Mutation of CarO participates in drug resistance in imipenem‐resistant Acinetobacter baumannii

Abstract Objective Acinetobacter baumannii has become an important problem because of the high drug resistance rate. The aim of this study was to assess the antimicrobial resistance profile and explore the role of membrane porin in imipenem resistance of A baumannii. Methods A total of 63 isolates of imipenem‐resistant A baumannii (IRAB) and 21 of imipenem‐sensitive A baumannii (ISAB) were collected. Susceptibility testing to 16 kinds of antimicrobial agents was conducted by K‐B method. PCR technique was used to detect carO and oprD genes, and sequencing was performed to compare the sequence between IRAB and ISAB. Three‐dimensional structure model of CarO protein was established. Results While ISAB isolates presented sensitive to most classes of antibiotics, isolates of IRAB displayed much higher resistance rate except tigecycline (3.2%), cefoperazone/sulbactam (28.6%), and minocycline (30.2%). All 84 isolates were observed carrying both carO and oprD genes. Further sequencing revealed important mutations of carO gene existed in IRAB in comparison with ISAB. Meanwhile, significant differences in three‐dimensional structure of carO protein molecule were also found between IRAB and ISAB. Conclusions The drug resistance profile of IRAB is increasingly severe in clinical settings. Mutation of CarO was identified as one of the molecular mechanisms involved in imipenem resistance in A baumannii.

proteins OMPs) loss or modification. 3 Carbapenemase was considered the main cause of imipenem resistance in the previous study, although it was reported that there was no carbapenemase detected in some IRAB. 4 The OMPs have been a hot research topic in recent years. The major OMPs associated with imipenem resistance include CarO and OprD. 5 CarO protein, generally considered as an eight-stranded β-barrel protein of 29 KDa, 6 plays a key role in the influx of imipenem (but not meropenem) into A baumannii. It could be divided into two groups, CarOa and CarOb, in which CarOb was showed to be twice more specific for imipenem than CarOa. 7 Mutations in the carO gene would alter the structure, decrease or delete the expression of the porin, resulting in reduction of antibiotics entry into the bacteria. OprD, a 43 KDa porin, is the main and specific porin for uptake of carbapenems into A baumannii. 8 A baumannii also possesses an OprD homologue, which is normally considered to be involved in carbapenems resistance. 9,10 However, a few studies suggested that OprD homologue in A baumannii does not really associate with resistance to carbapenem. 11,12 The role of CarO and OprD in A baumannii is still in controversy. This study aims to assess the antimicrobial resistance profile and explore the role of CarO and OprD in imipenem resistance of A baumannii.

| Bacterial isolates
From January to June 2016, a total of 84 nonduplicate isolates of A baumannii, including 63 IRAB and 21 ISAB, were randomly collected from many kinds of specimens such as sputum, pus, drainage fluid, urine, blood, and pharyngeal swabs in Shanxi Dayi Hospital, a teaching hospital with over 1000 beds. All strains selected were confirmed by Vitek2 compact system (bioMerieux Co., French). The strain of A baumannii ATCC25922 purchased from the National Center of Clinical Laboratory served as a control.

| Sequencing analysis
PCR products from both IRAB and ISAB isolates were sequenced by Invitrogen Ltd. Co., Shanghai, China. The carO and oprD gene sequences were analyzed by DNASTAR software. Additionally, amino acid sequences translated from these gene sequences were also analyzed.

| Three-dimensional structural modeling of CarO protein
CarO amino acid sequences from one strain of IRAB and one strain of ISAB were submitted to SWISS-MODEL workspace for three-dimensional structure modeling (PBD:4rlb as a template), and the similarity analysis was performed by molecular visual software Cn3D.

| Antimicrobial susceptibility patterns
The susceptibility profiles of total 84 isolates are presented in

| Detection of OMPs genes
All 84 isolates were observed carrying both carO and oprD genes.
Electrophoresis of these genes is shown in Figure 1.

| Three-dimensional structural modeling
Amino acid sequences translated from carO gene sequences exhibited about 80.9% similarity coefficient between IRAB and ISAB. The In summary, the resistance profile of IRAB is increasingly severe in clinical settings. Conformation change in CarO porin was identified as one of the important molecular mechanisms involved in imipenem resistance in A baumannii.