ABSTRACT
Multidrug-resistant (MDR) Acinetobacter spp. have been globally disseminated in association with the successful clonal lineage Acinetobacter baumannii international clone II (IC II). Because the prevalence of MDR Acinetobacter spp. in Japan remains very low, we characterized all Acinetobacter spp. (n = 866) from 76 hospitals between October 2012 and March 2013 to describe the entire molecular epidemiology of Acinetobacter spp. The most prevalent species was A. baumannii (n = 645; 74.5%), with A. baumannii IC II (n = 245) accounting for 28.3% of the total. Meropenem-resistant isolates accounted for 2.0% (n = 17) and carried ISAba1-blaOXA-23-like (n = 10), blaIMP (n = 4), or ISAba1-blaOXA-51-like (n = 3). Multilocus sequence typing of 110 representative A. baumannii isolates revealed the considerable prevalence of domestic sequence types (STs). A. baumannii IC II isolates were divided into the domestic sequence type 469 (ST469) (n = 18) and the globally disseminated STs ST208 (n = 14) and ST219 (n = 4). ST469 isolates were susceptible to more antimicrobial agents, while ST208 and ST219 overproduced the intrinsic AmpC β-lactamase. A. baumannii IC II and some A. baumannii non-IC II STs (e.g., ST149 and ST246) were associated with fluoroquinolone resistance. This study revealed that carbapenem-susceptible A. baumannii IC II was moderately disseminated in Japan. The low prevalence of acquired carbapenemase genes and presence of domestic STs could contribute to the low prevalence of MDR A. baumannii. A similar epidemiology might have appeared before the global dissemination of MDR epidemic lineages. In addition, fluoroquinolone resistance associated with A. baumannii IC II may provide insight into the significance of A. baumannii epidemic clones.
KEYWORDS: Acinetobacter spp., Acinetobacter baumannii international clone II, MLST, blaADC
INTRODUCTION
The prevalence of multidrug-resistant (MDR) Acinetobacter spp. has increased globally during the last 2 decades, and high rates of carbapenem resistance, as high as more than 50% of Acinetobacter species isolates, have been reported from Asian countries (1–3). The emergence and dissemination of several clonal lineages contribute to the increasing prevalence of MDR Acinetobacter spp. (4). One of the most successful clonal lineages, Acinetobacter baumannii international clone II (IC II), corresponds to clonal complex 92 (CC92) according to the multilocus sequence typing (MLST) Oxford scheme developed by Bartual et al. (5) and to sequence type 2 (ST2) of the alternative Pasteur MLST scheme. A. baumannii IC II is associated with nosocomial outbreaks and multidrug resistance (6) and has spread globally (7).
In Japan, A. baumannii IC II is also reported to be strongly associated with multidrug or carbapenem resistance (8–10) and has caused nosocomial outbreaks (11–13). However, the prevalence of MDR Acinetobacter species isolates (criteria are described in Materials and Methods section) remains low in Japan. According to the annual open report 2015 of the clinical laboratory division of Japan Nosocomial Infections Surveillance (JANIS) (14), a national surveillance system, MDR Acinetobacter species isolates accounted for only 0.5% of Acinetobacter species isolates, and the rates of imipenem and meropenem resistance were 3.2% and 1.8%, respectively (https://janis.mhlw.go.jp/english/report/open_report/2015/4/1/ken_Open_Report_Eng_201500_clsi2012.pdf). Therefore, previous studies mainly focused on MDR or carbapenem-nonsusceptible isolates cannot elucidate the entire molecular epidemiology of Acinetobacter spp. and the reason for the low levels of resistance in Japan. Thus, in this study, we characterized clinical Acinetobacter species isolates, including carbapenem-susceptible isolates, collected nationwide in Japan.
RESULTS
Bacterial isolates and species identification.
A total of 932 isolates from 78 hospitals were characterized. Of the 932 isolates, 886 isolates from 76 hospitals were identified as Acinetobacter spp. and subjected to further investigation. Distributions of species and specimen types are shown in Table 1. A. baumannii was the most prevalent (n = 645; 74.5%), followed by Acinetobacter nosocomialis and Acinetobacter pittii. Among A. baumannii isolates, 245 (38.0%) belonged to IC II, accounting for 28.3% of all Acinetobacter spp.
TABLE 1.
Species identifications and specimen types
| Identification | No. of isolates (% of total) | Specimen type |
GenBank accession no. of rpoB sequence of reference straina | |||||
|---|---|---|---|---|---|---|---|---|
| Respiratory | Urine | Wound | Blood | Stool | Other | |||
| A. baumannii | 645 (74.5) | 498 | 57 | 34 | 13 | 8 | 35 | DQ207471 |
| IC II | 245 (28.3) | 215 | 21 | 6 | 0 | 1 | 2 | |
| Non-IC II | 400 (46.2) | 283 | 36 | 28 | 13 | 7 | 33 | |
| A. nosocomialis | 83 (9.6) | 73 | 1 | 4 | 1 | 1 | 3 | EU477118 |
| A. pittii | 60 (6.9) | 38 | 2 | 1 | 3 | 2 | 14 | EU477114 |
| A. seifertii | 18 (2.1) | 13 | 2 | 2 | 1 | 0 | 0 | EU477126 |
| A. bereziniae | 13 (1.5) | 13 | 0 | 0 | 0 | 0 | 0 | DQ207475 |
| A. junnii/A. grimontii | 12 (1.4) | 8 | 0 | 1 | 0 | 1 | 2 | DQ207483/DQ207486 |
| A. soli | 11 (1.3) | 10 | 0 | 0 | 0 | 0 | 1 | HQ148175 |
| A. ursingii | 8 | 7 | 0 | 0 | 0 | 0 | 1 | DQ231239 |
| Genomic sp. between 1 and 3 | 5 | 2 | 1 | 0 | 1 | 1 | 0 | EU477122 |
| A. radioresistens | 2 | 1 | 1 | 0 | 0 | 0 | 0 | DQ207489 |
| A. guillouiae | 2 | 0 | 1 | 1 | 0 | 0 | 0 | DQ207476 |
| A. calcoaceticus | 1 | 1 | 0 | 0 | 0 | 0 | 0 | DQ207474 |
| A. baylyi | 1 | 1 | 0 | 0 | 0 | 0 | 0 | EU477155 |
| A. beijerinckii | 1 | 1 | 0 | 0 | 0 | 0 | 0 | EU477124 |
| A. johnsonii | 1 | 1 | 0 | 0 | 0 | 0 | 0 | DQ207485 |
| Genomic sp. 15BJ/genomic sp. 16 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | EU477133/EU477135 |
| Acinetobacter spp.b | 2 | 2 | 0 | 0 | 0 | 0 | 0 | |
| Total (% of total) | 866 | 669 (77.3) | 65 (7.5) | 44 (5.1) | 19 (2.2) | 13 (1.5) | 56 (6.5) | |
The following Acinetobacter species (GenBank accession no. of rpoB sequence of reference strain) were not isolated in this study: A. gerneri (DQ207482), A. venetianus (EU477136), A. towneri (DQ207493), A. tandoii (DQ207491), A. haemolyticus (DQ207484), A. schindleri (DQ207490), A. bouvetii (DQ207473), A. lwoffii (DQ207487), A. tjernbergiae (EU477153), A. gyllenbergii (EU477148), A. parvus (DQ207488), A. brisouii (KJ124836), A. albensis (KR611814), A. gandensis (KJ569689), A. kookii (KM821031), A. guangdongensis (KR611818), A. indicus (KJ124838), A. bohemicus (KJ124834), A. harbinensis (KF803234), A. rudis (KJ124837), A. nectaris (KJ124840), A. qingfengensis (KC631629), A. puyangensis (JX499272), genomic sp. 6 (DQ207480), genomic sp. 9 (DQ207481), genomic sp. 13BJ (EU477132), genomic sp. 14BJ (EU477147), genomic sp. 15TU (EU477119), genomic sp. 17 (EU477134).
Isolates were unidentifiable at the species level because the partial rpoB sequences had low identity with the closest reference sequences (91.9% and 85.5%), though they were identified as Acinetobacter spp. using 16S rRNA sequencing and Vitek2.
Only 19 Acinetobacter species isolates were isolated from blood. Interestingly, none of these was A. baumannii IC II, while 13 (68.4%) were A. baumannii non-IC II. Respiratory specimens were the most frequent specimens from which Acinetobacter spp. were isolated. Unlike isolates from blood, A. baumannii IC II tended to be isolated from respiratory specimens more often than A. baumannii non-IC II, with 87.8% (215/245) of A. baumannii IC II isolates derived from respiratory specimens and 70.8% (283/400) of A. baumannii non-IC II isolates derived from these specimens (P < 0.001).
The 866 isolates were obtained from 698 patients, and demographic information was available for 676 patients. There were 407 males (60.2%) and 269 females (39.8%). The age distribution of the 676 patients ranged between 0 and 97 years with a median age of 65 and was similar between males and females. The age and sex distributions were similar across patients who carried A. baumannii IC II, A. baumannii non-IC II, and non-baumannii Acinetobacter species isolates.
Differences in antimicrobial susceptibilities among A. baumannii IC II, A. baumannii non-IC II, and non-baumannii Acinetobacter species isolates.
The rates of antimicrobial resistance were compared among isolates categorized as A. baumannii IC II (n = 245), A. baumannii non-IC II (n = 400), and non-baumannii Acinetobacter spp. (n = 221) (Table 2). A. baumannii IC II exhibited a higher proportion of resistant isolates than the other two groups for all antimicrobial agents except colistin and polymyxin B. Notably, all A. baumannii IC II isolates were resistant to ciprofloxacin, whereas only 18.4% of the isolates in the other two groups were resistant. There were six MDR isolates, of which two were A. baumannii IC II and four A. baumannii non-IC II. All MDR isolates were susceptible to minocycline, colistin, and polymyxin B. Colistin-resistant isolates were identified as A. baumannii non-IC II (n = 1), Acinetobacter beijerinkii (n = 1), Acinetobacter bereziniae (n = 2), and Acinetobacter seifertii (n = 1).
TABLE 2.
Rates of antimicrobial resistance among isolates collected in this study and comparison with JANIS data
| Antimicrobial agent(s) | % of isolates collected in this study resistant among: |
% of Acinetobacter species isolates resistant (no. of isolates tested) in JANIS annual report 2013b | |||
|---|---|---|---|---|---|
| IC II (n = 245a) | Non-IC II (n = 400) | Non-baumannii (n = 221) | All isolates (n = 866) | ||
| Piperacillin-tazobactam | 24.5 | 0.8 | 0.9 | 7.5 | 7.8 (4,953) |
| Ampicillin-sulbactam | 8.2 | 0.0 | 0.0 | 2.3 | 5.8 (4,498) |
| Ceftazidime | 69.8 | 2.0 | 0.9 | 20.9 | 10.0 (20,856) |
| Cefepime | 48.2 | 4.0 | 0.0 | 15.5 | 9.2 (15,394) |
| Imipenem | 3.7 | 1.3 | 0.0 | 1.6 | 3.6 (16,947) |
| Meropenem | 4.9 | 1.3 | 0.0 | 2.0 | 3.7 (17,027) |
| Gentamicin | 51.0 | 6.0 | 2.7 | 17.9 | 9.5 (19,422) |
| Amikacin | 24.5 | 0.0 | 0.0 | 6.9 | 3.5 (20,863) |
| Levofloxacin | 87.8 | 13.0 | 2.7 | 31.5 | 8.3 (20,040) |
| Ciprofloxacin | 100 | 24.8 | 6.8 | 41.5 | No data |
| Minocycline | 13.5 | 0.0 | 0.0 | 3.8 | No data |
| Colistin | 0.0 | 0.3 | 1.8 | 0.6 | No data |
| Polymyxin B | 0.0 | 0.0 | 0.9 | 0.2 | No data |
To better characterize the epidemiology of these isolates throughout Japan, the prevalence of antimicrobial-resistant isolates in this study was compared with data from JANIS (Table 2) (https://janis.mhlw.go.jp/english/report/open_report/2013/3/1/ken_Open_Report_Eng_201300.pdf) (15). The proportions of isolates resistant to ceftazidime, cefepime, gentamicin, amikacin, and levofloxacin were higher in this study than in JANIS. A. baumannii IC II isolates exhibited significantly higher rates of resistance to these antimicrobial agents than did the other two groups. This suggests a higher proportion of A. baumannii IC II isolates in this study than in isolates from the hospitals participating in JANIS.
MIC90s and MIC50s for the three groups described above are shown in Table 3. It is notable that A. baumannii IC II isolates had higher MIC50s and MIC90s than the other two groups, even for some antimicrobial agents for which A. baumannii IC II isolates exhibited low rates of resistance, such as ampicillin-sulbactam, imipenem, and meropenem.
TABLE 3.
Distribution of MIC90s and MIC50s for A. baumannii IC II, A. baumannii non-IC II, and non-baumannii Acinetobacter species isolates
| Antimicrobial agent(s) | MIC range (μg/ml)a | Value(s) (μg/ml) for isolates in indicated group |
|||||
|---|---|---|---|---|---|---|---|
| MIC90 |
MIC50 |
||||||
| IC II (n = 245b) | Non-IC II (n = 400) | Non-baumannii (n = 221) | IC II (n = 245) | Non-IC II (n = 400) | Non-baumannii (n = 221) | ||
| Piperacillin-tazobactam | ≤8/4 to ≥512/4 | 256/4 | ≤8/4 | ≤8/4 | ≤8/4 | ≤8/4 | ≤8/4 |
| Ampicillin-sulbactam | ≤1/0.5 to 64/32 | 16/8 | 2/1 | 2/1 | 4/2 | ≤1/0.5 | ≤1/0.5 |
| Ceftazidime | ≤1 to ≥64 | ≥64 | 8 | 8 | ≥64 | 2 | 4 |
| Cefepime | ≤1 to ≥64 | ≥64 | 16 | 8 | 16 | 2 | 2 |
| Imipenem | ≤0.125 to 64 | 2 | 0.5 | 0.5 | 1 | 0.25 | 0.25 |
| Meropenem | ≤0.125 to 64 | 2 | 1 | 0.5 | 1 | 0.25 | 0.25 |
| Gentamicin | ≤1 to ≥128 | ≥128 | 8 | 2 | 16 | ≤1 | ≤1 |
| Amikacin | ≤2 to ≥256 | ≥256 | 8 | 4 | 4 | ≤2 | ≤2 |
| Levofloxacin | ≤0.25 to ≥64 | 16 | 8 | 0.5 | 8 | ≤0.25 | ≤0.25 |
| Ciprofloxacin | ≤0.25 to ≥64 | ≥64 | ≥64 | 1 | ≥64 | ≤0.25 | ≤0.25 |
| Minocycline | ≤1 to ≥64 | 16 | ≤1 | ≤1 | 4 | ≤1 | ≤1 |
| Colistin | ≤0.5 to ≥8 | ≤0.5 | ≤0.5 | 1 | ≤0.5 | ≤0.5 | ≤0.5 |
| Polymyxin B | ≤0.5 to ≥8 | ≤0.5 | ≤0.5 | 1 | ≤0.5 | ≤0.5 | ≤0.5 |
Distribution of MICs for all isolates.
n = number of isolates.
Distribution of acquired carbapenem resistance genes among Acinetobacter spp.
The distribution of acquired carbapenem resistance genes is shown in Table 4. None of the isolates were positive for blaOXA-40-like, blaOXA-58-like, blaVIM group, or blaTMB group genes. blaOXA-51-like and blaOXA-23-like, encoding naturally occurring oxacillinases, were detected in all 645 A. baumannii and both Acinetobacter radioresistens isolates, respectively. Among 17 meropenem-resistant isolates (MIC of ≥8 μg/ml), the most prevalent gene was ISAba1-blaOXA-23-like (n = 10). Three isolates had ISAba1 upstream from blaOXA-51-like, all of which were blaOXA-80. In contrast, the other 47 isolates with ISAba1-blaOXA-51-like were not resistant to meropenem, and the blaOXA-51-like sequences were blaOXA-66 in all but two isolates that carried blaOXA-80 and blaOXA-104, respectively. Overall, ISAba1-blaOXA-23-like and ISAba1-blaOXA-51-like were mainly detected in A. baumannii IC II and blaIMP was detected in A. baumannii non-IC II isolates.
TABLE 4.
Distribution of acquired carbapenem resistance genes
| Gene | No. of isolates with meropenem MIC of: |
Total | |||||||
|---|---|---|---|---|---|---|---|---|---|
| 8 μg/ml (n = 17a) |
≤4 μg/ml (n = 849) |
||||||||
| A. baumannii IC II | A. baumannii non-IC II | Non-baumannii Acinetobacter spp. | Subtotal | A. baumannii IC II | A. baumannii non-IC II | Non-baumannii Acinetobacter spp. | Subtotal | ||
| blaIMP | 4 | 4 | 4 | ||||||
| ISAba1-blaOXA-23-like | 9 | 1 | 10 | 10 | |||||
| ISAba1-blaOXA-51-like | 3 | 3 | 46 | 1 | 47 | 50 | |||
| No tested genes | 0 | 187 | 394 | 221 | 802 | 802 | |||
| Total | 12 | 5 | 0 | 17 | 233 | 395 | 221 | 849 | 866 |
n = number of isolates.
MLST.
A total of 110 nonduplicate A. baumannii isolates, consisting of 37 A. baumannii IC II and 73 A. baumannii non-IC II isolates from 70 hospitals, were subjected to MLST (Table 5). All IC II isolates belonged to ST2 according to the Pasteur scheme, and they were further classified into four STs, ST469, ST208, ST219, and ST1353, based on the Oxford scheme. A. baumannii non-IC II isolates belonged to diverse STs, with the 73 isolates being assigned to 46 and 50 different STs using the Pasteur and Oxford schemes, respectively. Most of these STs have been previously reported in Japan or represented novel STs identified in this study. Four isolates from three hospitals were identified as ST25, which is a successful lineage other than international clones I to III (4), using the Pasteur scheme.
TABLE 5.
MLST data and rates of ciprofloxacin resistance among STs
| Group (no. of isolates) | Pasteur ST (no. of isolates) | Country(ies) with isolates belonging to ST in databasea | Oxford ST (no. of isolates) | Country(ies) with isolates belonging to ST in databasea | No. (%) of ciprofloxacin-resistant isolatesb |
|---|---|---|---|---|---|
| IC II (n = 37) | ST2 (37) | Multiple countries | ST469 (18) | None | 18 (100) |
| ST208 (14) | Multiple countries | 14 (100) | |||
| ST219 (4) | Japan | 4 (100) | |||
| ST1353 (1) | Japanc | 1 | |||
| Non-IC II (n = 73) | ST149 (5) | Japan | ST862 (5) | None | 5 (100) |
| ST406 (5) | Japan, USA | ST310 (5) | USA | 2 (40) | |
| ST25 (4) | Multiple countries | ST229 (4) | Brazil, Mexico, USA | 2 (50) | |
| ST34 (4) | Sweden, Czech Republic | ST432 (2) | China, Czech Republic | 0 | |
| ST1352 (2) | Japanc | 0 | |||
| ST213 (4) | Japan | ST1351 (4) | Japanc | 0 | |
| ST151 (3) | Japan | ST1365 (2) | Japanc | 0 | |
| ST1381 (1) | Japanc | 0 | |||
| ST152 (3) | Japan | ST1373 (3) | Japanc | 0 | |
| ST246 (3) | Japan | ST1362 (3) | Japanc | 3 (100) | |
| ST33 (2) | Japan, USA | ST1364 (1) | Japanc | 0 | |
| ST1372 (1) | Japanc | 0 | |||
| ST40 (2) | China, Czech Republic, Japan | ST373 (1) | China, Czech Republic, Japan | 0 | |
| ST528 (1) | None | 0 | |||
| ST138 (2) | Taiwan, Germany | ST1354 (2) | Japanc | 0 | |
| ST448 (2) | Japanc | ST1361 (2) | Japanc | 0 | |
| ST22 (1) | The Netherlands | ST1382 (1) | Japanc | 1 | |
| ST106 (1) | China | ST605 (1) | None | 0 | |
| ST108 (1) | USA | ST105 (1) | Argentina, USA | 0 | |
| ST113 (1) | China, USA, Brazil | ST873 (1) | USA | 0 | |
| ST120 (1) | Japan | ST1383 (1) | Japanc | 0 | |
| ST150 (1) | Japan | ST718 (1) | None | 0 | |
| ST154 (1) | Spain, Saudi Arabia, USA | ST943 (1) | USA | 0 | |
| ST193 (1) | Brazil, Poland | ST1377 (1) | Japanc | 0 | |
| ST240 (1) | Japan, Switzerland | ST1370 (1) | Japanc | 0 | |
| ST241 (1) | Japan, Brazil, USA | ST613 (1) | USA | 0 | |
| ST267 (1) | Australia, Spain | ST942 (1) | Australia | 1 | |
| ST331 (1) | China | ST1366 (1) | Japanc | 0 | |
| ST412 (1) | Iraq, USA | ST1349 (1) | Japanc | 0 | |
| ST439 (1) | Japanc | ST1380 (1) | Japanc | 0 | |
| ST441 (1) | Japanc | ST1350 (1) | Japanc | 0 | |
| ST442 (1) | Japanc | ST1355 (1) | Japanc | 0 | |
| ST443 (1) | Japanc | ST1356 (1) | Japanc | 0 | |
| ST444 (1) | Japanc | ST1357 (1) | Japanc | 0 | |
| ST445 (1) | Japanc | ST1358 (1) | Japanc | 0 | |
| ST446 (1) | Japanc | ST1359 (1) | Japanc | 0 | |
| ST447 (1) | Japanc | ST1360 (1) | Japanc | 0 | |
| ST449 (1) | Japanc | ST1367 (1) | Japanc | 0 | |
| ST450 (1) | Japanc | ST1368 (1) | Japanc | 0 | |
| ST451 (1) | Japanc | ST1369 (1) | Japanc | 0 | |
| ST452 (1) | Japanc | ST1371 (1) | Japanc | 0 | |
| ST453 (1) | Japanc | ST1374 (1) | Japanc | 0 | |
| ST454 (1) | Japanc | ST1375 (1) | Japanc | 0 | |
| ST455 (1) | Japanc | ST1376 (1) | Japanc | 0 | |
| ST456 (1) | Japanc | ST1384 (1) | Japanc | 0 | |
| ST874 (1) | Japanc | ST1363 (1) | Japanc | 0 | |
| ST875 (1) | Japanc | ST1342 (1) | None | 0 | |
| ST876 (1) | Japanc | ST1378 (1) | Japanc | 1 | |
| ST877 (1) | Japanc | ST1379 (1) | Japanc | 0 | |
| ST897 (1) | Japanc | ST1116 (1) | None | 0 |
Country(ies) where the isolates corresponding to the STs were submitted to the PubMLST Acinetobacter baumannii isolate database (http://pubmlst.org/perl/bigsdb/bigsdb.pl?db=pubmlst_abaumannii_isolates:id1%96id3421).
Percentages are indicated only for STs including more than two resistant isolates.
Novel registration in this study.
Differences in antimicrobial susceptibilities among STs.
Among A. baumannii IC II isolates, isolates belonging to ST208 and its single-locus variant (SLV) ST219 exhibited significantly higher rates of resistance to piperacillin-tazobactam, ceftazidime, and cefepime than those belonging to ST469 (Table 6). Notably, all ST208 and ST219 isolates were resistant to ceftazidime and all ST469 isolates were susceptible to it. It has been reported that the overproduction of the intrinsic AmpC cephalosporinase ADC results from the insertion of ISAba1 sequences upstream from blaADC and is responsible for cephalosporin resistance in A. baumannii (16). To clarify the differences in antimicrobial susceptibilities among STs, β-lactamase genes and upstream ISs were identified (Table 7). The blaADC from ST208 and ST219 isolates encoded ADC-30 or the single-amino-acid variant ADC-73 (see Fig. S1a in the supplemental material). In all of these isolates, the strong promoter sequence presumed to derive from ISAba1 was detected upstream from blaADC, suggesting the overproduction of the intrinsic AmpC β-lactamase (Fig. S1b). In contrast, the blaADC gene in all but one ST469 isolate encoded ADC-25 and the original promoter sequence, rather than the promoter derived from the upstream insertion, was detected (Fig. S1a and b). There were no notable differences in other β-lactamase genes among ST208, ST219, and ST469 isolates.
TABLE 6.
Comparison of rates of antimicrobial resistance among ST208, ST219, and ST469
| Antimicrobial agent(s) | No. (%) of resistant isolates belonging to: |
P value | |
|---|---|---|---|
| ST208 and ST219 (n = 18 isolates) (allele profiles 1-3-3-2-2-97-3 and1-3-3-2-2-101-3) | ST469 (n = 18 isolates) (allele profile 1-12-3-2-2-103-3) | ||
| Piperacillin-tazobactam | 8 (44.4) | 0 (0.0) | 0.003 |
| Ampicillin-sulbactam | 2 (11.1) | 1 (5.6) | 1 |
| Ceftazidime | 18 (100) | 0 (0.0) | <0.001 |
| Cefepime | 14 (77.8) | 1 (5.6) | <0.001 |
| Imipenem | 1 (5.6) | 0 (0.0) | 1 |
| Meropenem | 1 (5.6) | 0 (0.0) | 1 |
| Gentamicin | 13 (72.2) | 7 (38.9) | 0.092 |
| Amikacin | 6 (33.3) | 0 (0.0) | 0.019 |
| Levofloxacin | 16 (88.9) | 14 (77.8) | 0.658 |
| Ciprofloxacin | 18 (100) | 18 (100) | |
| Minocycline | 3 (16.7) | 3 (16.7) | 1 |
| Colistin | 0 (0.0) | 0 (0.0) | |
| Polymyxin B | 0 (0.0) | 0 (0.0) | |
TABLE 7.
β-Lactamase-encoding genes detected in ST208, ST219, and ST469 isolates
| ST, allele profile | No. of isolates | blaOXA-51-like | blaADCa | Other β-lactamase-encoding gene(s) |
|---|---|---|---|---|
| ST208, 1-3-3-2-2-97-3 | 6 | blaOXA-66 | IS-blaADC-30 | |
| 4 | blaOXA-66 | IS-blaADC-30 | blaTEM-1 | |
| 2 | ISAba1-blaOXA-66 | IS-blaADC-30 | ||
| 1 | blaOXA-66 | IS-blaADC-30 | blaPER-1, blaTEM-1 | |
| 1 | blaOXA-66 | IS-blaADC-73 | blaTEM-1 | |
| ST219, 1-3-3-2-2-101-3 | 3 | blaOXA-66 | IS-blaADC-30 | |
| 1 | blaOXA-66 | IS-blaADC-30 | ISAba1-blaOXA-23 | |
| ST469, 1-12-3-2-2-103-3 | 9 | blaOXA-66 | blaADC-25 | |
| 8 | blaOXA-66 | blaADC-25 | blaTEM-1 | |
| 1 | blaOXA-66 | blaADC-153 (novel) |
For ISs of blaADC-30 and blaADC-73, the strong promoter sequence (−35, TTAGAA, and −10, TTATTT) presumed to derive from ISAba1 was detected upstream from blaADC genes. blaADC-153, a novel variant of blaADC, was deposited in GenBank under accession number KY997074.
Among the 73 non-IC II isolates, 58 (79.5%) were susceptible to all antimicrobial agents tested. Fifteen (20.5%) were resistant to ciprofloxacin, and among these, only four were also resistant to antimicrobial agents other than fluoroquinolones. Fluoroquinolone resistance appeared to be associated with specific STs, such as ST149 and ST246 in the Pasteur scheme (Table 5). Four isolates that were resistant to several classes of antimicrobial agents belonged to ST149, ST246, ST25, and ST406.
DISCUSSION
As carbapenem resistance among Acinetobacter spp. remains below 5% in Japan, we predicted a similar low prevalence of A. baumannii IC II. However, in this study, A. baumannii IC II isolates accounted for 28.3% of clinically isolated Acinetobacter spp. and were distributed among about half of the participating hospitals. This means that A. baumannii IC II is already widespread in Japan but is carbapenem susceptible. In our previous study in 2001, which collected 264 Acinetobacter species isolates from 88 hospitals, A. baumannii IC II accounted for only 3% (unpublished data). Although it is difficult to directly compare the data from these studies, as the isolate collection methods were not strictly the same, A. baumannii IC II might have gradually become disseminated in Japan without notice.
The isolates in this study appear to include a relatively higher proportion of A. baumannii IC II than did the isolates from hospitals participating in JANIS. This difference in the proportion of A. baumannii IC II isolates might be related to the characteristics of the participating hospitals. The JANIS annual report in 2013 (15) consisted of data from 745 hospitals, most of which are acute care hospitals (https://janis.mhlw.go.jp/english/report/open_report/2013/3/1/ken_Open_Report_Eng_201300.pdf). In contrast, the participating hospitals in this study included those that have specific beds for long-term care. Thus, our data are consistent with a previous report that long-term-care facilities are potential reservoirs of MDR organisms, including carbapenem-resistant A. baumannii (17).
The dissemination of carbapenem-susceptible A. baumannii IC II shown in this study suggests that epidemic clonal lineages of A. baumannii did not emerge and disseminate as carbapenem-resistant clones. Instead, this lineage may have evolved into a carbapenem-resistant or MDR lineage by acquiring various antimicrobial resistance genes during or after dissemination. This hypothesis is supported by previous reports. During the 1980s and 1990s, A. baumannii international clones I, II, and III (previously European clones I, II, and III) spread throughout Europe and caused outbreaks there, but they were usually susceptible to carbapenem (18, 19), like the A. baumannii IC II isolates in this study. Then, the emergence and proliferation of carbapenemase-producing European clones I and II with blaOXA-23-like, blaOXA-40-like, or blaOXA-58-like genes resulted in increasing rates of carbapenem resistance throughout Europe in the early 2000s (20). In this study, A. baumannii isolates with blaOXA-23-like, which is the major carbapenemase gene in A. baumannii worldwide, remained rare, which may be one reason for the low prevalence of carbapenem-resistant A. baumannii isolates in Japan. However, A. baumannii IC II with ISAba1 upstream from blaOXA-51-like accounted for 5.7% (n = 50) of the total isolates. Clones with ISAba1 upstream from blaOXA-51-like, such as the SE clone, spread in Europe before the dissemination of the OXA-23-producing clone (21). Therefore, it may be necessary to monitor the prevalence of the carbapenemase gene, not just rates of carbapenem resistance.
MLST using the Oxford scheme revealed that ST469, previously ST76 (10), and ST1353 are putative domestic STs of A. baumannii IC II in Japan, as to our knowledge they have been reported only from Japan, with the exception of the first registered ST469 isolate from China. Unlike ST208 and ST219, ST469 isolates were susceptible to most of the antimicrobial agents tested. Interestingly, the intrinsic AmpC cephalosporinase ADC-25 detected in ST469 was also detected in A. baumannii strain A320 (GenBank accession number JN247441), an old IC II isolate from the Netherlands in 1982. ADC-30 with ISAba1, detected in ST208 and ST219 isolates, has been reported more recently and frequently, such as in A. baumannii strain MDR-ZJ06 (China in 2006) (22), A. baumannii strain A91 (Australia in 2005) (23), and A. baumannii strain AB78 (United States in 2007) (24). Some research groups have investigated the genetic diversity of A. baumannii IC II using whole-genome sequencing and divided isolates into various clades (25, 26). Though detailed investigation and comparison of ST469 and ST208 isolates is a subject for a future study, the domestic IC II, ST469, might be closely related to the carbapenem-susceptible ancestral IC II that was disseminated in Europe in the 1980s and 1990s. That is, the molecular epidemiology of Acinetobacter spp. in Japan might be similar to that in Europe in previous decades. This may represent another reason why MDR A. baumannii remains rare in Japan.
The discriminatory power of MLST using the Oxford scheme has been considered to be too high in some cases (27, 28); however, our data suggest that the Oxford scheme is useful for classifying A. baumannii IC II into meaningful groups.
Among A. baumannii non-IC II isolates, most of the other STs were novel in this study or previously identified in Japan (10, 29), suggesting the presence of domestic types of A. baumannii in addition to ST469.
Fluoroquinolone resistance was associated with not only A. baumannii IC II but also some specific STs in the A. baumannii non-IC II isolates, such as ST149 and ST246, according to the Pasteur scheme. Notably, all IC II isolates were resistant to ciprofloxacin. Fluoroquinolone resistance is considered an important factor in the success of the epidemic clone A. baumannii IC II. In regard to A. baumannii non-IC II, ST149 was first identified by our group as an MDR isolate (10). Later, ST149 was reported as a quinolone-resistant isolate in Japan (29), suggesting the possibility of a novel epidemic clonal lineage, according to Shrestha et al. (30). Also, ST246 was first identified as a quinolone-resistant isolate in Japan (29), and ST246 harboring blaOXA-23-like caused a nosocomial outbreak in a hospital (31). Further monitoring of the STs will be required, considering the possibility of novel epidemic clones. The presence of these quinolone-resistant isolates and likely quinolone-resistant STs resulted in the discrepancy between the MIC50s and MIC90s of A. baumannii non-IC II isolates for fluoroquinolones shown in Table 3.
In conclusion, the low prevalence of acquired carbapenemase genes and the presumptive domestic STs of A. baumannii IC II might contribute to the maintenance of low rates of antimicrobial resistance among A. baumannii. The contrast in the epidemiological features of these isolates in Japan compared to those in other countries suggests that our control measures against Acinetobacter spp. have achieved a measure of success. In the “National Action Plan on Antimicrobial Resistance,” we set out to “enhance global multidisciplinary countermeasures against antimicrobial resistance” (http://www.mhlw.go.jp/file/06-Seisakujouhou-10900000-Kenkoukyoku/0000138942.pdf). Japan may therefore provide an international contribution toward antimicrobial resistance control measures around the world. In addition, our results suggest that fluoroquinolones may hold the key to overcoming epidemic clones.
MATERIALS AND METHODS
Collection of bacterial isolates and relevant information.
A total of 86 National Hospital Organization hospitals in Japan participated in this study on a voluntary basis. The 86 hospitals are located in 42 prefectures among the 47 prefectures across Japan (see Fig. S2 in the supplemental material) and include both acute care and long-term care hospitals. Between October 2012 and March 2013, all clinical isolates identified as Acinetobacter spp. were collected, regardless of antimicrobial susceptibility. Duplicate isolates from multiple specimens from the same patient were included. Patient information (age, gender, day of isolation, specimen type, and history of hospitalization abroad) was listed in an information sheet at each participating hospital, and the information sheets were sent to our laboratory with the isolates.
Species level identification and discrimination of A. baumannii IC II.
Species level identification was confirmed by partial rpoB sequence (nucleotide positions 2955 to 3775), with sequences compared with those of reference strains using the neighbor-joining method (32, 33). When the partial rpoB sequence yielded ≥97.5% identity with the closest reference sequence (Table 1), the isolate was identified as the same species as the reference strain. In this regard, the interspecies identities of the partial rpoB sequences of genomic sp. 15BJ (GenBank accession number EU477133) and genomic sp. 16 (GenBank accession number EU477135) and those of Acinetobacter junnii (GenBank accession number DQ207483) and Acinetobacter grimontii (GenBank accession number DQ207486) are 99.0% and 98.4%, respectively, and A. grimontii has been reported as a later heterotypic synonym of A. junnii (34). Therefore, we did not differentiate between these species. Isolates unidentifiable by rpoB sequencing were confirmed as species other than Acinetobacter spp. by 16S rRNA sequencing and the Vitek2 system (bioMérieux, Marcy l'Etoile, France) and excluded from further investigation. Discrimination of A. baumannii IC II was performed by pyrosequencing single-nucleotide polymorphism (SNP) analyses of blaOXA-51-like genes (35).
Antimicrobial susceptibility testing.
MICs for 13 antimicrobial agents, including piperacillin-tazobactam, ampicillin-sulbactam, ceftazidime, cefepime, imipenem, meropenem, gentamicin, amikacin, levofloxacin, ciprofloxacin, minocycline, colistin, and polymyxin B, were determined by the broth microdilution method using dry plate Eiken (Eiken Chemical Co., Tokyo, Japan). Resistance was determined based on the recommended breakpoints of CLSI document M100-S25 (36). Multidrug resistance was based on the criteria of the Infectious Diseases Control Law in Japan and defined as satisfying all three of the following MIC criteria: (i) imipenem and/or meropenem MIC of ≥16 μg/ml for carbapenems, (ii) ciprofloxacin MIC of ≥4 μg/ml and/or levofloxacin MIC of ≥8 μg/ml for fluoroquinolones, and (iii) amikacin MIC of ≥32 μg/ml.
Detection of carbapenem resistance genes.
Carbapenemase genes and the upstream insertion sequence (IS) were detected by PCR using primers specific for blaIMP, blaTMB, blaVIM, blaOXA-51-like, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and ISAba1F (37–39). The primers for blaTMB were designed in this study (forward, 5′-GTCATTTCGCTTTTGCCAACGAAG-3′, and reverse, 5′-CAGCGGTCGCCGTGATTGGCCTTG-3′). blaOXA-51-like and blaOXA-23-like were reported as naturally occurring oxacillinase genes in A. baumannii and A. radioresistens, respectively (40, 41). Therefore, in this study, the isolates in which ISAba1 was present upstream from blaOXA-51-like or blaOXA-23-like were regarded as isolates with carbapenem resistance genes. The entire nucleotide sequences of blaOXA-51-like genes were determined by PCR and sequencing using OXA-69A and OXA-69B primers (40).
MLST and identification of β-lactamase genes from whole-genome sequencing data.
A total of 110 nonduplicate A. baumannii isolates were analyzed using MLST. The 110 isolates were selected as follows. One representative isolate was selected from each hospital (70 isolates from 70 hospitals). In addition, isolates with antimicrobial susceptibility patterns or blaOXA-51-like sequences that were different from the first representative isolate were also selected from each hospital (40 isolates from 32 hospitals). Genomic DNA was subjected to whole-genome sequencing using the Illumina MiSeq sequencer (Illumina, San Diego, CA, USA). De novo assembly of derived paired-end sequence reads was performed with CLC Genomics Workbench version 8.5.1 (CLC bio, Aarhus, Denmark). Sequence types (STs) in both the Oxford and Pasteur schemes were identified based on assembled contigs using MLST1.8 provided by the Center for Genomic Epidemiology (42). Novel allele sequences were reconfirmed by Sanger sequencing. Novel alleles and novel ST profiles were submitted to the MLST database (http://pubmlst.org/abaumannii/) for assignment.
β-Lactamase-encoding genes were detected based on assembled contigs using ResFinder 2.1 with a threshold of 90% identity (43). The nucleotide sequences of β-lactamase-encoding genes were confirmed by comparison with the reference sequence listed on the Lahey β-lactamase site (http://www.lahey.org/Studies/) and in NCBI's β-Lactamase Data Resources (https://www.ncbi.nlm.nih.gov/pathogens/beta-lactamase-data-resources/). The promoter sequences of blaADC were identified by reference to a previous report (16).
This study was reviewed and approved by the Ethical Review Board of the National Institute of Infectious Diseases (Tokyo, Japan).
Statistical analysis.
All calculations were computed with SAS software.
Accession number(s).
A novel variant of blaADC has been deposited in NCBI's β-Lactamase Data Resources under GenBank accession number KY997074 as blaADC-153.
Supplementary Material
ACKNOWLEDGMENTS
We are grateful to National Hospital Organization and all of the participating hospitals for providing bacterial isolates, to Kumiko Kai and Yoshie Taki for technical assistance, and to Paul Higgins and curators of the Acinetobacter baumannii MLST database for their assistance in submitting our MLST data.
This work was supported by the Ministry of Health, Labor and Welfare of Japan (grant number H24-Shinkou-Ippan-010), the Research Program on Emerging and Re-emerging Infectious Diseases of the Japan Agency for Medical Research and Development (AMED), and a grant-in-aid for young scientists (B) from the Japan Society for the Promotion of Science (grant number 16K19208).
Footnotes
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02190-17.
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