Molecular characterization and clonal dynamics of nosocomial blaOXA-23 producing XDR Acinetobacter baumannii

Abstract

The emergence of infections associated to new antimicrobial resistance in Acinetobacter baumannii (Ab) genotypes represents a major challenge. In this context, this study aimed to determine the diversity of resistance mechanisms and investigate clonal dissemination and predominant sequence types (STs) in multidrug-resistant Ab strains of clinical (tracheal aspirate, n = 17) and environmental (surface, n = 6) origins. Additionally, the major clones found in clinical (A) and environmental (H) strains had their complete genomes sequenced. All strains were submitted to polymerase chain reactions (PCR) for the detection of the ISAba1/blaOXA-51-like and ISAba1/blaOXA-23-like genes, while the expression of genes encoding the carO porin, AdeABC (adeB), AdeFGH (adeG), and AdeIJK (adeJ) efflux pumps was determined by real time PCR (qPCR). Most of the strains were characterized as extensively drug-resistant (XDR) with high minimal inhibitory concentrations (MICs) detected for tigecycline and carbapenems. Associations between ISAba1/OXA-51 and ISAba1/OXA-23 were observed in 91.3% and 52.2% of the strains, respectively. Only the adeB gene was considered hyper-expressed. Furthermore, most of the strains analyzed by the MuLtilocus Sequence-Typing (MLST) were found to belong to the clonal complex 113 (CC113). In addition, a new ST, ST1399, belonging to CC229, was also discovered herein. Strains analyzed by whole genome sequencing presented resistance genes linked to multidrug-resistance phenotypes and confirmed the presence of Tn2008, which provides high levels carbapenem-resistance.

About the Authors:

Sabrina Royer

Roles Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

Affiliation: Institute of Biomedical Sciences (ICBIM), Laboratory of Molecular Microbiology, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil

ORCID logo http://orcid.org/0000-0002-2678-227X

Paola Amaral de Campos

Roles Methodology, Visualization, Writing – original draft

Affiliation: Institute of Biomedical Sciences (ICBIM), Laboratory of Molecular Microbiology, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil

Bruna Fuga Araújo

Roles Methodology

Affiliation: Institute of Biomedical Sciences (ICBIM), Laboratory of Molecular Microbiology, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil

Melina Lorraine Ferreira

Roles Methodology

Affiliation: Institute of Biomedical Sciences (ICBIM), Laboratory of Molecular Microbiology, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil

Iara Rossi Gonçalves

Roles Methodology

Affiliation: Institute of Biomedical Sciences (ICBIM), Laboratory of Molecular Microbiology, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil

Deivid William da Fonseca Batistão

Roles Visualization, Writing – original draft

Affiliation: School of Medicine (FAMED), Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil

Rebecca Tavares e Silva Brígido

Roles Methodology

Affiliation: National Reference Center for Sanitary Dermatology and Leprosy (CREDESH) Clinical Hospital Federal University of Uberlândia, Uberlândia, Minas Gerais, Brazil

Louise Teixeira Cerdeira

Roles Methodology

Affiliation: Department of Clinical Analysis, School of Pharmacy, University of São Paulo, São Paulo, Brazil

Luiz Gustavo Machado

Roles Methodology

Affiliation: Institute of Biomedical Sciences (ICBIM), Laboratory of Molecular Microbiology, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil

Cristiane Silveira de Brito

Roles Methodology

Affiliation: Institute of Biomedical Sciences (ICBIM), Laboratory of Molecular Microbiology, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil

Paulo Pinto Gontijo-Filho

Roles Supervision, Writing – original draft, Writing – review & editing

Affiliation: Institute of Biomedical Sciences (ICBIM), Laboratory of Molecular Microbiology, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil

Rosineide Marques Ribas

Roles Conceptualization, Supervision, Writing – original draft, Writing – review & editing

Affiliation: Institute of Biomedical Sciences (ICBIM), Laboratory of Molecular Microbiology, Federal University of Uberlandia, Uberlandia, Minas Gerais, Brazil

Introduction

Acinetobacter baumannii (Ab) is one of the most successful pathogens responsible for hospital-acquired infections worldwide, mainly in immunocompromised patients and those admitted to intensive care units (ICUs) [1,2].

Carbapenem resistance in Ab is most frequently mediated by intrinsic (OXA-51-like) or acquired (mainly OXA-23-like) oxacillinases [3]. The hyper-expression of oxacillinases is often associated to insertion sequences (ISs), such as the ISAba1sequence, located upstream of the OXA genes [4–6]. In addition, the increased expression of chromosomal genes of resistance-nodulation-cell division (RND)-type efflux systems [2,7] and the decreased expression of certain outer membrane channel-forming proteins [8,9] play an important role in Ab multidrug resistance. The relative contribution of these resistance mechanisms remains poorly assessed in clinical contexts [8].

Regarding the clonal nature and the spread of different sequence types (STs), both Brazilian and Latin American studies conducted between 2011 and 2016 demonstrated that the most widespread Ab strains belong to CC113 and CC109 [10–16], characterized by MuLtilocus Sequence-Typing—University of Oxford (MLST-UO). Other studies worldwide indicate that multidrug-resistant Ab is related to CC92 [17,12,18–19]. Tracking the evolution and clonal dissemination of carbapenem resistance in Ab isolates is important to support the implementation of control strategies, which is only possible through a comprehensive understanding of the complete genome of these strains.

In this context, the aim of the present study was to investigate the clonal dissemination and genetic basis of resistance among multidrug-resistant Ab strains recovered from an adult ICU in Brazil. Additionally, one clinical and one environmental strain belonging to the prevalent clones had their complete genomes sequenced.

Material and methods

Bacterial strains and setting

The origin and epidemiological characteristics of the strains used herein are described in Table 1. Clinical and environmental A. baumannii isolates were obtained from patients with ventilator-associated pneumonia (VAP) and the environments around their beds (bedside table, bed rail and door knob), respectively. The strains were obtained from April 2011 to June 2012, in a 30-bed clinical-surgical intensive care unit (ICU) at the Clinical Hospital belonging to the Federal University of Uberlandia, Minas Gerais, Brazil. The 23 isolates investigated herein were selected according to their resistance profile and pulsotype, obtained through Pulsed Field Gel Electrophoresis (PFGE), as published previously [20].

[Figure omitted. See PDF.]

Table 1. Molecular characterization by polymerase chain reaction (PCR) of resistance determinants and distribution of MICs to carbapenems and tigecycline in 23 strains of A. baumannii recovered from endotracheal aspirate and environment in an adult intensive care unit.

https://doi.org/10.1371/journal.pone.0198643.t001

Clinical microbiology

Resistance to tigecycline, imipenem and meropenem was determined by the Etest® method according to the manufacturer’s guidelines (AB Biodisk, Solna, Sweden). To confirm the results of Etest® for tigecycline, the broth microdilution technique was performed. All the resistance tests and the quality control protocols were done in accordance with the Clinical and Laboratory Standards Institute recommended practices [21]. Since there were no breakpoints available for tigecycline for Acinetobacter spp., US Food and Drug Administration (FDA) tigecycline breakpoints listed for Enterobacteriaceae (≤ 2, 4 and ≥ 8 μg/ml for susceptible, intermediate and resistant strains, respectively) were applied. Multidrug-resistant (MDR) was defined as resistance to three or more categories of antibiotics while extensively drug-resistant (XDR) was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories, according to Magiorakos and collaborators [22].

Polymerase chain reaction

The DNA extraction was performed by thermal lysis and a conventional PCR assay was performed for 23 isolates to detect the following genes: ISAba1/blaOXA-51-like, ISAba1/blaOXA-23-like, omp33-36, carO (29 kDa), adeB, adeG and adeJ, using previous published primers (S1 Table) [23,4,24,25,26,2]. The amplified PCR products were visualized by electrophoresis on 1.5% agarose gels using the photo documentation System L-Pix EX (Loccus Biotechnology, Brazil).

Relative quantification of CarO porin and adeB, adeG and adeJ efflux system genes by real time PCR (qPCR)

Out of a total of 23 strains with their clonals profiles previously evaluated [20] 10 were selected for analysis by qPCR. The carO, adeB, adeG and adeJ transcription was evaluated by qPCR using Power-SYBR Green PCR Master Mix (Applied Biosystems). The single-copy housekeeping rpoB gene from Ab was used as endogenous gene for normalization (S1 Table) [2,27]. The relative quantification (RQ) results were presented as ratios of normalized target gene transcription between the Ab isolates and the Type Strain ATCC 19606 (calibrator), which were obtained according to the following equation: RQ = 2-ΔΔCT, where CT is the value corresponding to the crossing point of the amplification curve with the threshold; ΔCT = target CT or calibrator CT–endogenous CT; and ΔΔCT = target ΔCT—calibrator ΔCT. Reduced carO differential transcription of strains relative to that of ATCC 19606 was considered significant when the ratios obtained between RQ values (RQ value of calibrator/RQ value of strains) were ≥2.0 [28], and the overexpression of adeB, adeG and adeJ was considered significant when the ratios obtained between RQ values were ≥4.0 [2]. Each experiment was performed in triplicate in two independent assays.

Multilocus Sequence Typing (MLST)

The same 10 strains selected for the qPCR reaction were selected for genotyping by MultiLocus Sequence Typing (MLST) as described [29]. The methodology was carried out following the guidelines of the website <https://pubmlst.org/abaumannii/info/primers_Oxford.shtml>. The sequence of amplified internal fragments of housekeeping genes gltA, gyrB, gdhB, recA, cpn60, gpi and rpoD was determined and compared with those in the Ab MLST database of Oxford scheme [29]. Clonal complexes (CCs) were formed by Sequence Types (STs) with five or more identical alleles by goeBURST (goeburst.phyloviz.net).

Whole-genome sequencing

Two strains, one clinical and other environmental, representing prevalent clones previously evaluated by PFGE, were selected for whole-genome sequencing. The total genomic DNA of selected isolates was sequenced, using Illumina NexSeq 500 sequencer (Illumina, San Diego, CA), and the sequence reads were de novo assembled using Velvet pipeline. The pairwise alignment was performed by BLASTn homology searches (https://blast.ncbi.nlm.nih.gov), and in silico comparative analysis using the Center for Genomic Epidemiology (CGE) pipelines. The amino acid sequences of the CarO were aligned using ClustalW with sequences available from GenBank.

Statistical analysis

Statistical analyses were performed using GraphPad Prism v.5 (GraphPad Software, San Diego, CA). Quantitative assays were compared using one-way analysis of variance (ANOVA) and Bonferroni multiple comparison test. All tests were performed with a confidence level of 95% and statistical significance was defined as P≤0.05.

Ethical considerations

The data and the samples analyzed in the present study were obtained in accordance with the norms and approved by the Federal University of Uberlandia Ethics Committee (UFU), through license number 228/11.

Results

The majority (18/23; 78.3%) of strains included in this study were XDR. The presence of associations between ISAba1/OXA-51 and ISAba1/OXA-23 was observed in 21 (91.3%) and 12 (52.2%) strains, respectively. All analyzed environmental strains displayed the ISAba1 insertion element linked to both the blaOXA-51 and the blaOXA-23 genes (Table 1).

The presence of outer membrane proteins (OMP) genes carO and omp33-36, were detected at 82.6% (19/23) and 100%, frequencies, respectively. The presence of genes encoding the AdeABC (adeB), AdeFGH (adeG), and AdeIJK (adeJ) efflux pumps was evidenced in all analyzed strains. Most exhibited very high MICs for both carbapenems and tigecycline when evaluated by the Etest® method. For the latter antimicrobial drug, the results were confirmed by broth microdilution, and all strains were classified as resistant (Table 1).

The expression of the adeB, adeG, and adeJ genes, which encode RND type efflux systems components, and the gene encoding the OMP of 29 kDa (carO), were measured by qPCR. The relative gene expression of the tested strains compared to the reference is displayed Fig 1. Based on the transcription levels determined as cut-offs for the hyperexpression of the efflux systems and decreased OMP expression, only the adeB gene, which codifies the production of the AdeABC efflux pump, was considered as hyper-expressed. Significant differences in the expression levels of most of the strains in relation to the adeB gene were observed (Fig 1B), while the opposite was detected when analyzing the carO, adeG, and adeJ genes (Fig 1A, 1C and 1D).

[Figure omitted. See PDF.]

Fig 1. Relative gene expression of the carO gene and the genes related to the three efflux pumps (AdeABC, AdeFGH and AdeIJK), determined by qPCR.

The results are shown in relation to strain ATCC 19606 used as reference. Each sample was tested in triplicate in two independent assays. Results represent means plus standard deviation (error bars). *P<0.01; ** P<0.001; ***P<0.0001, using one-way ANOVA and Bonferroni multiple comparison test.

https://doi.org/10.1371/journal.pone.0198643.g001

MLST revealed eight STs and five CCs (Fig 2), distributed as follows: 1) CC113, ST227 (n = 2), ST233 (n = 1), and ST258 (n = 1); 2) CC229, ST1399, and ST1489; 3) CC109 and ST405 (n = 2); 4) ST/CC235; and 5) ST/CC231. ST1399 is described for the first time herein and was deposited in the MLST database (https://pubmlst.org/bigsdb?db=pubmlst_abaumannii_oxford_seqdef). The three environmental strains included in this analysis belonged to distinct CCs: CC229, CC113, and CC109. CC109 and CC113 were the only CCs presenting strains with the same ST in both environmental and clinical specimens.

[Figure omitted. See PDF.]

Fig 2. Diagram constructed using the goeBURST algorithm and displayed on phyloviz software (PHYLOVIZ Online) indicating the similarity among sequence types (STs).

The Clonal Complexes (CCs) and STs observed in the present study are enlarged and highlighted by color.

https://doi.org/10.1371/journal.pone.0198643.g002

The total genome belonging to the Ab13 and Ab15 strains exhibited sizes of 3.76 Mb and 3.69 Mb, respectively, and generated a total of 7,694,708 and 9,944,440 reads, respectively, yielding approximately 290X and 370X sequence coverages. The whole genome sequencing analysis confirmed that the Ab13 strain (tracheal aspirate, clone A) belongs to ST/CC231, while the Ab15 strain (environmental, clone H) belongs to ST405/CC109. Resistance to carbapenems was explained by the presence of the Tn2008 (ISAba1-blaOXA-23) transposon, located in plasmids (Fig 3A). Both strains exhibited the blaOXA-69 gene, a variant of the intrinsically encoded blaOXA-51-like gene, while third-generation cephalosporin resistance occurs by increased transcription of the ampC gene (blaADC-25) when associated with ISAba1 (Fig 3B).

[Figure omitted. See PDF.]

Fig 3.

(A) Schematic representation of Tn2008 with ISAba1 located upstream of the blaOXA-23 gene in the analyzed strains. (B) Schematic representation of the ISAba1 localization upstream to the blaADC-25 gene.

https://doi.org/10.1371/journal.pone.0198643.g003

In the Ab13 strain, resistance to aminoglycosides is justified by the presence of an enzyme that modifies this drug (aacA4). In the Ab15 strain, the genes associated to aminoglycoside resistance are aacA4, strA, and strB, and floR and sul2 for chloramphenicol and sulfonamides, respectively. The plasmid-mediated quinolone resistance gene (PMQR) aac(6')Ib-cr, was identified in both strains.

Sequencing of the carO gene revealed that both analyzed strains exhibit an isoform protein different from those already registered in the NCBI database (CarOa and CarOb) but with a similarity of 99% to the SJ22 strain registered by Sen and Joshi [30] (Accession number in GenBank KP658474.1), differing only in a change at position 218 (substitution of glutamine for lysine, Q218K) (Fig 4). The strains displayed 93% identity when compared to the reference ATCC 19606 strain (CarOa; accession number in GenBank KP658473), which is susceptible to carbapenems due to the presence of point mutations. In addition, no genetic disruption by ISAba1 insertion was observed; therefore, no protein inactivation occurred.

[Figure omitted. See PDF.]

Fig 4. Sequence alignment of carO gene for two representative Acinetobacter baumannii strains from this study (Ab13 and Ab15) with SJ22 (GenBank: KP658474.1) and ATCC 19606 (GenBank: KP658473.1) strains, indicating homology and difference in amino acids.

The alignment was performed using ClustalW. The dotted line indicates the point mutation region (Q218K).

https://doi.org/10.1371/journal.pone.0198643.g004

Discussion

Resistance to antibiotics in Ab has reached alarming levels worldwide, particularly for carbapenems, and strains have been shown to be susceptible only to polymyxins [31–35]. In Brazil, unfortunately, resistance rates to carbapenems are very high (80.7%) [36] and, according to a study carried out by Rossi et al. (2017)[37], a variation of 30 to 70% in resistance to carbapenems was detected in Acinetobacter species between 2010 and 2014.

The increase in resistance to carbapenems in clinical Ab strains is mainly associated to the dissemination of OXA-23-producing strains [38–43,26,44], which can be explained by the fact that this gene can also be allocated in plasmids, partly justifying its global reach [45–48]. Allied to this, the presence of specific ISs, such as ISAba1, located adjacent to the blaOXA genes, leads to an increase of their expression, resulting in a further decrease in susceptibility to carbapenems [49,4,50–52].

In the present study, approximately half of the analyzed strains were associated to ISAba1/OXA-23, related to high MICs for carbapenems. Viana et al. (2016) [53] observed similar data (i.e., an increase in this association from 22 to 73% between 2009 and 2013, also correlated to elevated MICs for carbapenems. In some of the evaluated strains, no association with either ISAba1/OXA-51 or ISAba1/OXA-23 was observed, despite high MICs, which can be justified by the coexistence of other resistance mechanisms in these strains. These results suggest that the ISAba1/OXA-23 combination may be one of the most significant resistance mechanisms in Ab associated to high XDR phenotypes frequencies and high MICs for carbapenems.

Carbapenem resistance in Ab [54–55] may also result from modifications in the primary structure or loss of outer membrane proteins (OMPs) (porins), such as the 33–36 kDa and the 29 kDa protein named CarO [9]. In the case of OMP CarO, modifications are mostly a result from the rupture of the gene by several insertion elements [56]. From these results, we can infer that the CarO protein is not responsible for carbapenem resistance in the evaluated strains, since no reduced expression levels of this protein were observed. However, the sequence analysis of the carO gene revealed a protein isoform different from those already registered in the NCBI database (CarOa and CarOb). A point mutation in the protein (Q218K) has not been reported so far, so it was not possible to determine its exact participation in imipenem resistance.

Another antimicrobial resistance mechanism present in this microorganism is the hyper-expression of efflux pumps, such as those belonging to the RND family (AdeABC, AdeFGH, and AdeIJK) [8]. In addition, AdeABC and AdeFGH play an important role in acquired resistance [57,2], while AdeIJK contributes to intrinsic resistance [58]. Only the adeB gene was hyper-expressed based on the qPCR results. According to the literature [7], strains containing the AdeABC pump confer resistance to various antibiotics, including most β-lactams, aminoglycosides, fluoroquinolones, tetracyclines, tigecycline, macrolides, lincosamides, and chloramphenicol. Thus, alongside the presence of ISAba1/OXA-23, this pump is another important mechanism present in the strains analyzed herein.

Tigecycline is an interesting therapeutic option to treat infection by carbapenem-resistant Gram-negative bacteria [59]. However, the isolates evaluated herein demonstrated resistance to this antimicrobial by the Etest® and microdilution methods. A confirmatory evaluation of MICs ≥ 2 μg/ml determined by Etest® is mandatory, as these MICs values could be up to four-fold higher than those obtained by the microdilution method [60,61,62]. The results reported herein corroborate these findings and confirm tigecycline resistance of the strains investigated in this study.

In addition to the genes responsible for carbapenem resistance and high MIC values for tigecycline, most of the strains analyzed by MLST belonged to CC113, confirming its dissemination in Brazil and Latin America [12,63,13–14,64,15–16]. Additionally, a new ST, ST1399, belonging to CC229, is reported. In Brazil, deserves attention another CC identified in carbapenem-resistant Ab strains, CC109, corresponding to the international clone 1 [11,12,64].

Whole genome sequencing of the Ab13 and Ab15 strains demonstrated that carbapenem-resistance is mainly due to the presence of Tn2008, which is easily propagated among strains and includes different clones (as evidenced herein) disseminated worldwide [65,66]. In addition, the intrinsic resistance to third generation cephalosporins is due to the increased transcription of the blaADC-25 gene, due to the presence of ISAba1, adjacent to the gene, acting as a strong promoter, as described in the literature [67–68,5]. Another detected mechanism was the presence of genes encoding aminoglycoside-modifying enzymes, such as aacA4 [68,5]. In addition, the PMQR gene aac(6')Ib-cr was also identified. This is relevant data, since this gene is well described in Enterobactericeae family members and in Pseudomonas aeruginosa [69–71], but has seldom been studied in Ab [72].

Conclusions

Although a new ST was detected herein in Ab, the present study also observed a wide variety of CCs related to XDR strains carrying the blaOXA-23 gene, frequently associated with ISAba1 (Tn2008) as the main carbapenem-resistance mechanism, in addition to the hyperexpression of the AdeABC efflux pump. Understanding resistance mechanisms and the pathogenic potential of this microorganism, both from environmental and clinical origins, aids in explaining its persistence in the hospital environment and can provide tools to improve the treatment of serious infections, as well as increase control and prevention regarding these infections.

Nucleotide sequence accession numbers

The nucleotide sequence data underlying this study have been uploaded to GenBank under the accession numbers NKKO00000000 (Ab13) and NKKP00000000 (Ab15).

Supporting information

[Figure omitted. See PDF.]

S1 Table. Primer sequences of genes confirmed with PCR and qPCR.

https://doi.org/10.1371/journal.pone.0198643.s001

(DOCX)

Acknowledgments

The authors wish to thank members of Laboratory of Bacterial Resistance and Therapeutic Alternatives, Department of Microbiology—ICBII / USP, especially PhD Nilton Licopan for assisting in the analysis of sequencing. We are grateful to PhD Ana Paula Guedes Frazzon (Federal University of Rio Grande do Sul) who kindly provided the control strain A. baumannii ATCC 19606. We thank FAPEMIG (Fundação de Amparo à Pesquisa de Minas Gerais), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the financial support and scholarships.

Citation: Royer S, de Campos PA, Araújo BF, Ferreira ML, Gonçalves IR, Batistão DWdF, et al. (2018) Molecular characterization and clonal dynamics of nosocomial blaOXA-23 producing XDR Acinetobacter baumannii. PLoS ONE 13(6): e0198643. https://doi.org/10.1371/journal.pone.0198643

References

1. Lee CR, Lee JH, Park M, Park KS, Bae IK, Kim YB, et al. Biology of Acinetobacter baumannii: Pathogenesis, antibiotic resistance mechanisms, and prospective treatment options. Front Cell Infect Microbiol. 2017: 13:7–55.

2. Yoon EJ, Courvalin P, Grillot-Courvalin C. RND-type efflux pumps in multidrug-resistant clinical isolates of Acinetobacter baumannii: major role for AdeABC overexpression and AdeRS mutations. Antimicrob Agents Chemother. 2013: 57:2989–2995. pmid:23587960

3. Evans BA, Amyes SG. OXA β-lactamases. Clin Microbiol Rev. 2014: 27:241–263. pmid:24696435

4. Turton JF, Ward ME, Woodford N, Kaufmann ME, Pike R, Livermore DM, et al. The role of IS Aba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii. FEMS Microbiol Lett. 2006: 258:72–7. pmid:16630258

5. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev. 2008: 21:538–82. pmid:18625687

6. Pagano M, Martins AF, Machado ABMP, Barin J, Barth AL. Carbapenem-susceptible Acinetobacter baumannii carrying the ISAba1 upstream blaOXA-51-like gene in Porto Alegre, southern Brazil. Epidemiol Infect. 2013: 141:330–3. pmid:22717017

7. Yoon EJ, Chabane YN, Goussard S, Snesrud E, Courvalin P, Dé E, et al. Contribution of resistance-nodulation-cell division efflux systems to antibiotic resistance and biofilm formation in Acinetobacter baumannii. MBio. 2015: 6:e00309–15. pmid:25805730

8. Vila J, Martí S, Sánchez-Céspedes J. Porins, efflux pumps and multidrug resistance in Acinetobacter baumannii. J Antimicrob Chemother. 2007: 59:1210–5. pmid:17324960

9. Novovic K, Mihajlovic S, Vasiljevic Z, Filipic B, Begovic J, Jovcic B. Carbapenem-resistant Acinetobacter baumannii from Serbia: Revision of CarO classification. PloS One. 2015: 10:e0122793. pmid:25822626

10. Grosso F, Carvalho KR, Quinteira S, Ramos A, Carvalho-Assef APDA, Asensi M D, et al. OXA-23-producing Acinetobacter baumannii: a new hotspot of diversity in Rio de Janeiro? J Antimicrob Chemother. 2011: 66:62–5. pmid:21051372

11. Martins N, Martins IS, Freitas WV, Matos JA, Magalhães ACG, Girão VBC, et al. Severe infection in a lung transplant recipient caused by donor-transmitted carbapenem-resistant Acinetobacter baumannii. Transplant Infectious Disease, 2012: 14:316–320. pmid:22168176

12. Clímaco EC, de Oliveira ML, Pitondo-Silva A, Oliveira MG, Medeiros M, Lincopan N, et al. Clonal complexes 104, 109 and 113 playing a major role in the dissemination of OXA-carbapenemase-producing Acinetobacter baumannii in Southeast Brazil. Infect Genet Evol. 2013: 19:127–33. pmid:23838284

13. Martins N, Martins IS, de Freitas WV, de Matos JA, Girão VBDC, Coelho-Souza T, et al. Imported and intensive care unit-born Acinetobacter baumannii clonal complexes: one-year prospective cohort study in intensive care patients. Microb Drug Resist. 2013: 19:216–23. pmid:23336529

14. Stietz MS, Ramírez MS, Vilacoba E, Merkier AK, Limansky AS, Centrón D, Catalano M. Acinetobacter baumannii extensively drug resistant lineages in Buenos Aires hospitals differ from the international clones I–III. Infect Genet Evol. 2013: 14:294–301. pmid:23313831

15. Ramirez MS, Montaña S, Cassini M, Centron D. Preferential carriage of class 2 integrons in Acinetobacter baumannii CC113 and novel singletons. Epidemiol Infect. 2015: 143:3118–21. pmid:25697643

16. Leite GC, Oliveira MS, Perdigão-Neto LV, Rocha CKD, Guimarães T, Rizek C, et al. Antimicrobial combinations against pan-resistant Acinetobacter baumannii isolates with different resistance mechanisms. PloS One, 2016: 11: e0151270. pmid:26998609

17. Karah N, Sundsfjord A, Towner K, Samuelsen Ø. Insights into the global molecular epidemiology of carbapenem non-susceptible clones of Acinetobacter baumannii. Drug Resist Updat. 2012: 15:237–47. pmid:22841809

18. Medeiros M, Lincopan N. Oxacillinase (OXA)-producing Acinetobacter baumannii in Brazil: clinical and environmental impact and therapeutic options. J. Bras. Patol. Med. Lab. 2013: 49:391–405.

19. Huang G, Yin S, Gong Y, Zhao X, Zou L, Jiang B, et al. Multilocus sequence typing analysis of carbapenem-resistant Acinetobacter baumannii in a Chinese burns institute. Front Microbiol. 2016: 7:1717. pmid:27881972

20. Royer S, Faria ALS, Seki LM, Chagas TPG, de Campos PA, Batistão DWF, et al. Spread of multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa clones in patients with ventilator-associated pneumonia in an adult intensive care unit at a university hospital. Braz J Infect Dis. 2015: 19:350–7. pmid:25997783

21. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Five Informational Supplement M100-S25. CLSI, Wayne, P.A. 2015.

22. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012: 18:268–81. pmid:21793988

23. Segal H, Garny S, Elisha BG. Is IS(ABA-1) customized for Acinetobacter? FEMS Microbiol Lett. 2005: 243:425–9. pmid:15686845

24. Mostachio AK, Levin AS, Rizek C, Rossi F, Zerbini J, Costa SF. High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil. Int J Antimicrob Agents. 2012: 39:396–401. pmid:22455794

25. Lu PL, Doumith M, Livermore DM, Chen TP, Woodford N. Diversity of carbapenem resistance mechanisms in Acinetobacter baumannii from a Taiwan hospital: spread of plasmid-borne OXA-72 carbapenemase. J Antimicrob Chemother. 2009: 63:641–7. pmid:19182237

26. Lin L, Ling BD, Li XZ. Distribution of the multidrug efflux pump genes, adeABC, adeDE and adeIJK, and class 1 integron genes in multiple-antimicrobial-resistant clinical isolates of Acinetobacter baumannii–Acinetobacter calcoaceticus complex. Int J Antimicrob Agents. 2009: 33:27–32. pmid:18790612

27. Cardoso JP, Cayô R, Girardello R, Gales AC. Diversity of mechanisms conferring resistance to β-lactams among OXA-23–producing Acinetobacter baumannii clones. Diagn Microbiol Infect Dis. 2016: 85:90–7. pmid:26971181

28. Fonseca EL, Scheidegger E, Freitas FS, Cipriano R, Vicente ACP. Carbapenem-resistant Acinetobacter baumannii from Brazil: role of carO alleles expression and blaOXA-23 gene. BMC Microbiol. 2013: 13:245. pmid:24195496

29. Bartual SG, Seifert H, Hippler C, Luzon MAD, Wisplinghoff H, Rodríguez-Valera F. Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii. J Clin Microbiol. 2005: 43:4382–90. pmid:16145081

30. Sen B, Joshi SG. Studies on Acinetobacter baumannii involving multiple mechanisms of carbapenem resistance. J Appl Microbiol. 2016: 120:619–29. pmid:26709119

31. Pendleton JN, Gorman SP, Gilmore BF. Clinical relevance of the ESKAPE pathogens. Expert Rev Anti Infect Ther. 2013: 11:297–308. pmid:23458769

32. Jones RN, Flonta M, Gurler N, Cepparulo M, Mendes RE, Castanheira M. Resistance surveillance program report for selected European nations (2011). Diagn Microbiol Infect Dis. 2014: 78: 429–436. pmid:24440509

33. Costello SE, Gales AC, Morfin-Otero R, Jones RN, Castanheira M. Mechanisms of Resistance, Clonal Expansion, and Increasing Prevalence of Acinetobacter baumannii Strains Displaying Elevated Tigecycline MIC Values in Latin America. Microb Drug Resist. 2016: 22: 253–258. pmid:26716768

34. Jasemi S, Douraghi M, Adibhesami H, Zeraati H, Rahbar M, Boroumand MA, et al. Trend of extensively drug-resistant Acinetobacter baumannii and the remaining therapeutic options: a multicenter study in Tehran, Iran over a 3-year period. Lett Appl Microbiol. 2016: 63:466–472. pmid:27626896

35. Gao L, Lyu Y, Li Y. Trends in Drug Resistance of Acinetobacter baumannii over a 10-year Period: Nationwide Data from the China Surveillance of Antimicrobial Resistance Program. Chin Med J (Engl). 2017: 130:659–664.

36. ANVISA—National Health Surveillance Agency. Newsletter—Patient Safety and Quality of Health Services. www.anvisa.gov.br/. 2014.

37. Rossi F, Girardello R, Cury AP, Di Gioia TSR, Almeida JND Jr, Duarte AJDS. Emergence of colistin resistance in the largest university hospital complex of São Paulo, Brazil, over five years. Braz J Infect Dis. 2017: 21:98–101. pmid:27832961

38. Dalla-Costa LM, Coelho JM, Souza HA, Castro ME, Stier CJ, Bragagnolo KL, et al. Outbreak of carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme in Curitiba, Brazil. J Clin Microbiol. 2003: 41:3403–6. pmid:12843104

39. Carvalho KR, Carvalho-Assef APDA, Peirano G, dos Santos LCG, Pereira MJF, Asensi MD. Dissemination of multidrug-resistant Acinetobacter baumannii genotypes carrying blaOXA-23 collected from hospitals in Rio de Janeiro, Brazil. Int J Antimicrob Agents. 2009: 34:25–8. pmid:19216059

40. Martins AF, Kuchenbecker R, Sukiennik T, Boff R, Reiter KC, Lutz L, et al. Carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme: dissemination in Southern Brazil. Infection. 2009: 37:474–6. pmid:19768380

41. Mostachio AK, van der Heidjen I, Rossi F, Levin AS, Costa SF. Multiplex PCR for rapid detection of genes encoding oxacillinases and metallo-β-lactamases in carbapenem-resistant Acinetobacter spp. J Med Microbiol. 2009: 58:1522–4. pmid:19574410

42. Higgins PG, Lehmann M, Seifert H. Inclusion of OXA-143 primers in a multiplex polymerase chain reaction (PCR) for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents. 2010: 35:305.

43. Chagas TPG, Carvalho KR, de Oliveira Santos IC, Carvalho-Assef APDA, Asensi MD. Characterization of carbapenem-resistant Acinetobacter baumannii in Brazil (2008–2011): countrywide spread of OXA-23–producing clones (CC15 and CC79). Diagn Microbiol Infect Dis. 2014: 79:468–72. pmid:24880823

44. Vasconcelos AT, Barth AL, Zavascki AP, Gales AC, Levin AS, Lucarevschi BR, et al. The changing epidemiology of Acinetobacter spp. producing OXA carbapenemases causing bloodstream infections in Brazil: a BrasNet report. Braz J Infect Dis. 2015: 19:350–7.

45. Brown S, Amyes S. OXA β-lactamases in Acinetobacter: the story so far. J Antimicrob Chemother. 2005: 57:1–3. pmid:16332731

46. Labarca JA, Salles MJC, Seas C, Guzmán-Blanco M. Carbapenem resistance in Pseudomonas aeruginosa and Acinetobacter baumannii in the nosocomial setting in Latin America. Crit Rev Microbiol. 2016: 42:276–92. pmid:25159043

47. Kamolvit W, Sidjabat HE, Paterson DL. Molecular epidemiology and mechanisms of carbapenem resistance of Acinetobacter spp. in Asia and Oceania. Microb Drug Resist. 2015: 21:424–34. pmid:25714653

48. Nigro SJ, Hall RM. Structure and context of Acinetobacter transposons carrying the oxa23 carbapenemase gene. J Antimicrob Chemother. 2016: 71:1135–47. pmid:26755496

49. Poirel L, Nordmann P. Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology. Clin Microbiol Infect. 2006: 12:826–36. pmid:16882287

50. Ruiz M, Marti S, Fernandez-Cuenca F, Pascual A, Vila J. High prevalence of carbapenem-hydrolysing oxacillinases in epidemiologically related and unrelated Acinetobacter baumannii clinical isolates in Spain. Clin Microbiol Infect. 2007: 13:1192–8. pmid:17850347

51. Zong Z, Lü X, Valenzuela JK, Partridge SR, Iredell J. An outbreak of carbapenem-resistant Acinetobacter baumannii producing OXA-23 carbapenemase in western China. Int J Antimicrob Agents. 2008: 31:50–4. pmid:18077141

52. Anh NT, Nga TVT, Tuan HM, Tuan NS, Chau NVV, Baker S, et al. Molecular epidemiology and antimicrobial resistance phenotypes of Acinetobacter baumannii isolated from patients in three hospitals in southern Vietnam. J Med Microbiol. 2017: 66:46–53. pmid:28198682

53. Viana GF, Zago MCB, Moreira RRB, Zarpellon MN, Menegucci TC, Cardoso CL, et al. ISAba1/blaOXA-23: A serious obstacle to controlling the spread and treatment of Acinetobacter baumannii strains. Am J Infect Control. 2016: 44:593–5. pmid:26804302

54. Kempf M, Rolain JM. Emergence of resistance to carbapenems in Acinetobacter baumannii in Europe: clinical impact and therapeutic options. Int J Antimicrob Agents. 2012: 39:105–14. pmid:22113193

55. Opazo A, Domínguez M, Bello H, Amyes SG, González-Rocha G. OXA-type carbapenemases in Acinetobacter baumannii in South America. J Infect Dev Ctries. 2011: 6:311–6.

56. Catel-Ferreira M, Coadou G, Molle V, Mugnier P, Nordmann P, Siroy A, et al. Structure–function relationships of CarO, the carbapenem resistance-associated outer membrane protein of Acinetobacter baumannii. J Antimicrob Chemother. 2011: 66:2053–6. pmid:21705362

57. Coyne S, Courvalin P, Périchon B. Efflux-mediated antibiotic resistance in Acinetobacter spp. Antimicrob Agents Chemother. 2011: 55:947–53. pmid:21173183

58. Damier-Piolle L, Magnet S, Brémont S, Lambert T, Courvalin P. AdeIJK, a resistance-nodulation-cell division pump effluxing multiple antibiotics in Acinetobacter baumannii. Antimicrob Agents Chemother. 2008: 52:557–62. pmid:18086852

59. Gerson S, Nowak J, Zander E, Ertel J, Wen Y, Krut O, et al. Diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with tigecycline resistance in Acinetobacter baumannii. J Antimicrob Chemother. 2018: 73:1501–1508. pmid:29554339

60. Zavascki AP, Carvalhaes CG, Picão RC, Gales AC. Multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii: resistance mechanisms and implications for therapy. Expert Rev Anti Infect Ther. 2010: 8:71–93. pmid:20014903

61. Rahal JJ. Novel antibiotic combinations against infections with almost completely resistant Pseudomonas aeruginosa and Acinetobacter species. Clin Infect Dis. 2006: 43:S95–S99. pmid:16894522

62. Casal M, Rodríguez F, Johnson B, Garduno E, Tubau F, de Lejarazu RO, et al. Influence of testing methodology on the tigecycline activity profile against presumably tigecycline-non-susceptible Acinetobacter spp. J Antimicrob Chemother. 2009: 64: 69–72. pmid:19451133

63. Coelho-Souza T, Reis JN, Martins N, Martins IS, Menezes AO, Reis MGD, et al. Longitudinal surveillance for meningitis by Acinetobacter in a large urban setting in Brazil. Clin Microbiol Infect. 2013: 19:E241–4. pmid:23398654

64. Martins N, Picão RC, Adams-Sapper S, Riley LW, Moreira BM. Association of class 1 and 2 integrons with multidrug-resistant Acinetobacter baumannii international clones and Acinetobacter nosocomialis isolates. Antimicrob Agents Chemother. 2015: 59:698–701. pmid:25348522

65. Wang X, Zong Z, Lü X. Tn2008 is a major vehicle carrying blaOXA-23 in Acinetobacter baumannii from China. Diagn Microbiol Infect Dis. 2011: 69:218–22. pmid:21251570

66. Chen Y, Gao J, Zhang H, Ying C. Spread of the blaOXA–23-Containing Tn2008 in Carbapenem-Resistant Acinetobacter baumannii Isolates Grouped in CC92 from China. Front Microbiol. 2017: 8:163. pmid:28220115

67. Hamidian M, Hall RM. ISAba1 targets a specific position upstream of the intrinsic ampC gene of Acinetobacter baumannii leading to cephalosporin resistance. J Antimicrob Chemother. 2013: 68:2682–3. pmid:23788477

68. Karah N, Dwibedi CK, Sjöström K, Edquist P, Johansson A, Wai SN, et al. Novel aminoglycoside resistance transposons and transposon-derived circular forms detected in carbapenem-resistant Acinetobacter baumannii clinical isolates. Antimicrob Agents Chemother. 2016: 60:1801–18. pmid:26824943

69. Rodríguez-Martínez JM, Cano ME, Velasco C, Martínez-Martínez L, Pascual Á. Plasmid-mediated quinolone resistance: an update. J Infect Chemother. 2011: 17:149–82. pmid:20886256

70. Jiang X, Yu T, Jiang X, Zhang W, Zhang L, Ma J. Emergence of plasmid-mediated quinolone resistance genes in clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa in Henan, China. Diagn Microbiol Infect Dis. 2014: 79:381–3. pmid:24805186

71. Araujo BF, Ferreira ML, de Campos PA, Royer S, da Fonseca Batistão DW, Dantas RCC, et al. Clinical and Molecular Epidemiology of Multidrug-Resistant P. aeruginosa carrying aac(6')-Ib-cr, qnrS1 and blaSPM Genes in Brazil. PLoS One. 2016: 11:e0155914. pmid:27219003

72. Lee CR, Lee JH, Park M, Park KS, Bae IK, Kim YB, et al. Biology of Acinetobacter baumannii: Pathogenesis, antibiotic resistance mechanisms, and prospective treatment options. Front Cell Infect Microbiol. 2017: 7:55. pmid:28348979