Isolation and Whole-genome Sequence Analysis of the Imipenem Heteroresistant Acinetobacter baumannii Clinical Isolate HRAB-85

Accepted Manuscript Title: Isolation and Whole-genome Sequence Analysis of the Imipenem Heteroresistant Acinetobacter baumannii Clinical Isolate HRAB-85 Authors: Puyuan Li, Yong Huang, Lan Yu, Yannan Liu, Wenkai Niu, Dayang Zou, Huiying Liu, Jing Zheng, Xiuyun Yin, Jing Yuan, Xin Yuan, Changqing Bai PII: DOI: Reference:

S1201-9712(17)30183-2 http://dx.doi.org/doi:10.1016/j.ijid.2017.07.005 IJID 2984

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International Journal of Infectious Diseases

Received date: Revised date: Accepted date:

21-4-2017 2-6-2017 4-7-2017

Please cite this article as: Li Puyuan, Huang Yong, Yu Lan, Liu Yannan, Niu Wenkai, Zou Dayang, Liu Huiying, Zheng Jing, Yin Xiuyun, Yuan Jing, Yuan Xin, Bai Changqing.Isolation and Whole-genome Sequence Analysis of the Imipenem Heteroresistant Acinetobacter baumannii Clinical Isolate HRAB-85.International Journal of Infectious Diseases http://dx.doi.org/10.1016/j.ijid.2017.07.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Isolation and Whole-genome Sequence Analysis of the Imipenem Heteroresistant Acinetobacter baumannii Clinical Isolate HRAB-85

Puyuan Lia#, Yong Huangb#, Lan Yuc#, Yannan Liua, Wenkai Niua, Dayang Zoud, Huiying Liua, Jing Zhenga, Xiuyun Yine, Jing Yuand*, Xin Yuana*, Changqing Baia*

a

Department of Respiratory and Critical Care Diseases, 307th Hospital of PLA, Beijing

100071, China b

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and

Epidemiology, Beijing 100071, China c

Department of Gastroenterology, Navy General Hospital, 6 Fucheng Road, Beijing 100048,

China d

Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing

100071, China e

Department of Clinical Laboratory, 307th Hospital of PLA, Beijing 100071, China

*Correspondence: Professor Changqing Bai Department of Respiratory and Critical Care Diseases, 307th Hospital of PLA, No. 8 Dongda Street, Fengtai District, Beijing 100071, China Email: [email protected]

Professor Xin Yuan Department of Respiratory and Critical Care Diseases, 307th Hospital of PLA, No. 8 Dongda

Street, Fengtai District, Beijing 100071, China Email: [email protected]

Professor Jing Yuan Institute of Disease Control and Prevention, Academy of Military Medical Sciences, No. 20 Dongda Street, Fengtai District, Beijing 100071, China Email: [email protected]

#

These authors contributed equally to this work.

Highlights  We isolated/characterised an imipenem heteroresistant A. baumannii strain (HRAB-85) 

Subpopulations grew in the presence of imipenem at concentrations of up to 64 μg/mL



The total length of strain HRAB-85 was 4,098,585 bp with a GC content of 39.98%



Whole-genome sequencing revealed four ISs and 19 antibiotic-resistance genes



Our results improve our understanding of heteroresistant A. baumannii

ABSTRACT Objectives: Heteroresistance is a phenomenon in which there are various responses to antibiotics from bacterial cells within the same population. Here, we isolated and characterised an imipenem heteroresistant Acinetobacter baumannii strain (HRAB-85). Methods: The genome of strain HRAB-85 was completely sequenced and analysed to understand its antibiotic resistance mechanisms. Population analysis and multilocus sequence typing were performed. Results: Subpopulations grew in the presence of imipenem at concentrations of up to 64

μg/mL, and the strain was found to belong to ST208. The total length of strain HRAB-85 was 4,098,585 bp with a GC content of 39.98%. The genome harboured at least four insertion sequences: the common ISAba1, ISAba22, ISAba24, and newly reported ISAba26. Additionally, 19 antibiotic-resistance genes against eight classes of antimicrobial agents were found, and 11 genomic islands (GIs) were identified. Among them, GI3, GI10, and GI11 contained many ISs and antibiotic-resistance determinants. Conclusions: The existence of imipenem heteroresistant phenotypes in A. baumannii was substantiated in this hospital, and imipenem pressure, which could induce imipenem-heteroresistant subpopulations, may select for highly resistant strains. The complete genome sequencing and bioinformatics analysis of HRAB-85 could improve our understanding of the epidemiology and resistance mechanisms of carbapenem-heteroresistant A. baumannii.

Keywords: Acinetobacter baumannii, heteroresistance, carbapenem, genomic island, insertion sequence

Introduction Acinetobacter baumannii is an opportunistic pathogen with increasing relevance in a variety of nosocomial infections, such as ventilator-associated pneumonia, central line-associated bloodstream infections, urinary tract infections, surgical-site infections, and other types of wound infections, particularly in immunocompromised patients or in patients in the intensive care unit (ICU) (Peleg et al. 2008). The ability of these organisms to accumulate diverse mechanisms of resistance limits the available therapeutic agents, makes the infection difficult to treat, and is associated with a greater risk of death (Kim et al. 2014). Although carbapenems, such as imipenem (IPM) and meropenem (MPM), are recommended

as last-resort antimicrobial therapies against A. baumannii infections, carbapenem resistance in A. baumannii (CRAB) has been emerging in many parts of the world, and the resistance rate has increased to about 30% or higher (Pogue et al. 2013). Previously, resistance to carbapenems and other antibiotics in A. baumannii or other bacteria was thought to be homogeneous within a culture. However, the phenomenon of heteroresistance (also known as “heterogeneous antibiotic resistance” or “heterogeneous resistance”) has been observed in staphylococci and has been applied to describe the phenomenon in infections where subpopulations of seemingly isogenic bacteria exhibit a range of susceptibilities to a particular antibiotic (El-Halfawy and Valvano 2015). This phenomenon is more common in gram-positive staphylococci, particularly in the vancomycin-susceptible Staphylococcus aureus (VSSA) and vancomycin-intermediate Staphylococcus aureus (VISA) (Hiramatsu et al. 1997; Wong et al. 1999; Marchese et al. 2000; Khosrovaneh et al. 2004). Very recently, this phenomenon was found to occur in some gram-negative bacterium, such as Escherichia coli (Baquero et al. 1985), Pseudomonas aeruginosa (Pournaras et al. 2007; Ikonomidis et al. 2008; Hermes et al. 2013), Klebsiella pneumoniae (Pournaras et al. 2010; Tato et al. 2010), and Acinetobacter baumannii (Pournaras et al. 2005; Ikonomidis et al. 2009; Savini et al. 2009; Lee et al. 2011). Since more resistant subpopulations may be selected during antimicrobial therapy, these subpopulations may lead to treatment failure and persistent infection with increased mortality rates. However, this phenomenon, which further complicates the study of antibiotic resistance, is poorly characterised, and its clinical relevance is uncertain. Accordingly, in this study, we isolated and characterised an IPM heteroresistant A. baumannii strain and aimed to improve our understanding of its antibiotic resistance mechanisms by sequencing and analysing the complete genome of this strain.

Methods Bacterial isolates and antimicrobial susceptibility testing The A. baumannii strain HRAB-85 was isolated from the sputum sample of an 83-year-old female in the 307th Hospital of PLA in Beijing, China, who underwent severe pneumonia and chronic obstructive pulmonary disease (COPD). Five days after the administration of Imipenem-Cilastatin, the consecutive derived A. baumannii strain lost susceptibility to Imipenem rapidly. The strain was identified using the Vitek 2 System (BioMerieux Vitek, Inc., Hazelwood, MO, USA) and cultured at 37°C in Luria-Bertani broth overnight. The minimum inhibitory concentration (MIC) was determined in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI) and was confirmed by the Vitek 2 System with 24 antibiotics: ampicillin, amikacin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, cefazolin, cefuroxime, cefuroxime ester, cefotetan, ceftazidime, aztreonam, IPM, MPM, gentamycin, ciprofloxacin, ceftriaxone, cefepime, cotrimoxazole, levofloxacin, nitrofurantoin, polymyxin B, tobramycin, sulfamethoxazole, and tigecycline. Pseudomonas aeruginosa ATCC 27853 was used as a control.

Population analysis and stability of the heterogeneous phenotype Population analyses were performed by the methods described by Ikonomidis et al. (2009) and Pournaras et al. (2010), with some modifications. Briefly, subpopulations were yielded by spreading approximately 108 bacterial CFU on Mueller-Hinton agar plates with IPM in serial dilutions at concentrations ranging from 0.5 to 64 mg/L and incubating the plates for 48 h. The analysis was performed three times, and the mean numbers of viable CFU were estimated and plotted on a semilogarithmic graph. P. aeruginosa ATCC 21636 was used as a control for the population analysis experiments. The frequency of appearance of heteroresistant subpopulations in the presence of the highest drug concentration was

calculated by dividing the number of colonies that grew on the antibiotic-containing plate by the colony counts from the same bacterial inoculum that grew on antibiotic-free plates (Pournaras et al. 2010). The stability of IPM MICs for three distinct colonies grown at the highest drug concentration was determined by agar dilution after seven daily subcultures in antibiotic-free medium.

Multilocus sequence typing (MLST) The MLST scheme described by Bartual was performed according to the Acinetobacter baumannii MLST (Oxford) database (http://pubmlst.org/abaumammii) (Bartual et al. 2005). The assembled sequences of seven housekeeping gene sequences (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) were aligned using BLAST, and the aligned sequences were then extracted by comparing them to allele profiles in the A. baumannii MLST (Oxford) database.

DNA extraction, whole-genome sequencing, and annotation The genomic DNA of HRAB-85 was extracted using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). Total DNA was then subjected to quality control by agarose gel electrophoresis and quantified by Qubit2.0 fluorometric quantification (Thermo Fisher Scientific, MA, USA). The genome of HRAB-85 was sequenced by Single Molecule, Real-Time (SMRT) technology. Sequencing was performed at the Beijing Novogene Bioinformatics Technology Co., Ltd. SMRT Analysis 2.3.0 was used to filter low quality reads, and the filtered reads were assembled to generate one contig without gaps. The HRAB-85 genome was assembled by using GeneMarkS (Besemer et al. 2001) (http://topaz.gatech.edu/). Gene prediction was performed with an integrated model combining the GeneMarkS generated (native) and heuristic model parameters. tRNA genes were predicted with tRNAscan-SE (Lowe and Eddy 1997), rRNA genes were predicted with

rRNAmmer (Lagesen et al. 2007), and sRNAs were predicted by BLAST against the Rfam database (Gardner et al. 2009). Repetitive sequences were predicted using RepeatMasker (Saha et al. 2008) (http://www.repeatmasker.org/). Tandem repeats were analysed using Tandem Repeat Finder (Benson 1999) (http://www.pathogenomics.sfu.ca/islandviewer/resources.php). Functional classification was performed by aligning predicted proteins to the Clusters of Orthologous Groups (COG) database (Tatusov et al. 1997; Tatusov et al. 2003). All predicted genes were compared to a nonredundant (nr) protein database in NCBI using BLASTX, with E values of ≤ 1e−5 and identity of ≥ 30%. Metabolic pathways were analysed by a single-directional best-hit method on the KEGG web server (http://www.genome.jp/kegg/). The mobile genetic elements in the HRAB-85 genome sequences were detected by the following online tools and/or open-access database and manual examinations: MobilomeFINDER for tRNA/tmRNA gene-related genomic islands (GIs) (Ou et al. 2007), IslandViewer for the island-like regions (Dhillon et al. 2013), and IS Finder for insertion sequence (IS) elements (Siguier et al. 2006). PHAST (Zhou et al. 2011) was used for prophage prediction (http://phast.wishartlab.com/), and CRISPRFinder (Grissa et al. 2007) was used for CRISPR identification.

Phylogenetic tree The phylogenetic tree was generated by TreeBeST (Nandi et al. 2010) using the method of PhyML, with 1,000 bootstraps for orthologous genes detected from gene family analysis. Genomic data used in the phylogenetic tree were downloaded from the NCBI database, including complete genome sequences of the A. baumannii isolates AB0057 (CP001182.1), AB307-0294 (CP001172.1), AYE (CU459141.1), ACICU (CP000863.1), XH386(CP010779.1), MDR-TJ (CP003500.1), MDR-ZJ06 (CP001937.1), TCDC-AB0715

(CP002522.2), TYTH-1 (CP003856.1), ATCC 17978 (CP000521.1), ZW85-1 (CP006768.1), SDF (CU468230.2), and ADP1 (NC_005966.1).

GenBank accession number The genome sequences of the HRAB-85 chromosome and plasmids (pHRAB85) have been submitted to the GenBank under accession numbers CP018143 and CP018144, respectively.

Results Isolation and susceptibility profiles of HRAB-85 During this study, 134 nonrepetitive A. baumannii isolates were obtained from patients hospitalised in the ICU with clinically suspected multiresistant infections in 307th Hospital of PLA in China and characterised by antibiotic susceptibility testing using both the VITEK2 System and disk diffusion assays (for IPM and MPM). During this procedure, one clinical A. baumannii isolate with resistant subcolonies present in the clear zone of inhibition around IPM discs (Supplementary Figure S1) was found and named heteroresistant A. baumannii-85 (HRAB-85). The MICs of 22 antimicrobial agents are listed in Table 1. This isolate exhibited resistance to all available antimicrobials except tigecycline and was also resistant to IPM.

Characterisation of the heteroresistant subpopulations and population analysis The minimal inhibitory concentrations (MICs) of IPM for the clinical isolate HRAB-85, as determined by microbroth dilution assay, was 8 μg/mL; however, colonies grown within the zone of inhibition around IPM discs at concentrations up to 16 μg/mL (Table 2). Population analysis assays (PAP) with IPM showed that the subcolonies of HRAB-85 could grow at concentrations ranging from 16 to 64 g/mL (2–8 times their MICs), while control

isolates (A. baumannii ATCC 22933 and P. aeruginosa 21636) could not (Figure 1). The heterogeneous growth in the presence of IPM was quite stable to some degree; after seven daily subcultures in drug-free medium, the dilution IPM MIC of the heteroresistant colonies was 16 μg/mL, which was slightly higher than that of HRAB-85 (Table 2).

Whole-genome sequences and general features of HRAB-85 To date, no complete genomic sequence of IPM heteroresistant A. baumannii has been reported; therefore, in this study, we determined the complete genomic sequence of HRAB-85. Basic whole-genome sequencing analysis showed that the HRAB-85 genome consisted of a circular chromosome of 4,021,072 bp and 1 plasmid with 77,513 bp. The GC content of the genomes was approximately 39.98%, as expected for this species. The general features of the HRAB-85 genome are summarized in Table 3. The annotated genes were assigned functions by a combination of BLAST analysis and KEGG annotation (Altschul and Lipman 1990; Altschul et al. 1997; Kanehisa et al. 2006) and then assigned to clusters of orthologous groups (COGs) (Tatusov et al. 1997). Approximately 72% of the genes were assigned to a COG functional category (Supplementary Figure S2), of which the largest group was “general function prediction only”(R), followed by the classes of genes involved in amino acid transport and metabolism (E), transcription (K), translation, ribosomal structure and biogenesis (J), and lipid transport and metabolism (I).This result was basically consistent with a previous functional analysis of the COG classification for multidrug-resistant (MDR) A. baumanni (Liu et al. 2016). Based on the KEGG pathway mapping analysis and annotation of all 2141 genes, 20 genes were successfully mapped to the pathways on human diseases, of which eight genes were annotated in pathways associated with drug resistance. Red-framed genes involved in encoding Amp C of the beta-lactam resistance (pathway map: ko01501) were found in

bacterial isolates HRAB-85 (Supplementary Figure S3). Six genes, including blaCMY-1, blaCMY-2, blaDHA, blaFOX, blaACC, and blaACT-MIR, were found in this pathway. Unfortunately, none of the genes were identified by PCR in vitro.

Genotype analysis and phylogenetic tree construction In order to elucidate the genotype of HRAB-85, an MLST scheme based on the Oxford database was subsequently employed to analyse this strain as well as the other 35 clinical isolates of A. baumannii (including 19 CSAB strains and 16 CRAB strains) obtained from the same hospital during the same period. The results revealed that the isolates were clustered into 14 different genotypes or STs. HRAB-85 belonged to ST208 genotype, which was shared by the majority of isolates (19.4%, 7/36; Supplementary Figure S4). ST208 is the most common type in China (Wang et al. 2013; Deng et al. 2014; Ying et al. 2015) and is one of the predominant A. baumannii genotypes causing nosocomial outbreaks in Beijing. A phylogenetic tree was constructed based on the single copy gene using the TreeBeST with maximum likelihood method, and strain ADP1 was used as the root (Figure 2). The phylogenetic tree showed that strain HRAB-85 was closest to A. baumannii XH386 (Fang et al. 2016), MDR-TJ06 (Zhou et al. 2011), and TCDC-AB0715 (Chen et al. 2011), which were all MDR-AB and reported in China mainland and Taiwan.

Genomic islands (GIs) and resistance genetic determinants of HRAB The insertion sequence (IS) elements in the HRAB-85 genome sequences were detected by IS Finder. Compared with other MDR-ABs, the IS number of HRAB-85 was quite small. Overall, four types of IS elements, including ISAba1, ISAba22, ISAba24, and ISAba26, were found in HRAB-85. There were 17 copies of ISAba1 in the genome, 15 in the chromosome, and two in plasmid. One ISAba1 element was found to be linked to an ampC gene blaADC-25,

and two were interrupted by blaOXA-23 in the plasmid. ISAba24 was near the end of GI03 (Figure 3). ISAba22 was located at the end of GI05, following a phage-related protein (1593793–1594722) and phage tail length tape-measure protein (1598760–1603154), implying that the IS elements had a role in the transfer of this GI. ISAba26 was recently assigned (Ou et al. 2015) and shares 72% transposase amino acid identity with ISEc39. The GIs in the HRAB-85 genome sequences were detected by IslandPath-DIOMB software. A total of 11 genomic islands were predicted in the HRAB-85 genome (Table 4). GI03 consisted of a newly assigned transposon Tn6279, containing one ISAba24, two IS26 elements, and a partial ISVsa3 element (Figure 3). Additionally, this genomic island harboured resistance genes for aminoglycoside (aph3ia, aac6ib, catb3, and ant3ia) and for sulphonamides (sul2); thus, this GI was a resistance island (Table 5). The A. baumannii HRAB-85 sequences were searched antibiotic-resistance genes against both the ResFinder database (Zankari et al. 2012) and the ARDB (Liu and Pop 2009). From those mobile elements, we speculated that resistance to carbapenems for HRAB-85 was mediated by blaOXA-23, which was carried on a typical Tn2006 transposon in the plasmid of pHRAB-85 (Supplementary Figure S4). Resistance to third-generation cephalosporins was associated with blaADC-25 and blaTEM-1D, and one ISAba1 was also found upstream of blaADC-25, enhancing expression and leading to ceftazidime resistance. Resistance to aminoglycoside was partly due to the acquisition of resistant genes, such as aph(3')-Ic, aacA4, aadA1, armA, strA, and strB. Additional genes, including adea, adeb, and adec, which encode for RND-type multidrug efflux pump proteins for resistance towards aminoglycoside and fluoramphenicol, were also detected in HRAB-85. Moreover, this isolate also carried a series of genes involved in resistance against sulphonamide, macrolide, lincosamide, streptogramin B, tetracycline, and phenicol (Table 5).

Discussion Multidrug-resistant isolates of A. baumannii have been reported increasingly during the last few decades, probably as a consequence of extensive use of antimicrobial agents, particularly carbapenems, in many regions, including China. This organism is well adapted to the hospital environment, with excellent biofilm-producing ability and intrinsic and acquired resistance to various antibiotic agents, all of which make it a nosocomial pathogen of particular clinical concern (Dijkshoorn et al. 2007). An even more worrisome observation is heteroresistance to carbapenems, which may have implications for the treatment of MDR A. baumannii infections (Ikonomidis et al. 2009). However, heteroresistance has not been well characterised, further complicating the study of antibiotic resistance, and its clinical relevance is uncertain (El-Halfawy and Valvano 2015). Some studies on carbapenem heteroresistance in A. baumannii have been published (Pournaras et al. 2005; Ikonomidis et al. 2009; Lee et al. 2011); however, no standard method for determining carbapenem heteroresistance has yet been established, and these studies were therefore not consistent. Moreover, to date, some researchers have described the genomic sequence of carbapenem heteroresistant A. baumannii. Accordingly, in this study, an isolate of IPM heteroresistant A. baumannii was selected by the traditional disk diffusion method and confirmed by the PAP method recommended by El-Halfawy and Valvano (2015); the complete genome sequence of this organism was then analysed. In the present study, only one clinical isolate among 134 A. baumannii was found with resistant subcolonies present in the clear zone of inhibition around IPM discs. We further investigated this strain with PAP, and the result revealed that the subpopulations grew in concentrations 8-fold higher than MICs for HRAB-85. The rate of carbapenem heteroresistant A. baumannii (HRAB) was much lower than that of some reports (Pournaras et al. 2005; Ikonomidis et al. 2009; Fernández-Cuenca et al. 2012). Among the 134 A.

baumannii isolates, 96 strains were CRABs (data not shown), and even IPM heteroresistant A. baumannii HRAB-85 exhibited resistance to IPM and MPM. In contrast, in other reports, more carbapenem-susceptible A. baumannii were investigated for heteroresistance (Ikonomidis et al. 2009). This may be the main reason for the significant difference in the rate of HRAB. Moreover, studies on HRAB in mainland China are very rare, possible because HRAB is not as prevalent as in Greece (Ikonomidis et al. 2009) or Taiwan (Lee et al. 2011). However, the reason for this is still not clear. By collecting more samples from hospitals in Beijing or extending the bacteria collection period, we may reach a better understanding of the trends in HRAB infections in Beijing. The term “heteroresistance” has been applied to describe infections with bacterial strains having different levels of resistance to an antibiotic, i.e., variations in resistance among members of a bacterial population (El-Halfawy and Valvano 2015). Heteroresistance can be divided into three forms showing different clinical significances and requiring different therapeutic interventions. In the first form, MICs of distinct subpopulations are different, whereas the entire population is completely sensitive to the antibiotic. This form is probably of the least clinically importance. In the second form, the majority of the population is susceptible to an antibiotic, with a few highly resistant subpopulations. This is the classical and commonly reported form of heteroresistance (Ikonomidis et al. 2009; Pournaras et al. 2010) and is more likely to lead to treatment failure due to selection of the resistant subpopulations by inappropriate doses of antibacterial drugs guided by traditional susceptibility testing. Special attention should be paid to this form of heteroresistance. Finally, in the third form, the entire population is resistant or intermediate, while a group of subpopulations showing various levels of resistance to the antibiotic emerge; HRAB-85 is the third form. The MIC of the isolate HRAB-85 was 8 μg/mL, and PAP result showed that some of the subcolonies could grow at concentrations of IPM ranging from 16 to 64 μg/mL. One

major concern about this form is that more resistant subpopulations may pass antibiotic resistance to less resistant subpopulations within the same population, resulting in a switch of heteroresistance to homogeneous high-level resistance. If this is possible, this third form of heteroresistance could pose a serious threat to antibiotic treatment. Accordingly, we performed a preliminary exploration of the mechanism of heteroresistance. By complete genome sequencing of HRAB-85, we were able to provide bioinformatic analyses of the resistance genetic determinants of this strain, which may contribute to heteroresistance to carbapenems. Compared with other whole-genome sequenced A. baumannii, the number of ISs was relatively small. However, one recently assigned IS element ISAba26 was identified in the HRAB-85 genome. This IS element was first identified in the LAC-4 genome, near the ends of a GI (Ou et al. 2015). Additionally, one novel identified transposon Tn6279, which contained a cluster of resistance genes and was first reported in a CRAB isolate in Sweden in June 2012 (Karah et al. 2016), was located in the GI03 of HRAB-85. Furthermore, a typical Tn2006 transposon containing ISAba1 upstream from blaOXA-23 was found in the plasmid. Thus, the mobile genetic elements of HRAB-85 were quite moderate and were more likely associated with resistance to carbapenems than heteroresistance to carbapenems. However, the mechanisms involved in heteroresistance are still not clear, particularly for carbapenem heteroresistant A. baumannii. Genetic mechanisms explain many cases of variations across a bacterial population in some species of bacteria (El-Halfawy and Valvano 2015); however, no such genetic changes had been reported in carbapenem heteroresistant A. baumannii until now. Some scientists have suggested that administration of carbapenems is a factor that likely affects antibiotic resistance; the pressure of IPM and MPM could produce selected heteroresistant subpopulations after application of suboptimal therapeutic drug dosages, giving rise to highly resistant strains (Ikonomidis et al. 2009; Superti et al. 2009;

Lee et al. 2011). Others observed that differences in transcript levels may also underlie heteroresistance to carbapenems in some gram-negative bacteria. For example, significantly increased expression of the mexB and mexY genes and decreased expression of the oprD gene were detected at the transcription level in heteroresistant P. aeruginosa (Ikonomidis et al. 2008). Thus, we speculate that the use of IPM may explain the heteroresistance to HRAB-85; however, further studies are needed to determine true changes at the nongenetic level. In the future, by isolating more strains, we will use epigenetic, metabolic, and transcriptomic approaches to investigate the mechanisms of carbapenem heteroresistance in A. baumannii.

Author Contribution Prof. Changqing Bai, Prof. Jing Yuan, and Puyuan Li helped conceive the project and design the study. Prof. Xiuyun Yin, Prof. Xin Yuan, and Huiying Liu collected the strains. Puyuan Li wrote the main manuscript text and prepared all the tables and figures. Yong Huang and Yannan Liu conducted all the bioinformatics analysis. Lan Yu, Wenkai Niu, Dayang Zou, and Jing Zheng executed the experiments. All the authors reviewed the manuscript.

Funding This present work was supported by the Beijing Natural Science Foundation (grant no. 7142118), the Capital Health Research and Development of Special (grant no. 2016-2-5062), and the Capital Characteristic Clinic Project of Beijing (grant no. Z161100000516181).

Conflict of Interest The authors declare no competing financial interests.

Acknowledgements

We thank Prof. Qiu Shaobo and Dr. Jian Wang from the Institute of Disease Control and Prevention, Academy of Military Medical Sciences (Beijing, China) for MLST analysis. We are grateful to Prof. Mi Zhiqiang for constructive comments on this research. We also thank Zhao Xiangna, Wei Xiao, Lihuan, Li Erna, and Liboxing for assistance during the course of this study. We would like to thank Editage (www.editage.com) for English language editing and Publication Support.

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Figure Legends Figure 1. Imipenem population analysis profiles of the HRAB-85 isolates and the control strains. Dots correspond to mean values of three replicates for each strain.

Figure 2. Phylogenetic tree of 14 A. baumannii isolates. A maximum likelihood tree was constructed based on the single copy gene in each genome. Bar, 0.01 substitution per nucleotide. “R” = multidrug resistance; “S” = drug sensitive; “-” = unknown.

Figure 3. Structure of GI03. This structure was drawn to scale according to the sequences of GI03 in the genome. This island was 13.1 kb in length and contained several antibiotic-resistance genes.

Tables Table 1. Antimicrobial susceptibility profile of HRAB-85 MIC

MIC Sensitivitya

Antibiotics

Sensitivitya

Antibiotics

(μg/mL)

(μg/ml)

Ampicillin

≥ 32

R

Aztreonam

32

R

Ampicillin/sulbactam

≥ 32

R

Imipenem

8

R

Piperacillin

≥ 128

R

Meropenem

≥ 16

R

64

I

Gentamicin

≥ 16

R

Cefazolin

≥ 64

R

Tobramycin

≥ 16

R

Cefuroxime

≥ 64

R

Ciprofloxacin

≥4

R

Cefuroxime ester

≥ 64

R

Levofloxacin

≥8

R

Cefotetan

≥ 64

R

Nitrofurantoin

≥ 512

R

Ceftazidime

≥ 64

R

Cotrimoxazole

≥ 320

R

Ceftriaxone

≥ 64

R

Gentamicin

≥ 16

R

Cefepime

32

R

Tigecycline

≤2

S

Amikacin

≥ 64

R

Polymyxin B

≥8

R

Piperacillin/tazobactam

a

S, susceptible; I, intermediately resistant; R, resistant.

Table 2. Characteristics of the tested isolates Isolate

HRAB-85 A. baumannii ATCC

IPM MIC

Highest IPM

IPM MIC of

(μg/mL)

concentration for

heterogeneous

growth in PAP

subpopulation*

(μg/mL)

(μg/mL)

8

64

16

0.25

0.5

NA

0.25

0.25

NA

22933 P. aeruginosa 21636

*These MICs were estimated after seven daily subcultures in antibiotic-free medium

Table 3. General features of the HRAB-85 genome Feature Genome size Plasmid

HRAB-85 genome 4,098,585 bp 1

GC content (%)

39.98

CDSs

3,979

Gene average length (bp)

904

Number of tRNAs

74

Number of rRNAs (5s, 16s, 23s)

18

Number of sRNAs

1

Number of genomic islands (GIs)

11

Number of insertion sequences (Grissa et al.)

18

Genes assigned to GO

2,672

Genes assigned to COGs

2,851

Genes assigned to KEGG

2,141

Genes assigned to PHI

73

Genes assigned to VFDB

68

Genes assigned to ARDB

14

Repeated regions (%)

0.8845

Phage regions

4

CRISPRs

0

Table 4. Genomic island-like regions identified in the HRAB-85 chromosome G+C GIs_id

Sequence: start–end

Length

Features (%) ABC-type transport system

GIs01

402,550–412,964

10,415

42

involved in resistance to organic solvents, ATPase component Major ribosomal proteins, which

GIs02

432,577–446,136

13,560

43

involved in translation, ribosomal structure, and biogenesis Containing the majority of a newly assigned transposon

GIs03

1,364,093–1,377,215

13,123

50

Tn6279 and a cluster of resistance genes: aph3ia, aac6ib, catb3, ant3ia, and sul1

GIs04

1,559,285–1,563,834

4,550

34

Integrase ISAba22, phage-related protein,

GIs05

1,592,968–1,613,796

20,829

38 transposase DNA-binding transcriptional

GIs06

2,436,978–2,443,624

6,647

33 regulator

GIs07

2,649,326–2,656,275

6,950

51

blaTEM-1D, transposase Prophage antirepressor, integrase,

GIs08

2,963,342–2,986,930

23,589

35 putative SOS response-associated

peptidase YedK, nucleotidyltransferase GIs09

2,989,461–2,997,055

7,595

35

Zn-dependent peptidase ImmA Two-component system, OmpR

GIs10

3,234,355–3,278,479

44,125

36

family, sensor histidine kinase RstB, and response regulator RstA

GIs11

3,762,790–3,770,522

7,733

47

aph33ib, aph6id

Table 5. Antimicrobial resistance-associated genes in the HRAB-85 genome Antimicrobi

Resistan

al agent class ce gene

Predicted

Enzyme class,

NCBI

phenotype

description

DNA

Source

Accessio n Aminoglycosi aph(3')-I

Aminoglycos Aminoglycoside

des

ide

c

X62115

O-phosphotransferase

pneumoni

resistance aacA4

ae

Aminoglycos Aminoglycoside

KM2781

E.

ide

99

fergusoni

N-acetyltransferase

resistance aadA1

K.

i

Aminoglycos Streptomycin

JQ41404

ide

1

3''-adenylyltransferase

E. coli

resistance armA

strA

Aminoglycos Aminoglycoside

AY2205

K.

ide

resistance 16S ribosomal

58

pneumoni

resistance

RNA methylase

Aminoglycos Streptomycin ide resistance Alternate name:

phosphotransferase

ae M96392

E. amylovor a

aph(3'')-Ib strB

Aminoglycos Streptomycin ide

M96392

phosphotransferase

E. amylovor

resistance

a

Alternate name: aph(6)-Id Beta-lactam

blaADC-

Beta-lactam

25

resistance

Class D β-lactamase

EF01635 A. 5

baumann ii

blaOXA-

Beta-lactam

66

resistance

Class D β-lactamase

FJ36053

A.

0

baumann ii

blaTEM-

Beta-lactam

1D

resistance

class A β-lactamase

AF1882

E. coli

00

Alternate name: RblaTEM-1

blaOXA-

Beta-lactam

23

resistance

Class D β-lactamase

HQ7003

A.

58

baumann ii

Fluoroquinol

aac(6')Ib

Fluoroquinol

Aminoglycoside

EF63646 K.

one

-cr

one and

N-acetyltransferase

1

aminoglycosi

pneumoni ae

de resistance MLS -

msr(E)

Macrolide,

Macrolide,

lincosamide,

lincosamide,

and

and

streptogrami

streptogramin

nB

B

resistance mph(E)

ABC transporter

EU2942

A.

28

baumann ii

Macrolide

Mph(E) macrolide

EU2942

A.

resistance

2'-phosphotransferase

28

baumann ii

Sulphonamid

sul1

e

Sulphonamid Sulphonamide-resistant

CP00215 unculture

e resistance

1

dihydropteroate synthase

d bacteriu m

Tetracycline

tet(B)

Tetracycline

Major facilitator super

AP0003

resistance

family transporter,

42

S.flexneri

tetracycline, efflux pump Phenicol

catB8

Phenicol

Chloramphenicol

AF2275

K.

resistance

acetyltransferase

06

pneumoni

ae Aminoglycosi

Efflux pump

RND

------

A.

de/

protein

(resistance-nodulation-di

baumann

vision) family

ii

adea fluorampheni col

adeb

adec

Efflux pump

RND

protein

(resistance-nodulation-di

baumann

vision) family

ii

RND

protein

(resistance-nodulation-di

baumann

vision) family

ii

No resistance genes found

Fosfomycin

No resistance genes found

Fusidic acid

No resistance genes found

Nitroimidazol No resistance genes found e No resistance genes found

e Rifampicin

No resistance genes found

Trimethoprim No resistance genes found Glycopeptide

No resistance genes found

------

A.

Efflux pump

Colistin

Oxazolidinon

------

A.