieces of foreign DNA into the chromosome of competent cells would frequently occur through homologous recombination. Which environmental conditions, stress factors or selective forces promote such lateral gene spread among Acinetobacter sp. warrants further investigations in order to be controlled or prevented.

Acknowledgements

We thank medical microbiologists for referring the CTX-M-115-positive strains to the French National Reference Center for Antibiotic Resistance (NRC-AR).

Funding

This study was funded internally. The NRC-AR is funded by the French Ministry of Health through the Santé publique France agency.

Transparency declarations

None to declare.

Supplementary data

Tables S1 and S2, Figures S1 and S2 and Appendix 1 are available as Supplementary data at JAC Online.