Antibiotic Susceptibility, Clonality, and Molecular Characterization of Carbapenem-Resistant Clinical Isolates of Acinetobacter baumannii from Washington DC

The occurrence of carbapenem-resistant (CR) strains of Acinetobacter baumannii is reported to contribute to the severity of several nosocomial infections, especially in critically ill patients in intensive care units. The present study aims to determine the antibiotic susceptibility, clonality, and genetic mechanism of carbapenem resistance in twenty-eight Acinetobacter baumannii isolates from four hospitals in Washington DC. The antibiotic susceptibility of the isolates was determined by VITEK 2 analyses, while PCR was used to examine the presence of antibiotic-resistant genes and mobile genetic elements. Trilocus multiplex-PCR was used along with pulsed-field gel electrophoresis (PFGE) for strain typing and for accessing clonal relationships among the isolates. Antimicrobial susceptibility testing indicated that 46% of the isolates were carbapenem-resistant and possessed MDR and XDR phenotypes. PFGE clustered the 28 isolates into seven clonal (C1–C7) complexes based on >75% similarity cut-off. Thirty-six percent of the isolates belonged to international clone II, while 29% were assigned to Group 4 by trilocus multiplex-PCR. Although the blaOXA-51-like gene was found in all the isolates, only 36% were positive for the blaOXA-23-like gene. PCR analysis also found a metallo-β-lactamase (MBL) gene (blaVIM) in 71% of the isolates. Of the 13 CR isolates, 8 were PCR positive for both blaVIM and blaOXA-23-like genes, while 5 harbored only blaVIM gene. This study revealed the emergence of VIM carbapenemase-producing A. baumannii isolates, which has not been previously reported in the United States.


Introduction
Acinetobacter baumannii has emerged as an important nosocomial pathogen associated with hospital-acquired infections worldwide [1][2][3]. Multidrug-resistant (MDR) A. baumannii strains are associated with infections such as ventilator-associated pneumonia, septicemia, urinary tract, soft skin, wound, and meningitis, especially in immunocompromised patients in ICU settings [4][5][6][7]. e success of A. baumannii as an emerging nosocomial pathogen is mostly due to its efficiency in acquiring new antibiotic-resistant determinants [8,9] which may have contributed to the high antibiotic index as classified by the World Health Organization (WHO). e carbapenem-resistant A. baumannii is now grouped among the leading causes of bacterial nosocomial infections throughout the world.
is special category of pathogens termed "ESKAPE" consists of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. e acronym ESKAPE was developed by culling the first letter of each of the genus names in the group [10]. Antibiotic resistance in A. baumannii is mediated by enzymatic degradation of antibiotics, mutations/modification of target sites, reduced expression of porins, and overexpression of multidrug efflux pumps [3,11,12]. However, resistance to carbapenem is often mediated by β-lactamases including carbapenem-hydrolyzing class D β-lactamases (CHDLs) and metallo-β-lactamases (MBLs). Resistance by class D β-lactamases (CHDLs), also known as oxacillinases, is mainly mediated by the production of carbapenemase enzymes encoded by genes of the bla OXA-23 ,
All isolates were identified as Acinetobacter baumannii (ACB) complex using a VITEK® 2 automated instrument ID system (Biomerieux, Inc., Hazelwood, MO). Isolates positive for bla OXA-51 gene were identified as A. baumannii by PCR method. Isolates were stored at −20°C in trypticase soy broth (TSB) supplemented with 20% glycerol until further analysis could be performed.

Pulsed-Field Gel Electrophoresis (PFGE)
. PFGE was performed according to the standard operating procedure for PulseNet PFGE of Escherichia coli O157:H7, Escherichia coli non-O157 (STEC), Salmonella serotypes, Shigella sonnei, and Shigella flexneri (http://www.cdc.gov/pulsenet/ protocols.htm) with the following modifications: the genomic DNA in the agarose plugs was digested with ApaI (50 U/μl; New England BioLabs, Ipswich, MA) for 3 hours at 37°C. e restricted genomic DNA fragments were separated in 1% SeaKem Gold Agarose gels in 0.5 × TBE buffer using CHEF mapper system (Bio-Rad Laboratories, Hercules, CA) at 14°C, 200 V (6V/cm), with initial pulse time of 2.2 s and final pulse time at 35 s. Salmonella strain 201 was digested with restriction enzyme Xba I (5 U/μl) and used as a DNA standard. PFGE band patterns were analyzed by the BioNumerics software, version 6.6.4.0 (Applied Maths, St-Martens-Latem, Belgium), using Unweighted Pair Group Method and Arithmetic Mean (UPGMA) with 1.5% tolerance and 1.5% optimization. All isolates with similarity index of >75% were grouped together in the same cluster and showed similar PFGE banding pattern [22].

Trilocus Sequence Typing (3LST).
Trilocus sequence typing method was used to assign international/global clonal groups to the isolates as described by Turton et al. [23]. Briefly, multiplex-PCR was performed using primers targeting alleles of ompA, csuE, and bla OXA-51 -like genes. Isolates were assigned to respective groups based on the different combinations of gene amplifications as described in [23,24].

EDTA-Disk Synergy
Test. Imipenem disk (BD BBL ™ , 10 μg) and filter paper disk with 10 µl of 0.5 M EDTA solution were placed 10 mm apart. e presence of enlarged/ synergistic zone of enhancement was interpreted as metalloβ-lactamase (IMP or VIM type) positive strain.

Combined Disk Diffusion Test.
ree Imipenem (BD BBL ™ , 10 μg) disks, one serving as control and the other two with 0.1 M EDTA (10 μl) and 0.5 M EDTA (10 μl), were placed 10 mm apart from each other on the Mueller-Hinton agar plates. After overnight incubation, increase of ≥4 mm in the zone diameters of imipenem disk with 0.1 M EDTA and increase of ≥7 mm in the zone diameters of imipenem disks with 0.5 EDTA were interpreted as a positive test for MBL producing strain.

Colony PCR for Detection of Carbapenemases Genes and
Mobile Genetic Elements. Colony PCRs were performed to detect the presence of four main class D OXA β-lactamase genes (bla OXA-23-like , bla OXA-24-like , bla OXA-51-like , and bla OXA-58-like ) and three class B, also known as metalloβ-lactamases, genes (bla IMP , bla VIM , and bla SIM ). Isolates confirmed positive for oxacillinases genes were further screened for the presence of ISAba1 element upstream of these genes [27]. Briefly, colony PCRs were performed in a 50 μl reaction mixture with 25 μl of EconoTaq PLUS GREEN 2X Master Mix (Lucigen, Middleton, WI), 0.5 μl each of forward primer (100 μM) and reverse primer (100 μM), and 25 μl of nuclease-free water. Small amount of bacterial colony was removed using the sterile toothpick and added to the master mix. Amplification was carried out under the following conditions: denaturation at 94°C (3 min), 35 cycles of denaturation at 94°C (45 s), annealing for 45 s as described previously [28], and extension at 72°C for 1 min, followed by a final extension of 72°C for 1 min. e Qiagen purification kit (Qiagen, USA) was used to purify amplified PCR products, and both strands were sequenced by automated AB13100 DNA sequencer (Applied Biosystems) system. e nucleotide sequences were analyzed using the basic local alignment search tool (BLAST).

Pulsed-Field Gel Electrophoresis (PFGE).
Pulsed-field gel electrophoresis (PFGE) was used to determine the genetic and clonal relationships among these clinical isolates using ApaI-digested DNA. PFGE analysis clustered the isolates into seven clonal complexes, C1-C7, using a cut-off value of >75% similarity ( Figure 1). All isolates grouped in cluster C3 were carbapenem-resistant and possessed the XDR phenotype. Isolates in clusters C1, C2, C4, and C6 were XDR and/or MDR, except isolates B8, B1, C5, C1, A14, and C22. However, the isolates in clusters C5 and C7 were susceptible to all the antibiotics tested. e largest cluster identified as C4 included seven isolates, out of which two (B16 and H11) were MDR and one (C24) possessed an XDR phenotype. Isolates A11, A3, and A7 could not be assigned to any clonal group by this method.

Trilocus Sequence-Based Typing (3LST).
A 3LST-based PCR typing method, targeting three genes (ompA, csuE, and bla OXA-51-like ), was employed to assign the global clonal lineage of the isolates. e gel image of PCR amplification for ompA, csuE, and bla OXA-51-like alleles for all the isolates tested for Group 1 primers is shown in Figure 2; however, there was no amplification observed for Group 2 primers (data not shown). Of the 18 isolates typed by this method, 10 (36%) were assigned to Group 1 or international clone II lineage (G1/ ICII), while the remaining eight were assigned to Group 4, based on the proposed schemes [23,24]. Ten isolates could not be assigned to any international clonal group due to lack of amplification in the PCR reaction. However, all the isolates (n � 10) belonging to the G1/ICII group were carbapenemresistant and exhibited either a MDR and/or XDR phenotype, whereas most of the isolates (n � 5) in Group 4 were susceptible to the tested antibiotics, and only three were carbapenem-resistant (Table 1 and Figure 1).

Discussion
Based on the recent criteria by World Health Organization (WHO), carbapenem-resistant A. baumannii (CRAB) is among the top priority pathogens [29]. ere are several reports of the emergence and rise of carbapenem-resistant A. baumannii strains in hospitals throughout United States [22,28,30,31], but limited information is available regarding the genetic basis of hospital-acquired carbapenem resistance in Washington DC region. To address this research gap, twenty-eight nosocomial strains of A. baumannii were included in this study. With regard to antibiotic susceptibility, the data revealed that 13 (46.4%) of the 28 isolates were carbapenem-resistant. Out of 13 carbapenem-resistant isolates, 5 (38%) were MDR, while 8 (61%) were of XDR phenotype (Table 1). e presence of XDR isolates is of great concern, since infections caused by XDR isolates are difficult to treat because they are not susceptible to the known commercially available drugs and, thus, pose considerable infection control issues [32]. In a recent study from the Harbor-UCLA Medical Center (HUMC), California, USA, 57% of 21 isolates of A. baumannii tested for antibiotic susceptibility were found to be carbapenem-resistant; and all isolates were sensitive to colistin and tigecycline; however, none of the isolates possessed an XDR phenotype. Similarly, there has been an increase in reports of carbapenem-resistant A. baumannii from hospitals in other regions of the United States [22,28,30,31].
Several genotypic methods are available for surveillance, strain typing, and epidemiological studies of A. baumannii outbreaks [33]; however, there is not a single ideal method. In this study, we used pulsed-field gel electrophoresis (PFGE) and trilocus sequence-based typing (3LST) methods for finding the genetic relatedness among the isolates. PFGE is a widely used method and is still considered as the "gold standard" for genotyping irrespective of the limitation of the interlaboratory reproducibility; on the other hand, 3LST method has an added advantage of providing information pertaining to pathogenicity [23,34]. e genomic profile of isolates determined by PFGE appears to be in correlation with the antibiotic susceptibility pattern with few exceptions. Isolates with similar antibiotic susceptibility were clustered together in their respective clonal complexes, suggesting that they might be closely related and changes in the genome could be possibly linked to their phenotype. All isolates with similar antibiotic susceptibility profile were grouped into three clonal complexes, C1 (except isolates B10 and B8), C2 (except isolate B1), and C3, and were defined as XDR. Similarly, isolates in cluster C4 (except isolates C5, C11, A14, and C22) and C6 possessed MDR phenotypes. e remaining isolates grouped in clusters C5 and C7 were susceptible to all of the tested antibiotics ( Figure 1 and Table 1).
Results from both PFGE and 3LST genotyping methods were in agreement ( Figure 1). All isolates (n � 10) belonging to clusters C1(except isolate B8), C2 (except B1), C3, and C6 were assigned to G1/ICII category, while all isolates in cluster C4 belong to Group 4. Isolates in clusters 5 and 7 (except B9) could not be assigned to any global lineage.  Similarly, isolates A11, A3, and A7 could not be genotyped by both methods. e analysis revealed that the largest clonal complex C4 (n � 7) included isolates from all the four hospitals irrespective of the origin. e results from the strain typing indicated that the isolates (n � 10) assigned to G1/ICII lineage were carbapenem-resistant and possessed MDR and/or XDR phenotypes. Recently, Warner and coworkers examined 38 clinical isolates of A. baumannii from two hospital outbreaks in Los Angeles County. In their report, nine isolates were identified as belonging to G1/ICII lineage and were typed as sequence type 2 (ST2) isolates using the Pasteur MLST scheme [30].
Regarding the mechanisms of resistance, there seems to be a general consensus that the enzymatic modification or degradation of β-lactams by different β-lactamases is a major mechanism for carbapenem resistance in A. baumannii isolates from different parts of the world [3,14,[35][36][37][38][39]. e screening for OXA-type β-lactamases confirmed the presence of intrinsic chromosomally located OXA-51-like gene in all of the A. baumannii isolates in this study (Figure 3(a)). Additionally, OXA-23-like gene was detected in 36% (n � 10) of the isolates (Figure 3(b)). All OXA-23 positive strains were carbapenem-resistant and associated with insertion sequences ISAba1 element present upstream in the promoter region of the gene, which may amplify the overexpression of the OXA-23 gene ( Figure 5(b)). e detection of VIM metallo-β-lactamases gene is noteworthy in this study.
irteen out of twenty VIMharboring isolates were carbapenem-resistant. e coexistence of OXA-23-like and VIM metallo-β-lactamases gene in eight isolates may have possibly contributed to carbapenem resistance in these isolates. Interestingly, OXA-23-like gene was not detected in the remaining five carbapenem-resistant isolates, B2, B3, B4, B10, and H8. Among these isolates, three (B2, B3, and H8) were resistant to agents in all eight antimicrobial categories and exhibited XDR phenotype, while isolates B4 and B10 were of MDR phenotype. is indicates that VIM metallo-β-lactamases might be the main contributor to carbapenem resistance in these five isolates. is is in contrast to the reports on A. baumannii outbreak investigation in the United States, where the expression of OXA-23-like gene was mainly attributed to carbapenem resistance [22,28,30,31,[40][41][42].
is study presents the antibiotic susceptibility and molecular analysis of the carbapenem-resistant isolates of A. baumannii strains collected from four Washington DC hospitals. PFGE and TLSTdata suggest that the carbapenemresistant strains mainly belonged to international clonal II lineage. To the best of our knowledge, this is the first report on the emergence of VIM metallo-β-lactamases gene in A. baumannii isolates in the USA. e data revealed prevalence of mobile genetic elements like insertion sequence (ISAba1) in all of the isolates. e results of this study suggest the need for continuous surveillance for carbapenem-resistant strains in Washington DC hospitals and the maintenance of proper infection control measures to prevent transmission of MDR and/or XDR strains.

Data Availability
e data used to support the findings of this study are included within the article, and any additional data are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.