Occurrence of Acinetobacter baumannii genomic resistance islands (AbGRIs) in Acinetobacter baumannii strains belonging to global clone 2 obtained from COVID-19 patients

Aim The Acinetobacter baumannii genomic resistance islands (AbGRIs), which were characterized in the genome of the global clone 2 (GC2) A. baumannii contain resistance genes. Here, we aimed to determine the occurrence of AbGRIs in GC2 A. baumannii obtained from COVID-19 patients in a referral hospital in Tehran, Iran. Methods A total of 19 carbapenem-resistant A. baumannii (CRAB) isolates belonging to GC2 and sequence type 2 (ST2), including 17 from COVID-19 patients and two from the devices used in the ICU that the COVID-19 patients were admitted, were examined in this study. Antibiotic susceptibility testing was performed by the disk diffusion method. PCR and PCR mapping, followed by sequencing, were performed to characterize the structure of AbGRI resistance islands in the isolates tested. Results The AbGRI3 was the most frequent resistance island (RI) detected, present in all the 19 isolates, followed by AbGRI1 (15 isolates; 78.9%) and AbGRI2 (three isolates; 15.8%). Notably, AbGRIs were identified in one of the A. baumannii strains, which was isolated from a medical device used in the ICU where COVID-19 patients were admitted. Furthermore, new structures of AbGRI1 and AbGRI3 resistance islands were found in this study, which was the first report of these structures. Conclusions The present study provided evidence for the circulation of the GC2 A. baumannii strains harboring AbGRI resistance islands in a referral hospital in Tehran, Iran. It was found that resistance to several classes of antibiotics in the isolates collected from COVID-19 patients is associated with the resistance genes located within AbGRIs. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-023-02961-3.

This study aimed to characterize the structure of AbGRI1, AbGRI2, and AbGRI3 resistance islands in GC2 isolates collected from COVID-19 patients and also, the GC2 isolates obtained from the medical devices used within the ICU where COVID-19 patients were admitted in the largest referral hospital in Tehran, Iran.

Identification of antibiotic resistance profiles
All isolates were resistant to streptomycin, spectinomycin, kanamycin, tobramycin, netilmicin, and cefotaxime.
The results of disk diffusion for GC2 A. baumannii isolates is presented in Table S 1.In the present study, all A. baumannii isolates have the MDR phenotype (Table S 1).

AbGRI1 resistance island
Four out of the 19 GC2 isolates tested (21.1%), did not contain AbGRI1 resistance island (Table 1, not highlighted).In the remaining 15 isolates (78.9%), three groups were distinguished according to their shared characteristics (Table 1, highlighted).The typical AbGRI1 genes, including strA, strB (conferring resistance to aminoglycosides), tetA(B), and tetR(B) (conferring resistance to tetracyclines) were co-located in all isolates.While the interrupted comM gene, J2 junction, orf4b adjacent to comM gene (indicating that they contained AbGRI1 resistance island), CR2 element, and oxa23 gene (conferring resistance to carbapenems) were present in all isolates containing AbGRI1, 11 isolates (57.9%) lacked J1 junction, Tn6022, Tn6022Δ1, and orf region (yellow in Table 1).Furthermore, one of the isolates carrying AbGRI1 lacked the sul2 gene (conferring resistance to sulfonamides) (blue in Table 1).Long-read sequencing technology such as PacBio or Oxford Nanopore will be required to determine the structure of AbGRI1 in the isolates containing this island.

AbGRI2 resistance island
Sixteen out of the 19 GC2 isolates tested (84.2%), did not contain AbGRI2 resistance island (Table 2, not highlighted).In the remaining three isolates, two groups were distinguished according to their shared characteristics (Table 2, highlighted).Two isolates (10.5%) contained AbGRI2-12b (yellow in Table 2) and one isolate (5.3%) contained AbGRI2 ABI257 (an AbGRI2 with a structure that is similar to AbGRI2-12 except for missing a segment from the right-hand side of the island) (green in Table 2).While the bla TEM (conferring resistance to beta-lactams) was present in all isolates containing AbGRI2, the aphA1b (conferring resistance to aminoglycosides) was only present in one isolate carrying AbGRI2 (yellow in Table 2).Furthermore, the aacC1, aadA1 genes (conferring resistance to aminglycosides), and sul1 genes (conferring resistance to sulfonamides) were not observed in any isolate examined (Table 2).

AbGRI3 resistance island
It was found that all the isolates carry an AbGRI3 resistance island.Seven groups were distinguished in the isolates according to their shared characteristics (highlighted in Table 3).While the armA gene (conferring resistance to aminoglycosides) was present in all isolates, the aacA4 and aphA1b genes (conferring resistance to aminoglycosides) were present in 11 (57.9%) and 12 (63.2%)isolates, respectively (Table 3).AbGRI3-4 was found in seven isolates (36.8%, yellow and green in Table 3); however, a long-read sequencing technique is required to determine the structure of AbGRI3 in the rest of the isolates.

Discussion
Several outbreaks of the CRAB isolates belonging to GC2 in COVID-19 patients in a tertiary referral hospital in Iran were demonstrated in our previous study [19].Since the isolates exhibited an MDR phenotype, the genetic basis for their phenotype was investigated.In 2013, the term AbGRI was proposed for the RIs that were found in GC2 isolates [10].The isolates belonged to GC2, therefore, the AbGRI resistance islands carrying resistance genes were studied.Although the features of AbGRI1, AbGRI2, and AbGRI3 resistance islands carrying antibiotic resistance genes were used as informative epidemiological markers to find the relationships between the isolates distributed within the hospital in a study from Iran [20], there was no study to investigate the presence of AbGRIs in GC2 isolates obtained from COVID-19 patients.In the current study, 15 isolates (78.9%) carried an AbGRI1 resistance island.Consistent with our finding, 82.4% of the isolates examined in South Korea [16] and 72.2% of the isolates examined in Iran [20], carried AbGRI1 resistance islands.Here, the isolates that carried AbGRI1 but lacked J1 junction, Tn6022, Tn6022Δ1, and orf region were found, which is the first report of this structure in A. baumannii.In the present study, only two isolates (10.5%) contained AbGRI2-12b, which is consistent with the results of a study by Blackwell, et al. that 15% of GC2 isolates from Singapore carried this island [9].However, AbGRI2-12b was found in 51.5% and 62.6% of the GC2 isolates investigated in two studies in Iran, respectively [20,21].While AbGRI2 ABI257 with a structure that is similar to AbGRI2-12 but has lost a segment from the right-hand side of the island was found only in one GC2 isolate in this study, Table 1 Characteristics of the GC2 isolates containing AbGRI1 resistance island a. E1 and E2 refer to the GC2 isolates that were obtained from the environment of the ICU where the COVID-19 patients were admitted Rows highlighted in the same colour indicate the same pattern PCRs in bold are the linkage PCRs that were performed for identification of AbGRI1s in this study ND Not determined, for the isolates without the orf4b-comM fragment, other PCRs were not performed it was found in 34% of the isolates in Iran, previously [20].It was revealed that the armA gene was located within the AbGRI3 resistance island in all the isolates containing this gene.Consistent with the results of the current study, Blackwell et al. detected the armA gene in 15 out of 20 GC2 A. baumannii isolates from Singapore, and they revealed that the armA gene is located within AbGRI3 in all the isolates containing this gene [11].Also, Blackwell et al. found the armA in AbGRI3-4 in 46.7% of the GC2 isolates [9], which it is consistent with the presence of AbGRI3-4 in 36.8% of the isolates examined here.However, AbGRI3-4 was found in 80.7% and 77.7% of the GC2 isolates investigated in two studies in Iran, respectively [20,21].In addition, five new structures of AbGRI3 resistance islands were found in the current study (patterns three to seven in Table 3), which it is the first report of these structures in A. baumannii.This study provided evidence for the circulation of the GC2 A. baumannii isolates, which contained at least one AbGRI resistance island, between different ICU wards of a referral hospital.Hence, it is likely that the AbGRIs play a significant role in conferring resistance to various antibiotics in GC2 isolates from Iran.Furthermore, new structures of AbGRI1 and AbGRI3 resistance   islands were found in the GC2 isolates obtained from COVID-19 patients in the current study, which were not reported previously.As a limitation, long-read sequencing technology such as PacBio or Oxford Nanopore will be required to determine the structure of AbGRIs in all the isolates containing these islands.

Bacterial isolates
Among the isolates examined in our previous study [19], 17 CRAB isolates belonging to GC2 and ST2, which were collected from COVID-19 patients, were chosen to examine in this study [19].In addition, two isolates that were obtained from the medical devices used in the ICU where the COVID-19 patients were admitted (henceforth these isolates referred E1 and E2), were also included in the present study.
To investigate the structures of AbGRI1 resistance islands, PCR and PCR mapping experiments were performed to determine interrupted comM gene; J1 and J2 boundaries of the AbGRI1; orf4b adjacent to comM on the right-hand side of AbGRI1; backbone transposon, resistance region, and orf region (Table 4).To investigate the structures of AbGRI2 resistance islands, PCR and PCR mapping experiments were used to determine the arrangement of the segments including AB57_1175-tnpR 1 , bla TEM− tnpA 1000, tnpR 5393 c-aphA1b, aphA1-sul1, tnpA 21 -AB57_1209, TE32_13140-tnpR1, aphA1b-ABA1_01228, and tnpR 5393 c-ABA1_01228 (Table 6).To investigate the structures of AbGRI3 resistance islands, PCR and PCR mapping experiments were used to determine the armA gene, interrupted atr gene; and determine the arrangement of the segments including Δatr-repAciN, aphA1b-Δasr, armA-asrΔ, Δatr-ISAba24, intI1-aphA1b, intI1-Δasr, and atrΔ-asrΔ (Table 5).The identity of PCR amplicons was confirmed by DNA sequencing.• fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

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Table 2
Characteristics of the GC2 isolates containing AbGRI2 resistance island a. E1 and E2 refers to the GC2 isolates that were obtained from the environment of the ICU where the COVID-19 patients were admittedRows highlighted in the same colour indicate the same patternThe PCR in bold is a new linkage PCR that was performed for identification of AbGRI2 in this study.The predicted size for AbGRI2-12a and AbGRI2-12b is 1581 and 1772 bp, respectively

Table 3
Characteristics of the GC2 isolates containing AbGRI3 resistance island a. E1 and E2 refers to the GC2 isolates that were obtained from the environment of the ICU where the COVID-19 patients were admitted Rows highlighted in the same colour indicate the same pattern

Table 4
Primer pairs used for mapping of AbGRI1

Table 4
(continued).Predicted sizes based on Tn6022Δ1.For Tn6022 is 3410 bp PCRs in bold are the linkage PCRs that were performed for the identification of AbGRI1s in this study a

Table 5
Primer pairs used for mapping of AbGRI2

Table 6
Primer pairs used for mapping of AbGRI3