Dissemination of the Acinetobacter baumannii isolates belonging to global clone 2 containing AbGRI resistance islands in a referral hospital

ABSTRACT Acinetobacter baumannii strains belonging to global clone 2 (GC2) contain resistance islands (AbGRIs), which are composed of genes conferring resistance to older and newer antibiotics. Here, to locate these genes in AbGRIs, the GC2 strains from Tehran, Iran were examined. Among the 170 A. baumannii, 90 isolates were identified as GC2. Of the genes that confer resistance to older antibiotics, tetA(B), tetR(B) (tetracyclines), strA, and strB (aminoglycosides) were located in AbGRI1 of 65 GC2 isolates (72.2%). Of the other aminoglycosides, the aphA1b was located in AbGRI2-12b (63.6%), AbGRI2-12a (21.2%), or AbGRI2-1 (15.1%). The aacC1 and aadA1 genes were co-located within AbGRI2-1 (5.5%). The armA was located in AbGRI3-4 (77.7%) and AbGRI3ABI221 (22.2%). Of sulfonamides, the sul1 was located within AbGRI2-1 (5.5%). Of beta-lactams, the blaTEM was located in AbGRI2-12b (42%), AbGRI2-12a (14%), AbGRI2-1 (10%), or AbGRI2ABI257 (34%). The oxa23 gene conferring resistance to newer antibiotics (carbapenems) was located in AbaR4 (81.1%); of them, the AbaR4 was located within AbGRI1 in 45.2% of the isolates. This study showed that the GC2 isolates, which contained at least one AbGRI, disseminate in the hospital. Hence, it is likely that the AbGRIs play a significant role in conferring resistance to older and newer antibiotics in GC2 isolates from Iran. IMPORTANCE The majority of Acinetobacter baumannii isolates that are resistant to multiple antibiotics belong to one of the two major global clones, namely global clone 1 (GC1) and global clone 2 (GC2). The resistance islands, which contain variable assortments of transposons, integrons, and specific resistance genes, have been characterized in the genome of these GCs. In GC2 A. baumannii, the chromosomally located A. baumannii genomic resistance islands (AbGRIs) carry the genes conferring resistance to older and newer antibiotics. In this context, we tested whether GC2 isolates collected from a referral hospital carry the AbGRIs containing these genes. This study provided evidence for the circulation of the GC2 A. baumannii strains harboring AbGRI resistance islands between different wards of a referral hospital.

The high levels of resistance to older and newer antibiotics were reported in the previous study from Iran (10).However, little is known regarding the role of AbGRIs in conferring resistance to older and newer antibiotics from Iran and other Middle East countries (11).Here, we aimed to locate these genes in AbGRIs in GC2 strains from Tehran, Iran.

Identification of GC2 isolates and antibiotic resistance profiles
Of the isolates examined, 90 isolates belonged to GC2.All isolates were resistant to carbapenems (imipenem, meropenem, and doripenem); however, resistance to older antibiotics, including tetracyclines, aminoglycosides, and sulfonamides, varied among the isolates.The antibiotic resistance profile of the isolates is shown in Table 1.The results of disk diffusion for GC2 A. baumannii isolates are presented in Table S1.
In the isolates found to carry AbGRI1, four groups were identified based on their backbone transposon and the presence of the sul2 gene.The isolates in the first group contained both Tn6022 and Tn6022Δ1 (with a specific deletion in respect to Tn6022); however, the isolates in groups 2 and 3 contained Tn6022Δ1 and Tn6022, respectively.While the sul2 gene was present in the isolates in groups 1-4, it was absent in the isolates in group 4 (Table S4).In the isolates containing the sul2 gene, the primers linking sul2 to comM gene (RH928 and sul2F) failed to amplify a product of the predicted size of 16,826 bp, which is not surprising due to the predicted large amplicon size.Furthermore, since AbGRI1-1 and AbGRI1-6 are the only known AbGRI1s without the sul2 gene, the mapping strategies for AbGRI1-1 and AbGRI1-6 were attempted for the isolates without the sul2 gene.As all isolates were positive for the PCR used to link the oxa23 to tetA(B), they were similar to AbGRI1-6; however, they did not contain Tn6022Δ1.Therefore, it was not possible to determine the genetic structure of AbGRI1 in both the isolates containing sul2 and the isolates without this gene.

The genes conferring resistance to newer antibiotics carried by AbGRI1 resistance islands
The oxa23 gene (carbapenems), which confers resistance to newer antibiotics, was detected in all GC2 isolates.The oxa23 gene was located in AbaR4 (81.1%); of them, 45.2% were positive for the PCR used to link the oxa23 to tetA(B), indicating that the AbaR4 was located within AbGRI1 in these isolates (Table S2).

DISCUSSION
A. baumannii has attracted significant attention due to the occurrence of strains that are resistant to almost all the available antimicrobial agents (1).The A. baumannii strains that are resistant to older antibiotics, including tetracycline, sulfonamides, and aminogly cosides, began to be noticed since the 1970s (13), and the strains that are resistant to newer antibiotics, such as carbapenems, were recognized in 1985 (14).In the previous studies, the genes conferring resistance to older and newer antibiotics have been found in GC2 isolates harboring AbGRIs (4-7); however, much less is known about them in the Middle East region, including Iran (11).Here, to locate the genes conferring resistance to older and newer antibiotics in AbGRIs, the GC2 strains from Tehran, Iran, were examined.
Of the genes which confer resistance to older antibiotics, the tetA(B), tetR(B) (tetracycline), strA, and strB (aminoglycosides) genes were located in AbGRI1 in more than two-thirds of the GC2 isolates tested.In consistent with this finding, 82.4% of the isolates examined in South Korea carried AbGRI1-type resistance islands (9).In addition, all 62 GC2 isolates examined in Australia contained AbGRI1 resistance island (15).In 2013, the term AbGRI was proposed for the RIs that were found in GC2 isolates (6).What are currently called AbGRI1 variants were previously named as AbaR or Tn, or (incorrectly) as derivatives of AbaR4 in the studies conducted in the countries such as Lithuania ( 16), South Korea (17,18), Latvia (19), and Australia (20).The AbGRI2 resistance islands, which usually contain some or all of the aphA1b, aacC1, aadA1, bla TEM , and sul1 genes conferring resistance to antibiotics used clinically in the 1970s ( 5), were identified in the genomes of GC2 isolates in 2013 ( 6) and later, a number of variants were characterized in the isolates from Australia and Singapore and also in the sequences of published genomes (8,15,21).While only 15% of GC2 isolates from Singapore contained AbGRI2-12b (8), this island was detected in nearly half of the isolates examined in this study.The AbGRI2-1 harboring a class 1 integron was detected in 5.5% of the isolates tested in this study; however, it was present in 29% and 25% of GC2 isolates examined in Australia and Singapore, respectively (8,15).Although Blackwell et al. found the armA in AbGRI3-4 in 46.7% of the GC2 isolates (7), this gene was located in AbGRI3-4 in more than two-thirds of the isolates examined here.In addition, a new structure of AbGRI3 resistance island was found in this study.The bla TEM gene, which confers resistance to beta-lactams, was mostly located in AbGRI2-12b (42%) and with lower frequency in AbGRI2-12a or AbGRI2-1.Furthermore, a new structure of AbGRI2 resistance island that carries bla TEM was found in this study (Fig. 1).The sul1 gene which confers resistance to sulfonamides was located in a class 1 integron within AbGRI2-1 in only five GC2 isolates.Of the carbapenem-resistant GC2 isolates examined here, all carried the oxa23 gene.The oxa23 gene conferring resistance to newer antibiotics was located in AbaR4 (81.1%), which the AbaR4 was located within AbGRI1 in 45.2% of the isolates.In a study conducted in Singapore (8), the AbaR4 was located in AbGRI1 in 40% of the GC2 isolates.In consistent with this study, the AbaR4 was located in AbGRI1 in 45.2% of our isolates.In this study, the sul2 gene was only absent in the isolates in group 4 (20%) (four groups were identified in the isolates to carry AbGRI1 based on their backbone transposon and the presence of the sul2 gene).In consistent with this study, 13.84% of the isolates examined in Singapore did not contain the sul2 gene (8).In the isolates containing the sul2 gene in this study, the primers linking sul2 to comM gene (RH928 and sul2F) failed to amplify a product of the predicted size of 16,826 bp, which is not surprising due to the predicted large amplicon size.Conse quently, sequencing using long-read technology, such as PacBio or Oxford Nanopore, will be required to determine the genetic structure of AbGRI1 in these isolates.
It is noteworthy that in this study, two sets of isolates were recovered from the patients admitted to a single hospital at different time periods of 2011-2012 (set 1) and 2018-2020 (set 2).Hence, it was possible to compare these two sets of isolates in terms of the location of genes conferring resistance to older and newer antibiotics.Based on the presence or absence of AbGRI resistance islands in the GC2 isolates, 23 patterns were identified.Of the 23 patterns detected, 22 were identified in the isolates collected from 2018 to 2020.In addition, six patterns were detected in the isolates collected earlier during 2011-2012; of them, five patterns were shared between two sets of isolates.When using the Institut Pasteur scheme to perform multi-locus sequence typing (MLST) for the representative isolates of each pattern (according to the type of AbGRI resistance islands carried by the isolates), three STs were identified (Table 1), including ST2 (28/30, 93.3%), ST10 (1/30, 3.3%), and ST113 (1/30, 3.3%).It is demonstrated that ST2 is the most dominant ST sequence type in GC2 strains (22).Although the ST of isolates was mostly the same according to the Institut Pasteur scheme, several patterns were identified according to the type of AbGRI resistance islands carried by the isolates.These patterns were identified in the isolates recovered from the patients in the different wards of the hospital, indicating their dissemination between the hospital wards.It was found that the isolate belonging to ST10 (Institut Pasteur scheme) contains unique pattern based on both the type of AbGRI resistance islands carried by the isolates and also antibiotic resistance profile.In addition, the isolate belonging to ST113 (Institut Pasteur scheme), which was the only GC2 isolate with the uninterrupted chromosomal comM gene, contained a unique antibiotic resistance profile.As little intra-clonal variability has been observed using the Institut Pasteur MLST scheme, it was not possible to differenti ate the circulating strains in different wards of the hospital based on this scheme (23).However, the isolates could be tracked according to the type of AbGRI resistance islands carried by the isolates.The Oxford MLST scheme is more capable to differentiate closely related isolates, and there has been much more intra-clonal diversity observed using this scheme (24).Using the Oxford scheme, four different STs were detected (ST1624, ST452, and two new STs (ST2868, ST2869) (Table 1).Most of this diversity is due to changes in the gpi allele, which is caused by capsule locus replacement and would cause a change in ST (25).As the STs of limited isolates were determined using the Oxford scheme in this study, the diverse STs that were observed were correlated with the diverse patterns identified based on the type of AbGRI resistance islands carried by the isolates.Although two isolates belonged to the same ST1624 according to the Oxford scheme, they belonged to the different patterns based on the type of AbGRI resistance islands carried by the isolates and also based on the antibiotic resistance profile.
This study provides evidence for the dissemination of GC2 isolates carrying AbGRI resistance islands in a referral hospital in Tehran, Iran.In fact, the features of AbGRI1, AbGRI2, and AbGRI3 resistance islands, alongside antibiotic resistance profile, were informative epidemiological markers to find the relationships between the isolates, which were distributed within the hospital.This study demonstrated that the strains carrying the AbGRI resistance islands are not restricted to a particular geographical region, and they are globally distributed.The results obtained in this study will underpin future studies of GC2 strains from the Middle Eastern countries.

Bacterial isolates
A total of 170 non-repetitive A. baumannii isolates were collected from a single referral hospital in Tehran, Iran in different time periods of November 2011 to September 2012 (n = 45; set 1) and between December 2018 and January 2020 (n = 125; set 2), except for the 3 months interval in 2019.The ethics committee of Tehran University of Medical Sciences approved the study "IR.TUMS.SPH.REC.1397.291." All patients have signed the informed consent for giving the required specimens for research.Parents/legal guardians provided written-informed consent for their children under the age of 18 years to participate in this study.All isolates were initially identified using conventional microbiological methods (26) and were further confirmed by the detection of oxaAb gene by PCR (27).

Identification of GC2 isolates
To identify the A. baumannii isolates belonging to GC2, two multiplex PCR assays were performed as described previously (29).

Identification of the genes conferring resistance to older and newer antibiotics carried by AbGRI1 resistance islands
To locate the genes conferring resistance to older antibiotics in AbGRI1, including tetA(B), tetR(B), strA, and strB, PCR and PCR mapping experiments, followed by sequenc ing confirmation were performed using the primers listed in Table S5.The sul2 gene was detected, and whenever possible, it was linked to the AbGRI1 using the primers described previously (8).To examine whether the oxa23 gene conferring resistance to newer antibiotics is located in AbaR4 and/or AbaR4Δ1 and also, to examine whether the AbaR4 and AbaR4Δ1 are located within AbGRI1, PCR mapping experiments were performed using the primers listed in Table S5.The linkage PCRs, which were performed to map the backbone of AbGRI1 resistance island, are shown in Fig. 3.

Identification of the genes conferring resistance to older antibiotics carried by AbGRI2 and AbGRI3 resistance islands
To locate the genes conferring resistance to older antibiotics in AbGRI2 (aphA1b, sul1, aadA1, aacC1, bla TEM ) and AbGRI3 (armA, aphA1b, sul1, aadA1, aacA4), PCR and PCR mapping experiments, followed by sequencing confirmation were performed using the primers listed in Table S5.The PCR product of Δatr-ISAba24 segment was sequenced by primer walking strategy (primers are listed in Table S6). e

FIG 1
FIG 1 Schematic of the AbGRI2 ABI257 .The central dark gray line represents the resistance island.The light gray line indicates the adjacent chromosomal sequence.Arrows indicate extent and orientation of genes and open reading frames.The resistance genes are red with the name below.Boxes represent IS, and the internal arrows indicate the transposase.The vertical arrows indicate the location of deletion (Δ) found in the AbGRI2 ABI257 in this study.Dashed lines in AbGRI2 ABI257 represent the extent of deletions relative to AbGRI2-12.The primers used to link sequences are indicated beneath each green line, along with the predicted amplicon size.The figure is adapted from reference (8) with permission from Dr. Blackwell.

FIG 2
FIG 2 Schematic of the dissemination of GC2 isolates between hospital wards.The symbols indicate different patterns identified in the isolates (according to the type of AbGRIs carried by the isolates) as shown in the key below the figure.

FIG 3
FIG 3 The linkage PCRs to map the backbone of AbGRI resistance islands.The central dark gray line represents the resistance island.The light gray line indicates the adjacent chromosomal sequence.Arrows indicate extent and orientation of genes and open reading frames.The resistance genes are red with the name below.Boxes represent IS and CR elements, and the internal arrows indicate the transposase or the rolling circle replicase.Vertical bars indicate IRs, and the triangles next to the genes indicate the deletion in the genes.The location of the Tn6022∆1, orf region, and AbaR4 is shown by black box around them.The figure is adapted with from reference 8 with permission from Dr. Blackwell.

TABLE 1
The patterns identified in the isolates, according to the type of AbGRI1, AbGRI2, and AbGRI3 resistance islands carried by the isolates i (Continued on next page) Research Article Microbiology Spectrum September/October 2023 Volume 11 Issue 5 10.1128/spectrum.05373-223

TABLE 1
The patterns identified in the isolates, according to the type of AbGRI1, AbGRI2, and AbGRI3 resistance islands carried by the isolates i (Continued) The profile is relevant to the genes conferring resistance to older and newer antibiotics, which are located on AbGRI1, AbGRI2, and AbGRI3 resistance islands.The chromosomal comM gene was found to be uninterrupted, and the J1 and J2 junction PCRs failed to amplify in this isolate.
a c