{'fraction_non_alphanumeric': 0.0408726625111309, 'fraction_numerical': 0.018788958147818342, 'mean_word_length': 5.0740941049215795, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Epidemiological Characterizations of Class 1 Integrons from Multidrug-Resistant Acinetobacter Isolates in Daejeon, Korea

Background Multidrug-resistant (MDR) Acinetobacter spp. acquire antimicrobial agent-resistance genes via class 1 integrons. In this study, integrons were characterized to investigate the antimicrobial resistance mechanisms of MDR Acinetobacter isolates. In addition, the relationship between the integron type and integron-harboring bacterial species was analyzed by using epidemiological typing methods. Methods Fifty-six MDR Acinetobacter spp.-A. baumannii (N=30), A. bereziniae (N=4), A. nosocomialis (N=5), and A. pittii (N=17)-were isolated. The minimum inhibitory concentrations (MICs) were determined on the basis of the results of the Epsilometer test (Etest). PCR and DNA sequencing was performed to characterize the gene cassette arrays of class 1 integrons. Multilocus sequence typing (MLST) and repetitive extragenic palindromic sequence (REP)-PCR were performed for epidemiological typing. Results Class 1 integrons were detected in 50 (89.3%) of the 56 isolates, but no class 2 or 3 integron was found within the cohorts. The class 1 integrons were classified into 4 types: 2.3-kb type A (aacA4-catB8-aadA1), 3.0-kb type B (aacA4-blaIMP-1-blaOXA-2), 3.0-kb type C (blaVIM-2-aacA7-aadA1), and 1.8-kb type D (aac3-1-blaOXA-2-orfD). Type A was most prevalent and was detected only in A. baumannii isolates, except for one A. bereziniae isolate; however, type B was amplified in all Acinetobacter isolates except for A. baumannii isolates, regardless of clone and separation time of the bacteria. Conclusions Although class 1 integron can be transferred horizontally between unrelated isolates belonging to different species, certain types of class 1 integrons tend to transfer horizontally and vertically among A. baumannii or non-baumannii Acinetobacter isolates.


INTRODUCTION
Acinetobacter spp. are aerobic, glucose-nonfermenting gramnegative bacteria that are widely distributed in soil and water environments [1,2]. Isolates belonging to the A. calcoaceticus-A. baumannii (Acb) complex of the genus Acinetobacter are important nosocomial pathogens. Finding an appropriate antimicrobial therapy against these pathogens is a serious concern.
Genetic elements like integrons frequently transfer antimicrobial resistance determinants, and are widely disseminated among MDR Acinetobacter spp.; however, scarce data is available about the epidemiological characterization of integron-harboring bacteria. In this study, integrons were characterized to investigate the antimicrobial resistance mechanisms of MDR Acinetobacter spp. identified in a university hospital in Daejeon, Korea over a period of 7 yr. In addition, multilocus sequence typing (MLST) and repetitive extragenic palindromic sequence (REP)-PCR were performed to analyze the relationship between the integron types and MDR Acinetobacter isolates harboring integrons.

Bacterial isolates and rpoB gene analysis
A total of 56 consecutive and MDR Acinetobacter isolates were collected from a university hospital laboratory in Daejeon, Korea between January 2006 and December 2012. Acinetobacter spp. were identified by using the Vitek 2 Automated Instrument ID System (bioMérieux; Marcy l'Etoile, France) and by sequencing the partial rpoB housekeeping gene as described previously [17].

Antimicrobial agents and minimum inhibitory concentration (MIC) determinations
The MICs of Acinetobacter isolates for amikacin, gentamicin, ceftazidime, cefepime, imipenem, meropenem, and ciprofloxacin were determined by the Epsiolon test (Etest; bioMérieux). The data were interpreted as per the criteria approved by CLSI [18]. Escherichia coli ATCC 25922 was used as a reference strain.

Identification of Acinetobacter spp.
A total of 4 Acinetobacter spp. were identified: A. baumannii (N = 30), A. bereziniae (N = 4), A. nosocomialis (N = 5), and A. pittii (N = 17). The distribution of MICs for the 7 antimicrobial agents tested was similar among the 4 Acinetobacter isolates. Most of the Acinetobacter isolates showed high-level resistance to the antimicrobial agents, except for ciprofloxacin. MICs of ciprofloxacin were diverse, ranging from 0.19 mg/L to ≥ 32 mg/L (Table 1).

Molecular typing
The A. baumannii isolates were grouped by MLST into 6 distinct . ST138 (n = 11) was the most prevalent ST, followed by ST137 (n = 10). In this study, the A. All A. baumannii and A. pittii isolates were typed by REP-PCR. Although most A. baumannii isolates displayed the same REP-PCR type regardless of the STs, A. pittii isolates showed diverse patterns depending on the STs (Fig. 1).

DISCUSSION
In recent years, dissemination of the antimicrobial resistance genes through integrons in Acinetobacter spp. has become a major focus in the treatment of nosocomial infections by MDR bacteria [13]. Class 1 integrons have been found to be prevalent in Acinetobacter clinical isolates worldwide, which contain diverse gene cassettes that encode resistance factors to antimicrobial agents such as aminoglycosides, broad-spectrum β-lactams, and trimethoprims [14]. Therefore, our study was designed to determine the prevalence of various class 1 integrons of MDR Acinetobacter spp. In addition, we analyzed the relationship between the class 1 integron gene cassette array and Acinetobacter spp.
Most of the MDR Acinetobacter isolates (89.3%) analyzed in this study harbored class 1 integrons, and the frequency of these isolates in the study region was considerably higher than that in other geographical regions, including the United Kingdom (60%) and China (74.9%) [24]. However, the prevalence of class 1 integrons among MDR Acinetobacter spp. is similar to that documented previously showing the occurrence of class 1 integrase gene in only 15 of 123 Acinetobacter isolates (12%), with all of these 15 isolates showing MDR [25]. The higher detection rate of class 1 integrons was probably due to the pre-selection of clinical isolates showing the MDR phenotype. Our results indicate that class 1 integron is widely disseminated among MDR Acinetobacter spp. in the university hospital in Daejeon, Korea.
According to our results, most MDR Acinetobacter spp. carry class 1 integrons, but the specific integron type varies depending on the species. Type A is the most prevalent class 1 integron detected in Acinetobacter isolates. However, of the 4 Acinetobacter species tested, only an isolate belonging to A. baumannii harbored type A, except for one A. bereziniae isolate. Twentyfive A. baumannii strains harboring type A were collected between 2007 and 2012, and all of the strains belonged to 6 STs (ST69, ST75, ST92, ST137, ST138, and ST358). Our results suggest that type A is a dominant type of integron and that it has been conserved for many years in A. baumannii and other Acinetobacter species. The type A gene cassette array has been reported in A. baumannii as well as in other bacteria, including Acinetobacter spp. (92.0%; 126/137), Burkholderia cepacia (22.2%; 2/9), Klebsiella pneumonia (4.8%; 4/83), and E. coli (0.6%; 1/164) [24,26]. Although the type A detection rate was highest in Acinetobacter isolates, there is a relative paucity of data on the non-A. baumannii Acinetobacter isolates harboring type A. These earlier reports support our results that type A can usually be transmitted horizontally and vertically among A. baumannii isolates.
In contrast to type A, type B was amplified in A. bereziniae, A. nosocomialis, and A. pittii isolates, but not in A. baumannii. In particular, all A. bereziniae and A. pittii isolates contained type B, irrespective of the time sampled or the clone. This result was similar to that of previous studies that found type B in A. pittii, A. bereziniae, and A. nosocomialis strains isolated from Taiwan and South Korea [27,28]. Our results suggest that type B can usually be transmitted across species among non-A. baumannii Acineto-bacter isolates, and is conserved in the Acinetobacter isolates.
In this study, we performed MLST and REP-PCR for epidemiological typing of MDR A. baumannii and A. pittii isolates. The STs were not directly correlated with REP-PCR types in the A. baumannii isolates, but they were well correlated in A. pittii isolates. Previous studies have reported no correlation of STs with REP-PCR types, probably because of the REP-PCR methods used; however, the MLST results were correlated with the REP-PCR clusters [29,30]. Further studies are required to confirm the relationship between REP-PCR and MLST types. On the other hand, we confirmed that A. pittii exhibited a diverse REP-PCR pattern, which was less clonal than that of A. baumannii showing the same REP-PCR pattern. All class 1 integrons detected in our study contained aminoglycoside-resistance genes such as acetyltransferase (aac) and adenylyltransferase (aad), which have been typically found within integron gene cassette arrays [13]. All isolates harboring class 1 integrons showed a high-level resistance rate to aminoglycoside compounds such as amikacin and gentamicin. In addition, the class 1 integrons harbored β-lactamase genes (blaOXA-2, blaIMP-1, and blaVIM-2) and chloramphenicol-resistance gene (catB8). Consequently, the co-occurrence of several antimicrobial-resistance determinants via class 1 integrons can lead to the emergence of MDR Acinetobacter spp. In particular, type B and type C harboring metallo-β-lactamase (MBL) genes (blaIMP-1 and blaVIM-2) were detected in non-A. baumannii Acinetobacter isolates. These results suggest that a major mechanism of carbapenem resistance in non-A. baumannii Acinetobacter isolates involves class 1 integrons containing MBL genes. However, in this study, integrons containing carbapenemase genes were not detected in the A. baumannii isolates. This result suggests that acquisitions of class 1 integrons were not considered as major factors of carbapenem resistance in A. baumannii isolates. It has been reported that the carbapenem resistance of A. baumannii isolates is often associated with the presence of carbapenem-hydrolyzing class D β-lactamase (CHDL) genes located in chromosomes, plasmids, or transposons [31].
Despite the increasingly frequent discovery of MDR Acinetobacter isolates in Korea, not much information is available regarding the distributions of integrons of MDR Acinetobacter strains isolated in Daejeon, Korea. We confirmed that class 1 integrons harboring diverse gene cassettes are widely disseminated among MDR Acinetobacter isolates. Although class 1 integrons can be transferred horizontally between unrelated isolates belonging to different species, each type of the 4 gene cassette arrays evaluated in this study was found in isolates belonging to Acinetobacter spp., irrespective of the time sampled and STs. Our findings suggest that certain class 1 integrons tend to transfer horizontally and vertically among A. baumannii or non-A. baumannii Acinetobacter isolates. Accordingly, the present study emphasizes that continuous analyses of gene cassette arrays of class 1 integrons will provide useful information regarding antimicrobial resistance mechanisms specific to different Acinetobacter species.