Abstract
Carbapenem-resistant Acinetobacter baumannii (A. baumannii, CRAb) is an emerging global threat for healthcare systems, particularly in Southeast Asia. Next-generation sequencing (NGS) technology was employed to map genes associated with antimicrobial resistance (AMR) and to identify multilocus sequence types (MLST). Eleven strains isolated from humans in Vietnam were sequenced, and their AMR genes and MLST were compared to published genomes of strains originating from Southeast Asia, i.e., Thailand (n = 49), Myanmar (n = 38), Malaysia (n = 11), Singapore (n = 4) and Taiwan (n = 1). Ten out of eleven Vietnamese strains were CRAb and were susceptible only to colistin. All strains harbored ant(3”)-IIa, armA, aph(6)-Id and aph(3”) genes conferring resistance to aminoglycosides, and blaOXA-51 variants and blaADC-25 conferring resistance to ß-lactams. More than half of the strains harbored genes that confer resistance to tetracyclines, sulfonamides and macrolides. The strains showed high diversity, where six were assigned to sequence type (ST)/2, and two were allocated to two new STs (ST/1411-1412). MLST analyses of 108 strains from Southeast Asia identified 19 sequence types (ST), and ST/2 was the most prevalent found in 62 strains. A broad range of AMR genes was identified mediating resistance to ß-lactams, including cephalosporins and carbapenems (e.g., blaOXA-51-like, blaOXA-23, blaADC-25, blaADC-73, blaTEM-1, blaNDM-1), aminoglycosides (e.g., ant(3”)-IIa, aph(3”)-Ib, aph(6)-Id, armA and aph(3’)-Ia), phenicoles (e.g., catB8), tetracyclines (e.g., tet.B and tet.39), sulfonamides (e.g., sul.1 and sul.2), macrolides and lincosamide (e.g., mph.E, msr.E and abaF). MLST and core genome MLST (cgMLST) showed an extreme diversity among the strains. Several strains isolated from different countries clustered together by cgMLST; however, different clusters shared the same ST. Developing an action plan on AMR, increasing awareness and prohibiting the selling of antibiotics without prescription must be mandatory for this region. Such efforts are critical for enforcing targeted policies on the rational use of carbapenem compounds and controlling AMR dissemination and emergence in general.
Keywords: Acinetobacter baumannii, carbapenem-resistant, MDR, WGS, Vietnam, Southeast Asia
1. Introduction
Acinetobacter (A.) baumannii is a notorious Gram-negative pathogen associated with a multitude of severe nosocomial infections and high mortalities in intensive care units (ICUs) [1]. The pathogen is known to cause ventilator-associated pneumonia, bloodstream, skin and urinary tract infections and secondary meningitis [2,3]. A. baumannii is intrinsically resistant to many antibiotics and may acquire resistance via mutational changes in chromosomal structure and through horizontal gene transfer [4]. The spread and dissemination of multidrug-resistant (MDR) and extremely drug-resistant (XDR) A. baumannii have become a public health concern in both developing and developed countries. The prevalence of resistance towards last-resort antibiotics such as carbapenems and colistin is increasing globally [4,5]. The treatment of carbapenem-resistant A. baumannii (CRAb) is becoming a global challenge. CRAb is emerging worldwide, and the majority of these isolates often show MDR or XDR patterns [6,7]. They are also associated with an increased length of stay at hospital ICUs [8]. CRAb is associated with increased mortalities in patients with bloodstream infections in low- and middle-income countries [9]. It is considered one of the most common pathogens of nosocomial infections in Southeast Asia [10].
Vietnam is one of the Southeast Asian countries with the highest resistance prevalences amongst Gram-negative pathogens in the Asia-Pacific region [11]. It is considered the “hottest” spot of MDR A. baumannii in Asia. A. baumannii was considered one of the most frequent causes of ventilator-associated respiratory infection [12,13,14] and secondary meningitis [15] in Vietnamese ICUs. From 2008 to 2011, 101 clinical A. baumannii strains were isolated from ICU patients in two medical settings in Vietnam [16]. Between 2012 and 2014, 252 Acinetobacter spp. were isolated from patients admitted to three hospitals in southern Vietnam. Among them, 160 were confirmed as A. baumannii [17]. Between 2014 and 2015, A. baumannii posed serious therapeutic problems in ICU patients at a major tertiary hospital in Ho Chi Minh City [18]. Several separate studies have been carried out on A. baumannii in Southeast Asian countries and showed increases in the general distribution of A. baumannii in hospitals in Vietnam [12,17,18,19,20], Malaysia [21], Thailand [22], the Philippines [23] and Indonesia [24]. However, no comparison of the circulating strains and no multinational transboundary studies have been carried out to investigate the resistance profiles, MLST and the lineage of A. baumannii in the Southeast Asia region.
Thus, the current study aimed to analyze the whole-genome sequencing (WGS) data of carbapenem-resistant A. baumannii isolates obtained from Vietnamese patients and compare their AMR genes, MLST types and genetic diversity with published genomes of strains from Southeast Asia.
2. Results and Discussion
2.1. Whole-Genome Sequencing Data, MLST and cgMLST Analysis
Genome sequencing of 11 Vietnamese A. baumannii isolates yielded an average read length of 262,090 per isolate (range 1,137,862–2,537,360). The isolates’ mean coverage was 105.9-fold (range from 72-fold to 149-fold) (Table S1). To check for possible contamination and accurate species identification, the software Kraken2 was used, which classifies each read (or contig) [25]. At the genus level, the first match for all isolates was always “Acinetobacter”, on average, 97.61% of the reads (maximum 99.18%, minimum 89.61%). At the species level, the first match for all 11 isolates was always “Acinetobacter baumannii”, on average, 78.24% of the reads. Genome assembly yielded an average genome size of 4,070,357 bp with a minimum of 3,792,190 bp. The GC content was, on average, 38.97%. The mean N50 of the 11 assembled genomes was 88.347 bp (range 41,904–184,445 bp) (Table S2). MLST analyses of eleven Vietnamese A. baumannii strains based on the Pasteur scheme allocated nine strains into a distinct ST. Six strains were assigned to ST/2, two were assigned to ST/571 and one was assigned to ST/164. Two isolates were allocated into two new STs (ST/1411-1412) (Table 1).
Table 1.
Strain types (ST) of 108 A. baumannii strains isolated in Southeast Asia according to the Pasteur scheme.
| MLST | Frequency | Country |
|---|---|---|
| ST/2 | 62 | Vietnam (6), Myanmar (19), Thailand (33), Singapore (3), Malaysia (1) |
| ST/164 | 9 | Vietnam (1), Myanmar (5), Thailand (3) |
| ST/16 | 6 | Myanmar (2), Thailand (4) |
| ST/23 | 4 | Myanmar (3), Thailand (1) |
| ST/25 | 4 | Myanmar (3), Thailand (1) |
| ST/1 | 3 | Myanmar (3) |
| ST/215 | 3 | Thailand (3) |
| ST/571 | 3 | Vietnam (2), Thailand (1) |
| ST/374 | 2 | Malaysia (2) |
| ST/575 | 2 | Myanmar (2) |
| ST/46 | 1 | Malaysia, soil (1) |
| ST/52 | 1 | Thailand (1) |
| ST/109 | 1 | Myanmar (1) |
| ST/129 | 1 | Taiwan (1) * |
| ST/220 | 1 | Malaysia (1) |
| ST/360 | 1 | Malaysia (1) |
| ST/739 | 1 | Malaysia (1) |
| ST/1411 | 1 | Vietnam (1), new ST |
| ST/1412 | 1 | Vietnam (1), new ST |
| ND | 1 | Thailand (1) |
| 20 | 108 | Total |
* Taiwan is part of the area of Greater China and is used as an example of a trading country with Southeast Asian countries.
MLST analysis confirmed the considerable genetic diversity of A. baumannii in the investigated strains from Southeast Asia. Six strains were removed as they did not fit our quality criteria. MLST analyses based on the Pasteur scheme identified 19 sequence types (ST) for 107 strains, and a strain from Thailand could not be assigned to a distinct sequence type due to new alleles. ST/2 was the most prevalent sequence type circulating in the Southeast Asian countries. It was found in 62 strains isolated from humans from Vietnam, Myanmar, Thailand, Singapore and Malaysia, followed by ST/164 and ST/16, which were found in nine and six strains, respectively. A strain obtained from the soil in Malaysia was assigned to ST/46.
The MLST analyses showed that more than half of the human strains (62 out of 107) in the current study were assigned to ST/2 (Pasteur). ST/2 belongs to the international clone II and is the most dominant type globally [26]. In previous studies, most of the CRAb isolates were found belong to the ST2 lineage in strains isolated from Thailand [22] and Myanmar [27] and among strains isolated from all five countries that contributed to this study. From 2008 to 2011, 101 clinical A. baumannii strains were isolated from patients in two medical settings in Vietnam. Most of the A. baumannii isolates obtained from a hospital in Hanoi were ST/91 and ST/231, whereas almost all strains from a hospital in Ho Chi Minh City were ST/136, ST/195 and ST/254 [16]. ST/1, ST/575 and ST/109 were found only in Myanmar, while ST/46, 220, 360, 374 and ST/739 were found only in Malaysia. ST/52 and ST/215 were found only in Thailand, while ST/129 was found only in Taiwan (Table 1). The published knowledge on the general distribution of STs of A. baumannii in the population of Southeast Asia is scarce. These findings are, moreover, in agreement with those of Gaiarsa et al., who demonstrated that the Pasteur scheme is more appropriate for epidemiological studies of A. baumannii [28] and proposed it to be the scheme of choice in parallel with cgMLST.
In the current study, we applied the cgMLST scheme included in the SeqSphere+ Ridom software tools, using a gene-by-gene approach to compare and describe the geographical relationship and the lineage among A. baumannii strains from Vietnam and Southeast Asia. The Vietnamese strains showed high diversity. Five strains appeared in three different clusters, and six strains were unique singletons. WGS showed a higher discriminatory power than conventional typing methods [6]. It allows a more precise trackback analysis of the strains, particularly when analyzing carbapenem-resistant A. baumannii isolates, which threaten the global healthcare system. We observed 14 different clusters (1 to 14) using cgMLST SeqSphere+ analysis. Clusters 1 and 5 contained isolates from Thailand and Myanmar. However, isolates were obtained from different countries and clustered in two distinctive clusters but shared the same Pasteur sequence type (ST/2). Cluster 11 contained one strain from Thailand and one from Vietnam and shared the same Pasteur sequence type (ST/164). Our analyses demonstrate that the strains are highly diverse. However, there was a good correlation between MLST and cgMLST clusters. All strains in the same cluster were assigned to the same ST. The predominant type ST/2 based on the Pasteur scheme could be sub-divided into eleven different clusters and distinct lineages when cgMLST was employed. These results show a superior discriminatory ability of cgMLST compared to the conventional MLST [29].
2.2. Antibiotic Susceptibility Testing (AST) and AMR Determinants of Vietnamese Strains
Antibiotic resistance is a serious problem in the Asia-Pacific region, and A. baumannii strains are among the most accounted for Gram-negative bacteria (GNB) causing urinary tract infections in this region [30]. Ten out of eleven A. baumannii strains originating from Vietnam were MDR, displayed resistance to fluoroquinolones (ciprofloxacin and levofloxacin), carbapenems (imipenem, meropenem and ertapenem), third-generation cephalosporins (cefotaxime, ceftazidime, ceftazidime/avibactam and ceftolozane/tazobactam), fourth-generation cephalosporins (cefepime), piperacillin, piperacillin/tazobactam, chloramphenicol and fosfomycin and were susceptible only to colistin. Resistance to the tetracycline derivative tigecycline (MIC = 1 µg/mL) and trimethoprim/sulfamethoxazole (MIC > 4/76 µg/mL) was seen in nine strains (Table S3). All MDR strains (n = 10) were carbapenem-resistant with MIC values of >8 µg/mL to imipenem (IMP) and >0.5 µg/mL to ertapenem (ERT). The MIC values to meropenem (MER) ranged from 32 to 128 µg/mL, higher than the MIC value (16 µg/mL) reported for the A. baumannii DMS06669 strain isolated in a Vietnam hospital [31]. From 2012 to 2014, resistance to cephalosporins, fluoroquinolones and carbapenems was >90% among 904 A. baumannii isolates from patients with hospital-acquired or ventilator-associated pneumonia in Vietnam [13], while colistin (MIC90, ≤0.25 mg/L) and tigecycline (MIC90, 4 mg/L) showed appreciable activity against A. baumannii. MIC90 for meropenem and imipenem was >32 mg/L in more than 80% of 74 strains isolated from tracheal aspirate specimens taken from patients with suspected ventilator-associated pneumonia from January 2011 to June 2012 [32]. All MIC values of tested antibiotics in the current study are shown in Table 2. One strain showed susceptibility to almost all tested antibiotics. It may has been isolated from a patient from rural areas where the use of antibiotics is seldom because they always depend on medicinal plants in treatment, in contrast to other patients.
Table 2.
The MIC (µg/mL) for the 11 sequenced Vietnamese A. baumannii strains as evaluated with MICRONAUT software.
| ID | CIP | LEV | AMK | COL | CMP | FOS | TGC | T/S | PIP | PIT | CTX | CAZ | CAA | CTA | CEP | IMP | MER | ERT |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 18Y0059 | >2 | >2 | >32 | ≤1 | >16 | >64 | =1 | >4/76 | >16 | >64/4 | >2 | >128 | >16/4 | >8/4 | =128 | >8 | =64 | >0.5 |
| 18Y0060 | >2 | >2 | >32 | ≤1 | >16 | >64 | =1 | >4/76 | >16 | >64/4 | >2 | >128 | >16/4 | >8/4 | =128 | >8 | =64 | >0.5 |
| 18Y0061 | >2 | >2 | >32 | ≤1 | >16 | >64 | =1 | 4/76 | >16 | >64/4 | >2 | >128 | >16/4 | >8/4 | =64 | >8 | =64 | >0.5 |
| 18Y0064 | >2 | >2 | >32 | ≤1 | >16 | >64 | =1 | >4/76 | >16 | >64/4 | >2 | >128 | >16/4 | >8/4 | =64 | >8 | =128 | >0.5 |
| 18Y0065 | >2 | >2 | >32 | ≤1 | >16 | >64 | =1 | =2/38 | >16 | >64/4 | >2 | >128 | >16/4 | >8/4 | =128 | >8 | =64 | >0.5 |
| 18Y0066 | >2 | >2 | >32 | ≤1 | >16 | >64 | 0.5 | >4/76 | >16 | >64/4 | >2 | =64 | =16/4 | >8/4 | =64 | >8 | =128 | >0.5 |
| 18Y0067 | >2 | >2 | >32 | ≤1 | >16 | >64 | =1 | >4/76 | >16 | >64/4 | >2 | =64 | =16/4 | >8/4 | =64 | >8 | =64 | >0.5 |
| 18Y0068 | ≤0.25 | ≥0.5 | ≤4 | ≤1 | >16 | >64 | ≤0.25 | =4/76 | ≤8 | ≤4/4 | =2 | ≤1 | ≤1/4 | ≤1/4 | ≤1 | ≤1 | ≤0.125 | =0.25 |
| 18Y0072 | >2 | >2 | ≤4 | ≤1 | >16 | >64 | ≤0.25 | ≤1/19 | >16 | >64/4 | >2 | >128 | =16/4 | >8/4 | =128 | >8 | =32 | >0.5 |
| 18Y0074 | >2 | >2 | >32 | ≤1 | >16 | >64 | 0.5 | >4/76 | >16 | >64/4 | >2 | >128 | =16/4 | >8/4 | =32 | >8 | =64 | >0.5 |
| 18Y0075 | >2 | >2 | >32 | ≤1 | >16 | >64 | =1 | =4/76 | >16 | >64/4 | >2 | >128 | >16/4 | >8/4 | =128 | >8 | =64 | >0.5 |
The minimum inhibitory concentration (MIC), ciprofloxacin (CIP), levofloxacin (LEV), amikacin (AMK), colistin (COL), chloramphenicol (CMP), fosfomycin (FOS), tigecycline (TGC), trimethoprim/sulfamethoxazole (T/S), piperacillin (PIP), piperacillin/tazobactam (PIT), cefotaxime (CTX), ceftazidime (CAZ), ceftazidime/avibactam (CAA), ceftolozane/tazobactam (CTA), cefepime (CEP), imipenem (IMP), meropenem (MER) and ertapenem (ERT).
The genome of all strains (100%) harbored ant(3”)-IIa, which confers resistance to aminoglycosides, while the armA gene was found in eight (72.7%) isolates, and aph(6)-Id and aph(3”)-Ib were found in seven (63.6%) isolates. All isolates harbored at least one of the blaOXA-51 variants and blaADC-25, which confer resistance to ß-lactams, while blaOXA-23 and blaTEM-1 were found in nine (81.8%) and six (54.5%) isolates, respectively. More than half of the Vietnamese strains harbored genes that confer resistance to tetracyclines, sulfonamides and macrolides (Table S4).
The prevalence of MDR/CRAb was very high. The results are in agreement with previous reports: most CRAb strains display resistance to at least one compound in three or more antimicrobial categories and are designated as MDR [6,7]. Resistance against imipenem was seen in 91.6% of XDR A. baumannii strains isolated from patients in three hospitals in southern Vietnam between 2012 and 2014 [17]. A strain resistant to all tested classes of antibiotics except ciprofloxacin and colistin was isolated from the sputum of a patient with hospital-acquired pneumonia at the general hospital of Dong Nai [19]. Examination of 79 strains recovered from patients with pneumonia in Thong Nhat Dong Nai General Hospital showed carbapenem resistance and MDR in 80% and 90% of isolates, respectively [33]. This study highlights the very high prevalence of MDR/CRAb in the General Hospital of Phutho, Hanoi. Clinically, A. baumannii has become a notorious nosocomial pathogen worldwide [26], and the emergence of MDR/CRAb has serious consequences in the healthcare system in Southeast Asia [10]. The general distribution of A. baumannii is increasing in hospitals in Vietnam [19], Thailand [22,34], the Philippines [23], Malaysia [21], Indonesia [24] and Taiwan [35]. Our results support previous data from Southeast Asian hospitals, where a substantial increase in the MDR/CRAb isolation rate was demonstrated [10]. Identification of the genetic determinants associated with carbapenem resistance in A. baumannii is helping to explain the continuous selection and ongoing transmission within the healthcare system of Vietnam and other Southeast Asian countries.
2.3. Predicted Phenotype and AMR Determinants of A. baumannii from Southeast Asia
To compare the findings of AMR determinants in Vietnam to neighboring countries, we downloaded and analyzed A. baumannii sequence data from Southeast Asia. The frequency and percentage of resistance genes conferring specific antibiotic resistance were identified in 108 A. baumannii whole genomes. Our WGS approach identified different AMR genes, among which at least 47 genes confer resistance to ß-lactams, 18 to aminoglycosides, 8 to phenicoles, 4 to tetracyclines, 3 to sulfonamides and 3 to macrolides and lincosamide (Table S4).
2.3.1. Resistance to β-Lactams
In total, forty-seven AMR genes mediating resistance to ß-lactams, including cephalosporins and carbapenems, were identified. The Ambler class D β-lactamases were present in almost all strains. The variants of the intrinsic blaOXA-51-like carbapenemase gene were found in 103 (95.5%) strains; blaOXA-66 was the most frequent and was found in 68 (61%) isolates, followed by blaOXA-91, blaOXA-402 and blaOXA-64. Five isolates (4.5%) were devoid of blaOXA-51-like. The blaOXA-23 variant was found in 90 (83%) isolates, and blaOXA-58 was found in 13 (12%) isolates. In terms of Ambler class A β-lactamases, blaTEM-1 was found in 55 (51%) isolates, followed by bla-PER- in 8 (7.5%) isolates, bla-CARB-16 in 5 isolates, bla-VEB-21 in 4 isolates and bla-SHV-5 in 1 strain. Regarding Ambler class B β-lactamases, blaNDM-1 was found in nine (8%) isolates, and blaIMP-14 was found in two strains. Sixteen Acinetobacter-derived cephalosporinase blaADC variants of the Ambler class C β-lactamases were identified. The blaADC-25 variant was the most frequent variant and was detected in all isolates (100%), followed by blaADC-73 in 50 (46%) isolates, blaADC-52 in 8 isolates, blaADC-30 and blaADC-26 in 7 isolates and blaADC-76, blaADC-169 and blaADC-199 in 6 isolates (Table S4).
Four Ambler classes of β-lactamases (i.e., classes A, B, C and D) were identified in the current study. Various resistance genes conferring resistance to carbapenems and cephalosporins were found in A. baumannii isolated from Southeast Asia. The blaOXA-23 and blaOXA-51-like variants were among the most frequent AMR genes identified. Both are currently spreading on plasmids and associated with resistance to all β-lactam compounds, including carbapenems [21,36]. The ADC beta-lactamases are cephalosporinases with extended-spectrum resistance to cephalosporins. All strains harbored blaADC-25, and approximately half of the strains (46%) harbored blaADC-73, which are considered significant determinants responsible for cephalosporins resistance in A. baumannii [37]. More than half of the strains (51%) harbored blaTEM-1. It encodes a class A β-lactamase and has been found in CRAb strains from different regions worldwide [38,39,40].
2.3.2. Resistance to Aminoglycosides
In the 108 analyzed strains, 18 AMR genes conferring resistance to aminoglycosides were identified. Aminoglycoside-modifying enzymes (AMEs), including acetyltransferases (AACs), methyltransferase (armA), phosphotransferases (APHs) and nucleotidyltransferases (ANTs), were identified. The new subclass of intrinsic aminoglycoside nucleotidyltransferase, ANT(3”)-IIa, was widely distributed and was found in almost all strains (99%), followed by the intrinsic aminoglycoside O-phosphotransferase aph(6)-Id and aph(3”)-Ib found in 83 (77%) strains. To date, over a hundred AMEs have been described, and AACs represent the largest group of AMEs [41]. In the current study, AAC genes were detected in 12% of the isolates, while ANT and APH genes were distributed in almost all isolates (Table 3). Moreover, the intrinsic aminoglycoside methyltransferase (MET) armA was found in 73 (76.5%) isolates. ArmA is the most important class of plasmid-mediated MET enzymes that confer resistance to gentamicin in Enterobacteriaceae [42]. Aminoglycosides are broad-spectrum antibiotics used against a wide range of infections caused by Gram-negative bacteria in clinical settings. However, their efficacy has been reduced by resistance development [7,43]. This finding highlights the diversity of aminoglycoside-resistant A. baumannii strains mediated by ANTs, APHs, armA and AACs of AMEs in Southeast Asian countries.
Table 3.
AMR genes detected in 108 whole-genome sequences of A. baumannii originating from Southeast Asia.
| Antibiotic Class | AMR Resistance Genes | Mechanism | Predicted Phenotype | Origin of Strains | |
|---|---|---|---|---|---|
| Gene Family | Frequency (%) | ||||
| Aminoglycosides | ant(3“)-IIa | 107 (99%) | NUT: Nucleotidyltransferase | Streptomycin, spectinomycin | Viet, Myan, Thai, Sing, Mala, Tiaw |
| aph(3“)-Ib | 83 (77%) | PHT: Phosphotransferase | Streptomycin | Viet, Myan, Thai, Sing, Mala, Tiaw | |
| aph(6)-Id | 83 (77%) | PHT: Phosphotransferase | Streptomycin | Viet, Myan, Thai, Sing, Mala, Tiaw | |
| armA_1 | 73 (63.5%) | MET: Methyltransferase | Gentamicin | Viet, Myan, Thai, Sing, Mala, Tiaw | |
| aph(3’)-Ia | 53 (49%) | PHT: Phosphotransferase | Kanamycin | Viet, Myan, Thai, Sing, Mala, Tiaw | |
| aadA1 | 22 (20%) | NUT: Nucleotidyltransferase | Streptomycin | Viet, Myan, Thai, Sing, Tiaw | |
| aph(3’)-Via | 15 (14%) | PHT: Phosphotransferase | Amikacin, kanamycin | Viet, Myan, Thai, | |
| ant(2“)-Ia | 14 (13%) | NUT: Nucleotidyltransferase | Gentamicin, kanamycin | Viet, Myan, Thai | |
| aac(6’)-Ib | 12 (11%) | ACT: Acetyltransferase | Gentamicin | Viet, Myan, Thai, Tiaw | |
| aac(3)-IId | 11 (10%) | ACT: Acetyltransferase | Gentamicin | Myan, Thai | |
| β-lactams | blaOXA-51-like | 103 (95.5%) | Ambler class D β-lactamases | β-lactam (carbapenem) | Viet, Myan, Thai, Sing, Mala, Tiaw |
| blaOXA-66 | 68 (61%) | blaOXA-51 variant | β-lactam (carbapenem) | Viet, Myan, Thai, Sing, Mala, Tiaw | |
| blaOXA-91 | 10 (9%) | blaOXA-51 variant | β-lactam (carbapenem) | Viet, Myan, Thai | |
| blaOXA-23 | 90 (83%) | Ambler class D β-lactamases | β-lactam (carbapenem) | Viet, Myan, Thai, Sing, Mala, Tiaw | |
| blaOXA-58 | 13 (12%) | Ambler class D β-lactamases | β-lactam (carbapenem) | Viet, Myan, Thai, Mala | |
| blaTEM-1 | 55 (51%) | Ambler class A β-lactamases | β-lactam | Viet, Myan, Thai, Sing, Mala | |
| blaADC-25 | 108 (100%) | Ambler class C β-lactamases | β-lactam (cephalosporin) | Viet, Myan, Thai, Sing, Mala, Tiaw | |
| blaADC-73 | 50 (46%) | Ambler class C β-lactamases | β-lactam (cephalosporin) | Viet, Myan, Thai | |
| blaNDM-1 | 9 (8.5%) | Ambler class B β-lactamases | β-lactam (carbapenem) | Viet, Myan, Thai | |
| Phenicoles | catB8 | 14 (13%) | Enzymes inactivation | Chloramphenicol | Viet, Myan, Thai, Tiaw |
| Macrolide | mph.E. | 83 (77%) | Enzymes inactivation | Macrolide | Viet, Myan, Thai, Sing, Mala, Tiaw |
| msr.E. | 85 (79%) | Antibiotic efflux | Macrolide | Viet, Myan, Thai, Sing, Mala, Tiaw | |
| Sulfonamides | sul1 | 31 (29%) | Antibiotic target replacement | Sulfonamide | Viet, Myan, Thai, Sing, Mala, Tiaw |
| sul2 | 71 (66%) | Antibiotic target replacement | Sulfonamide | Viet, Myan, Thai, Sing, Mala, Tiaw | |
| Tetracyclines | tet.39. | 15 (14%) | Antibiotic efflux | Tetracycline | Viet, Myan, Thai |
| tet.B. | 79 (73%) | Antibiotic efflux | Tetracycline | Viet, Myan, Thai, Sing, Mala, Tiaw | |
| Rifamycin | arr-2 | 14 (13%) | Rifamycin | Myan, Thai, | |
Viet (Vietnam), Myan (Myanmar), Thai (Thailand), Sing (Singapore), Mala (Malaysia), Tiaw (Taiwan).
2.3.3. Resistance to Phenicoles, Tetracyclines, Macrolides, Sulfonamides and Rifamycin
At least eight genes encoding resistance to phenicoles (cmlA, catB8, cmlA1, floR, cmlA5, catB3, catA1 and cmlA6) were identified. CatB8 was the most frequent and found in 14 (13%) isolates, followed by cmlA in 6 isolates and floR in 4 isolates. Most A. baumannii isolates are intrinsically resistant to chloramphenicol [44]. However, genes encoding resistance to phenicoles were found only in 13% of the isolates. Four genes encoding resistance to tetracycline (tet.B, tet.39, tet.A, tet.M) were identified. Tet.B was identified in 79 (73%) isolates, and tet.39 was identified in 15 (14%) isolates. Tet.B is a tetracycline efflux protein that confers resistance to tetracycline but not to tigecycline [45]. In the current survey, it was found in the majority of strains. Two genes encoding macrolide resistance were identified. Mph.E and msr.E were identified in 77% and 79% of the isolates, respectively. Three genes encoding resistance to sulfonamides (sul1, sul2 and sul3) were identified. Sul2 and sul1 variants were found in 66% and 29% of the strains, respectively, while sul3 was found in one isolate. Both sul2 and sul1 are mediated by transposons and plasmids [46]. The presence of one or both genes in A. baumannii isolates might confer resistance to trimethoprim/sulfamethoxazole. The arr-2 gene confers resistance to rifampicin was found in 14 (13%) strains (Table 3). Several resistance mechanisms for different antibiotic classes exist in A. baumannii [47] and were found in the current study. Genes encoding resistance to macrolides, tetracyclines, sulfonamides and phenicoles were seen in 77%, 73%, 66% and 13% of the isolates, respectively. The circulation of genes at such high frequency in Southeast Asia is alarming and highlights the urgent need to take effective control measures.
2.3.4. Antibiotic Efflux Pumps
Four categories of efflux pumps were found in A. baumannii isolates of Southeast Asian origin, including the resistance-nodulation-division (RND) superfamily, the major facilitator superfamily (MFS), the multidrug and toxic compound extrusion (MATE) family and the small multidrug resistance (SMR) family transporters. Among these different pumps, the MFS transporter (amvA), RND (adeFGH, adeIJK and adeL), SMR (abeS) and MATE (abeM) were most frequent and found in almost all isolates (99–100%). RND efflux pump-coding genes (adeN, adeR, adeS and adeAB) were found in 92.2%–94.5% of the strains (Table S4). Resistance mediated by antibiotic efflux pump-encoding genes is well documented in A. baumannii [7,20]. Efflux pumps play significant roles in developing AMR in A. baumannii [48]. The chromosomally encoded tripartite efflux pump adeABC is a worldwide-distributed RND superfamily efflux pump in A. baumannii and was found in approximately 80% of the clinical strains. The overexpression of adeABC efflux pumps is mainly associated with reduced susceptibility to tigecycline [49], fluoroquinolones, tetracycline, chloramphenicol and erythromycin, confers resistance to aminoglycosides [50,51] and may contribute resistance to carbapenems [39]. The circulation of numerous efflux pumps with high frequency suggests a significant rise in A. baumannii antibiotic resistance in Southeast Asia. Acinetobacter baumannii AbaF was found in 104 (96%) strains isolated from all contributing countries. AbaF is a major facilitator superfamily (MFS) antibiotic efflux pump interfering with protein synthesis. Its expression in E. coli increases resistance to fosfomycin. The high frequency of resistance to fosfomycin was seen in the tested strains and in a previous study on A. baumannii [7]. It has been reported that the abaF gene is involved in fosfomycin resistance in A. baumannii and plays a role in biofilm formation and virulence mechanisms [52].
2.4. Acquired Resistance in A. baumannii of Southeast Asian Origin
Examination of all strains in the current study, either sequenced strains from Vietnam or downloaded genomes from PlasmidFinder [53] and Platon [54] as two different tools to investigate the potential presence of plasmids, failed to detect plasmids or plasmid replicons in all strains, except for one strain from Myanmar which harbored only two replicons (Col.MG828._1 and Col8282_1). This information is included in Table S5. Moreover, the comprehensive ResFinder server [55] was used to investigate the potential acquired AMR genes in the A. baumannii strains. ResFinder can identify acquired genes and chromosomal mutations mediating AMR in a total or partial DNA sequence. Several genes encoding resistance to aminoglycosides, β-lactams, tetracycline, sulfonamides and macrolides were found. The Acinetobacter-derived cephalosporinase blaADC.25 conferring resistance to cephalosporin was identified in all isolates (100%), and the two genes conferring resistance to carbapenems, blaOXA-23 and blaOXA-51-like (blaOXA-66 variant), were detected in 83% and 61% of the strains, respectively. Moreover, different variants of blaTEM, blaCARB, blaIMP, blaVEB, blaPER, aph.6.Id, aph.3.Ia, aph.3.Ib, tet, sul and armA were identified using the ResFinder database. The presence of various plasmids in the genome of A. baumannii [56] and its ability to acquire foreign DNA [57,58] enhance the acquisition of AMR genes. Several reports suggested that mobile genetic elements play significant roles in the horizontal transfer of AMR genes in A. baumannii, particularly genes that confer resistance to aminoglycosides, chloramphenicol and tetracycline [59,60,61]. Identification of a wide variety of AMR genes by ResFinder in the current study highlights the role of horizontal gene transfer in the development of resistance in A. baumannii in Southeast Asia.
3. Materials and Methods
3.1. Identification of Bacterial Isolates and Antibiotics Susceptibility Testing (AST)
In total, eleven non-repetitive A. baumannii strains isolated from Vietnamese patients at the General Hospital of Phutho, Hanoi, were received by the Institute of Bacterial Infections and Zoonoses (IBIZ, Jena) for species confirmation and typing. The strains were isolated from blood, sputum, CSF and abscess samples of patients admitted to the hospital in 2017. The agreement for receiving the genetic samples according to the Nagoya Protocol was obtained from the Natural Resources and Environment Minister. No additional ethical approval was required. All strains were identified at species level using a combination of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) with a log value of >2.300 and the intrinsic blaOXA-51-like-PCR [62]. The minimum inhibitory concentration (MIC) was determined by the broth microdilution method using an automated MICRONAUT-S system (Micronaut, MERLIN Diagnostics GmbH, Bornheim-Hersel Germany) according to the manufacturer’s instructions. The results were evaluated as susceptible, intermediate and resistant automatically with the built-in MICRONAUTS software. The MIC values for a panel of the 18 antibiotics were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) breakpoint guidelines available for A. baumannii as previously described [7].
3.2. Whole-Genome Sequencing and Collection of Sequence Data from Southeast Asia
DNA extraction was performed using the High-Pure template preparation kit (Roche Applied Sciences, Mannheim, Germany) according to the manufacturer’s instructions. The library preparation and paired-end sequencing on an Illumina MiSeq sequencer were performed for the 11 Vietnamese A. baumannii strains as previously described [7]. Briefly, the Nextera XT DNA Library Prep Kit (Illumina, Inc., San Diego, CA, USA) was utilized to prepare the sequencing library, followed by paired-end sequencing on an Illumina MiSeq sequencer (Illumina, San Diego, CA, USA).
Raw sequencing data were downloaded from NCBIs’ Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/?term, accessed on 1 November 2020) according to the following criteria: Search for species [Acinetobacter baumannii] with the geo_loc_name_country_continent [Asia] provided 1025 genomes. The results were filtered, and the search items following the Library Layout [paired], Library Source [genomic] and Platform [Illumina] were eligible for inclusion. Raw sequence data of all A. baumannii strains belonging to the search criteria from Southeast Asian countries were included in the study. Data of 103 A. baumannii strains were extracted. Of these, 49 A. baumannii genomes were from Thailand (BioProjects: PRJNA623108, PRJNA627433, PRJNA647677 and PRJNA389557], 38 were from Myanmar (BioProject: PRJDB8528), 11 were from Malaysian BioProjects (PRJNA565663 and PRJNA185400), four were from a Singapori BioProject (PRJNA627433), and one was from a Taiwanese BioProject (PRJNA627433). No genomes were found for Vietnam, Indonesia, the Philippines, Cambodia, Laos, Brunei and Timor-Leste (Table 4).
Table 4.
Numbers, geographical origin and source of A. baumannii genomes investigated in the current study from Southeast Asia.
| No. | Country | Geographical Location | No. of Strains | No. of Strains Analyzed | Source of Sequence |
|---|---|---|---|---|---|
| 1 | Thailand | Southeast Asia | 49 | 49 | NCBI |
| 2 | Myanmar | Southeast Asia | 38 | 37 | NCBI |
| 3 | Malaysia | Southeast Asia | 11 | 7 | NCBI |
| 4 | Vietnam | Southeast Asia | 11 | 11 | IBIZ/FLI |
| 5 | Singapore | Southeast Asia | 4 | 3 | NCBI |
| 6 | Indonesia | Southeast Asia | 0 | 0 | - |
| 7 | Philippines | Southeast Asia | 0 | 0 | - |
| 8 | Cambodia | Southeast Asia | 0 | 0 | - |
| 9 | Laos | Southeast Asia | 0 | 0 | - |
| 10 | Brunei | Southeast Asia | 0 | 0 | - |
| 11 | Timor-Leste | Southeast Asia | 0 | 0 | - |
| 12 | Taiwan | East Asia/Trade country | 1 | 1 | NCBI |
| Total | 114 | 108 | |||
3.3. Bioinformatic Data Analysis
Data analysis of sequences from eleven samples sequenced within this study and downloaded sequences was performed with the pipeline WGSBAC (v2.0.0) [7,63,64]. In short, raw sequencing data quality was controlled by WGSBAC with FastQC (v. 0.11.5) [65], and coverage was determined. Shovill (v. 1.0.4), based on SPAdes (v3.14.0) [25], was used for assembly. Assembly quality was checked with QUAST (v. 5.0.2) [66]. Kraken 2 (v. 2.0.7 beta) [67], in combination with the database MiniKraken (v2), was used to classify reads and assemblies and to check for contamination. In silico determination of classical multilocus sequence typing (MLST) was performed based on the assembled genomes using mlst software (v. 2.16.1) according to the Acinetobacter baumannii#2 scheme published by Diancourt and coworkers and referred to as the Pasteur scheme [68]. For antimicrobial resistance profiling and determination of AMR genes, the databases from NCBI AMR Finder Plus [69], ResFinder [55] and CARD [70] were used. Core-Genome MLST (cgMLST) was performed by employing the software Ridom SeqSphere+, version 5.1.0 (Ridom GmbH, Münster, Germany) [71]. PlasmidFinder [53] and Platon [54] were used to investigate the potential presence of plasmids and plasmid replicons.
4. Conclusions and the Way Forward
Southeast Asia encompasses eleven countries with a wide diversity in history, culture and religion: Brunei, Myanmar (Burma), Cambodia, Timor-Leste, Indonesia, Laos, Malaysia, the Philippines, Singapore, Thailand and Vietnam. According to the Southeast Asia Infectious Disease Clinical Research Network, a previous multinational, multicenter cross-sectional study showed that A. baumannii was among the causative pathogens of sepsis in South Asian countries [72] and that CRAb was the most common nosocomial pathogen associated with infection in ICUs in this region [10]. In silico analysis of 108 whole genomes of A. baumannii strains isolated from Southeast Asia successfully assigned 107 strains into distinct STs using the Pasteur scheme. MLST analysis confirmed the considerable diversity of A. baumannii, and ST/2 was the most prevalent sequence type. The strains harbored a wide variety of AMR genes mediating resistance mostly to β-lactams (including cephalosporins and carbapenems), aminoglycosides, phenicoles, tetracyclines, sulfonamides and macrolides. However, the strain resistance phenotype is unknown except for Vietnamese strains. Several antibiotic resistance mechanisms for various antibiotic classes were observed, including β-lactamases, aminoglycoside-modifying enzymes, permeability defects, alteration and replacement of antibiotic target sites, enzymatic inactivation, multidrug efflux pumps and acquisition of AMR genes. A. baumannii combines clonal spread with high genetic flexibility. The clonal spread of ST/2 is obvious, but there is a lot of evolutionary dynamic outside ST/2 and, most concerning, also within ST2. Aminoglycosides mediating resistance genes ant(3”)-IIa, aph(6)-Id, armA and aph(3”)-Ib, and blaOXA-64, blaOXA-23, blaTEM-1 and blaADC-73 genes mediating resistance to β-lactams, as well as tet.B mediating resistance to tetracyclines, are the highest variable genes within ST/2. These might be genes playing a role in improving the adaptation to the environment. Therefore, both decreased antibiotic consumption and infection prevention and control (IPC) (mainly tackling the clinical component of the spread) are required to counteract this pathogen’s spread.
In general, the prevalence of MDR increases due to limited infection control measures and the lack of antimicrobial stewardship teams in healthcare settings. Moreover, antibiotics are sold without a prescription in rural and urban pharmacies in many developing countries, including Southeast Asian countries [73]. Notably, awareness of antibiotic resistance is missing, particularly in rural areas [73]. Uncontrolled travel throughout this region and transboundary trade of animals and foodstuffs have contributed to increasing AMR prevalence. Resistance to carbapenems is increasingly being reported in Southeast Asia health facilities where the antibiotic is not routinely used and is emerging despite its restricted uses due to high costs. A way forward for this region is to design multinational collaborative efforts geared towards investigating the molecular epidemiology of CRAb and its burden on healthcare systems and understanding the underlying genetic mechanisms associated with resistance to carbapenems. In 2013, the Vietnamese Ministry of Health was the first ministry in the WHO Western Pacific region to develop a national action plan on AMR. The antimicrobial resistance reference laboratory and surveillance program were initiated in Vietnam in 2017 [74]. However, within a region characterized by open borders, an environment with high prevalences of AMR organisms and patient self-treatment, a multinational transboundary surveillance program is urgently needed [75]. Multinational unified antimicrobial stewardship and broad-scale collaboration would ultimately enable the persons in charge to identify existing hotspots, possible reservoirs and possible practices, cultures and attitudes that may predispose different communities to CRAb infections. Control of selling antibiotics without prescription and increasing awareness are required. Implementing the Vietnamese way, i.e., developing a national action plan on AMR and establishing an antimicrobial resistance reference laboratory, is a supreme priority. Such efforts would be critical in creating targeted policies on carbapenem compounds’ rational use and controlling AMR’s dissemination and emergence in general. This will be significant because carbapenems are expected to become cheaper and readily available in the future in most countries.
Acknowledgments
We thank Gernot Schmoock, Johannes Solle, Claudia Grosser and Birgit Schikowski for excellent technical assistance.
Supplementary Materials
The following are available online at https://www.mdpi.com/article/10.3390/antibiotics10050563/s1. Table S1: The average read length and the coverage of 108 A. baumannii isolates from Southeast Asia. Table S2: The data of genome assembly of 11 A. baumannii isolates from Vietnam. Table S3: The results of antibiotic susceptibility testing of Vietnamese A. baumannii isolates. Table S4: Metadata, MLST, AMR genes and the predicted antibiotic classes of 108 A. baumannii isolates from Southeast Asia. Table S5: The results of PlasmidFinder for all A. baumannii strains provide some information about the presence of replicons.
Author Contributions
G.W., H.N., L.D.S. and M.W.P. conceptualization, designed research, analyzed data and wrote the paper; J.L. downloaded sequences and performed the bioinformatic analysis; N.H.N. and T.N.M.N. collected the samples and made a preliminary identification of the Vietnamese strains; G.W. performed the work and wrote the first draft. All authors read and approved the final manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
All study data are included in the article and supporting information. The data have also been submitted to the European Nucleotide Archive (ENA). The project accession number is PRJEB43552.
Conflicts of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Footnotes
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Kurihara M.N.L., Sales R.O., Silva K.E.D., Maciel W.G., Simionatto S. Multidrug-resistant Acinetobacter baumannii outbreaks: A global problem in healthcare settings. Rev. Soc. Bras. Med. Trop. 2020;53:e20200248. doi: 10.1590/0037-8682-0248-2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Tian H., Chen L., Wu X., Li F., Ma Y., Cai Y., Song S. Infectious complications in severe acute pancreatitis: Pathogens, drug resistance, and status of nosocomial infection in a university-affiliated teaching hospital. Dig. Dis. Sci. 2019 doi: 10.1007/s10620-019-05924-9. [DOI] [PubMed] [Google Scholar]
- 3.Metan G., Zarakolu P., Otlu B., Tekin I., Aytac H., Bolek E.C., Metin B.C., Arsava E.M., Unal S. Emergence of colistin and carbapenem-resistant Acinetobacter calcoaceticus-Acinetobacter baumannii (CCR-Acb) complex in a neurological intensive care unit followed by successful control of the outbreak. J. Infect. Public Health. 2019 doi: 10.1016/j.jiph.2019.09.013. [DOI] [PubMed] [Google Scholar]
- 4.Pormohammad A., Mehdinejadiani K., Gholizadeh P., Mohtavinejad N., Dadashi M., Karimaei S., Safari H., Azimi T. Global prevalence of colistin resistance in clinical isolates of Acinetobacter baumannii: A systematic review and meta-analysis. Microb. Pathog. 2019 doi: 10.1016/j.micpath.2019.103887. [DOI] [PubMed] [Google Scholar]
- 5.Theriault N., Tillotson G., Sandrock C.E. Global travel and Gram-negative bacterial resistance; implications on clinical management. Expert Rev. Anti-Infect. Ther. 2020 doi: 10.1080/14787210.2020.1813022. [DOI] [PubMed] [Google Scholar]
- 6.Nodari C.S., Cayô R., Streling A.P., Lei F., Wille J., Almeida M.S., de Paula A.I., Pignatari A.C.C., Seifert H., Higgins P.G., et al. Genomic analysis of carbapenem-resistant Acinetobacter baumannii isolates belonging to major endemic clones in South America. Front. Microbiol. 2020;11:584603. doi: 10.3389/fmicb.2020.584603. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Wareth G., Linde J., Hammer P., Nguyen N.H., Nguyen T.N.M., Splettstoesser W.D., Makarewicz O., Neubauer H., Sprague L.D., Pletz M.W. Phenotypic and WGS-derived antimicrobial resistance profiles of clinical and non-clinical Acinetobacter baumannii isolates from Germany and Vietnam. Int. J. Antimicrob. Agents. 2020;56:106127. doi: 10.1016/j.ijantimicag.2020.106127. [DOI] [PubMed] [Google Scholar]
- 8.Niu T., Xiao T., Guo L., Yu W., Chen Y., Zheng B., Huang C., Yu X., Xiao Y. Retrospective comparative analysis of risk factors and outcomes in patients with carbapenem-resistant Acinetobacter baumannii bloodstream infections: Cefoperazone-sulbactam associated with resistance and tigecycline increased the mortality. Infect. Drug Resist. 2018;11:2021–2030. doi: 10.2147/IDR.S169432. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Stewardson A.J., Marimuthu K., Sengupta S., Allignol A., El-Bouseary M., Carvalho M.J., Hassan B., Delgado-Ramirez M.A., Arora A., Bagga R., et al. Effect of carbapenem resistance on outcomes of bloodstream infection caused by Enterobacteriaceae in low-income and middle-income countries (PANORAMA): A multinational prospective cohort study. Lancet. Infect. Dis. 2019;19:601–610. doi: 10.1016/S1473-3099(18)30792-8. [DOI] [PubMed] [Google Scholar]
- 10.Suwantarat N., Carroll K.C. Epidemiology and molecular characterization of multidrug-resistant Gram-negative bacteria in Southeast Asia. Antimicrob. Resist. Infect. Control. 2016;5:15. doi: 10.1186/s13756-016-0115-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Kiratisin P., Chongthaleong A., Tan T.Y., Lagamayo E., Roberts S., Garcia J., Davies T. Comparative in vitro activity of carbapenems against major Gram-negative pathogens: Results of Asia-Pacific surveillance from the COMPACT II study. Int. J. Antimicrob. Agents. 2012;39:311–316. doi: 10.1016/j.ijantimicag.2012.01.002. [DOI] [PubMed] [Google Scholar]
- 12.Phu V.D., Nadjm B., Duy N.H.A., Co D.X., Mai N.T.H., Trinh D.T., Campbell J., Khiem D.P., Quang T.N., Loan H.T., et al. Ventilator-associated respiratory infection in a resource-restricted setting: Impact and etiology. J. Intensive Care. 2017;5:69. doi: 10.1186/s40560-017-0266-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Biedenbach D.J., Giao P.T., Hung Van P., Su Minh Tuyet N., Thi Thanh Nga T., Phuong D.M., Vu Trung N., Badal R.E. Antimicrobial-resistant Pseudomonas aeruginosa and Acinetobacter baumannii From patients with hospital-acquired or ventilator-associated pneumonia in Vietnam. Clin. Ther. 2016;38:2098–2105. doi: 10.1016/j.clinthera.2016.07.172. [DOI] [PubMed] [Google Scholar]
- 14.Nhu N.T.K., Lan N.P.H., Campbell J.I., Parry C.M., Thompson C., Tuyen H.T., Hoang N.V.M., Tam P.T.T., Le V.M., Nga T.V.T., et al. Emergence of carbapenem-resistant Acinetobacter baumannii as the major cause of ventilator-associated pneumonia in intensive care unit patients at an infectious disease hospital in southern Vietnam. J. Med. Microbiol. 2014;63:1386–1394. doi: 10.1099/jmm.0.076646-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Trung N.T., Van Son T., Quyen D.T., Anh D.T., Sang V.V., Lam N.X., Manh N.D., Duong V.P., Cuong B.T., Tuyen Q.D., et al. Significance of nucleic acid testing in diagnosis and treatment of post-neurosurgical meningitis caused by multidrug-resistant Acinetobacter baumannii: A case report. J. Med. Case Rep. 2016;10:313. doi: 10.1186/s13256-016-1104-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Tada T., Miyoshi-Akiyama T., Kato Y., Ohmagari N., Takeshita N., Hung N.V., Phuong D.M., Thu T.A., Binh N.G., Anh N.Q., et al. Emergence of 16S rRNA methylase-producing Acinetobacter baumannii and Pseudomonas aeruginosa isolates in hospitals in Vietnam. BMC Infect. Dis. 2013;13:251. doi: 10.1186/1471-2334-13-251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Tuan Anh N., Nga T.V.T., Tuan H.M., Tuan N.S., Y D.M., Vinh Chau N.V., Baker S., Duong H.H.T. Molecular epidemiology and antimicrobial resistance phenotypes of Acinetobacter baumannii isolated from patients in three hospitals in southern Vietnam. J. Med. Microbiol. 2017;66:46–53. doi: 10.1099/jmm.0.000418. [DOI] [PubMed] [Google Scholar]
- 18.Tran G.M., Ho-Le T.P., Ha D.T., Tran-Nguyen C.H., Nguyen T.S.M., Pham T.T.N., Nguyen T.A., Nguyen D.A., Hoang H.Q., Tran N.V., et al. Patterns of antimicrobial resistance in intensive care unit patients: A study in Vietnam. BMC Infect. Dis. 2017;17:429. doi: 10.1186/s12879-017-2529-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Si-Tuan N., Ngoc H.M., Nhat L.D., Nguyen C., Pham H.Q., Huong N.T. Genomic features, whole-genome phylogenetic and comparative genomic analysis of extreme-drug-resistant ventilator-associated pneumonia Acinetobacter baumannii strain in a Vietnam hospital. Infect. Genet. Evol. 2020;80:104178. doi: 10.1016/j.meegid.2020.104178. [DOI] [PubMed] [Google Scholar]
- 20.Leus I.V., Weeks J.W., Bonifay V., Smith L., Richardson S., Zgurskaya H.I. Substrate specificities and efflux efficiencies of RND efflux pumps of Acinetobacter baumannii. J. Bacteriol. 2018:200. doi: 10.1128/jb.00049-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Rao M., Rashid F.A., Shukor S., Hashim R., Ahmad N. Detection of antimicrobial resistance genes associated with carbapenem resistance from the whole-genome sequence of Acinetobacter baumannii isolates from Malaysia. Can. J. Infect. Dis. Med. Microbiol. 2020;2020:5021064. doi: 10.1155/2020/5021064. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Thirapanmethee K., Srisiri A.N.T., Houngsaitong J., Montakantikul P., Khuntayaporn P., Chomnawang M.T. Prevalence of OXA-Type β-Lactamase genes among carbapenem-resistant Acinetobacter baumannii clinical isolates in Thailand. Antibiotics. 2020;9:864. doi: 10.3390/antibiotics9120864. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Velasco J.M., Valderama M.T., Margulieux K., Diones P.C., Peacock T., Navarro F.C., Liao C., Chua D., Macareo L., Crawford J., et al. Comparison of carbapenem-resistant microbial pathogens in combat and non-combat wounds of military and civilian patients seen at a tertiary military hospital, Philippines (2013–2017) Mil. Med. 2020;185:e197–e202. doi: 10.1093/milmed/usz456. [DOI] [PubMed] [Google Scholar]
- 24.Karuniawati A., Saharman Y.R., Lestari D.C. Detection of carbapenemase encoding genes in Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumanii isolated from patients at Intensive Care Unit Cipto Mangunkusumo Hospital in 2011. Acta Med. Indones. 2013;45:101–106. [PubMed] [Google Scholar]
- 25.Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., Nikolenko S.I., Pham S., Prjibelski A.D., et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. J. Comput. Mol. Cell Biol. 2012;19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Hamidian M., Nigro S.J. Emergence, molecular mechanisms and global spread of carbapenem-resistant Acinetobacter baumannii. Microb. Genom. 2019:5. doi: 10.1099/mgen.0.000306. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Tada T., Uchida H., Hishinuma T., Watanabe S., Tohya M., Kuwahara-Arai K., Mya S., Zan K.N., Kirikae T., Tin H.H. Molecular epidemiology of multidrug-resistant Acinetobacter baumannii isolates from hospitals in Myanmar. J. Glob. Antimicrob. Resist. 2020;22:122–125. doi: 10.1016/j.jgar.2020.02.011. [DOI] [PubMed] [Google Scholar]
- 28.Gaiarsa S., Batisti Biffignandi G., Esposito E.P., Castelli M., Jolley K.A., Brisse S., Sassera D., Zarrilli R. Comparative analysis of the two Acinetobacter baumannii multilocus sequence typing (MLST) schemes. Front. Microbiol. 2019;10:930. doi: 10.3389/fmicb.2019.00930. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Hua X., Zhang L., He J., Leptihn S., Yu Y. Population biology and epidemiological studies of Acinetobacter baumannii in the era of whole-genome sequencing: Is the oxford scheme still appropriate? Front. Microbiol. 2020;11:775. doi: 10.3389/fmicb.2020.00775. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Jean S.S., Coombs G., Ling T., Balaji V., Rodrigues C., Mikamo H., Kim M.J., Rajasekaram D.G., Mendoza M., Tan T.Y., et al. Epidemiology and antimicrobial susceptibility profiles of pathogens causing urinary tract infections in the Asia-Pacific region: Results from the Study for Monitoring Antimicrobial Resistance Trends (SMART), 2010–2013. Int. J. Antimicrob. Agents. 2016;47:328–334. doi: 10.1016/j.ijantimicag.2016.01.008. [DOI] [PubMed] [Google Scholar]
- 31.Si-Tuan N., Ngoc H.M., Hang P.T.T., Nguyen C., Van P.H., Huong N.T. New eight genes identified at the clinical multidrug-resistant Acinetobacter baumannii DMS06669 strain in a Vietnam hospital. Ann. Clin. Microbiol. Antimicrob. 2017;16:74. doi: 10.1186/s12941-017-0250-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Le Minh V., Nhu N.T.K., Phat V.V., Thompson C., Lan N.P.H., Nga T.V.T., Tam P.T.T., Tuyen H.T., Nhu T.D.H., Van Hao N., et al. In vitro activity of colistin in antimicrobial combination against carbapenem-resistant Acinetobacter baumannii isolated from patients with ventilator-associated pneumonia in Vietnam. J. Med. Microbiol. 2015;64:1162–1169. doi: 10.1099/jmm.0.000137. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Hoang Quoc C., Nguyen Thi Phuong T., Nguyen Duc H., Tran Le T., Tran Thi Thu H., Nguyen Tuan S., Phan Trong L. Carbapenemase genes and multidrug resistance of Acinetobacter baumannii: A cross-sectional study of patients with pneumonia in Southern Vietnam. Antibiotics. 2019;8:148. doi: 10.3390/antibiotics8030148. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Leungtongkam U., Thummeepak R., Wongprachan S., Thongsuk P., Kitti T., Ketwong K., Runcharoen C., Chantratita N., Sitthisak S. Dissemination of bla(OXA-23), bla(OXA-24), bla(OXA-58), and bla(NDM-1) Genes of Acinetobacter baumannii isolates from four tertiary hospitals in Thailand. Microb. Drug Resist. 2018;24:55–62. doi: 10.1089/mdr.2016.0248. [DOI] [PubMed] [Google Scholar]
- 35.Chen T.L., Lee Y.T., Kuo S.C., Hsueh P.R., Chang F.Y., Siu L.K., Ko W.C., Fung C.P. Emergence and distribution of plasmids bearing the blaOXA-51-like gene with an upstream ISAba1 in carbapenem-resistant Acinetobacter baumannii isolates in Taiwan. Antimicrob. Agents Chemother. 2010;54:4575–4581. doi: 10.1128/AAC.00764-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Khurshid M., Rasool M.H., Ashfaq U.A., Aslam B., Waseem M., Xu Q., Zhang X., Guo Q., Wang M. Dissemination of bla(OXA-23)-harbouring carbapenem-resistant Acinetobacter baumannii clones in Pakistan. J. Glob. Antimicrob. Resist. 2020;21:357–362. doi: 10.1016/j.jgar.2020.01.001. [DOI] [PubMed] [Google Scholar]
- 37.Lee S.Y., Oh M.H., Yun S.H., Choi C.W., Park E.C., Song H.S., Lee H., Yi Y.S., Shin J., Chung C., et al. Genomic characterization of extensively drug-resistant Acinetobacter baumannii strain, KAB03 belonging to ST451 from Korea. Infect. Genet. Evol. 2018;65:150–158. doi: 10.1016/j.meegid.2018.07.030. [DOI] [PubMed] [Google Scholar]
- 38.Vranić-Ladavac M., Bedenić B., Minandri F., Ištok M., Bošnjak Z., Frančula-Zaninović S., Ladavac R., Visca P. Carbapenem resistance and acquired class D beta-lactamases in Acinetobacter baumannii from Croatia 2009–2010. Eur. J. Clin. Microbiol. Infect. Dis. Off. Publ. Eur. Soc. Clin. Microbiol. 2014;33:471–478. doi: 10.1007/s10096-013-1991-9. [DOI] [PubMed] [Google Scholar]
- 39.Zhu L.J., Pan Y., Gao C.Y., Hou P.F. Distribution of carbapenemases and efflux pump in carbapenem-resistance Acinetobacter Baumannii. Ann. Clin. Lab. Sci. 2020;50:241–246. [PubMed] [Google Scholar]
- 40.Krizova L., Poirel L., Nordmann P., Nemec A. TEM-1 β-lactamase as a source of resistance to sulbactam in clinical strains of Acinetobacter baumannii. J. Antimicrob. Chemother. 2013;68:2786–2791. doi: 10.1093/jac/dkt275. [DOI] [PubMed] [Google Scholar]
- 41.Kim S.Y., Park Y.J., Yu J.K., Kim Y.S. Aminoglycoside susceptibility profiles of Enterobacter cloacae isolates harboring the aac(6’)-Ib gene. Korean J. Lab. Med. 2011;31:279–281. doi: 10.3343/kjlm.2011.31.4.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Fritsche T.R., Castanheira M., Miller G.H., Jones R.N., Armstrong E.S. Detection of methyltransferases conferring high-level resistance to aminoglycosides in Enterobacteriaceae from Europe, North America, and Latin America. Antimicrob. Agents Chemother. 2008;52:1843–1845. doi: 10.1128/AAC.01477-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Mortazavi S.M., Farshadzadeh Z., Janabadi S., Musavi M., Shahi F., Moradi M., Khoshnood S. Evaluating the frequency of carbapenem and aminoglycoside resistance genes among clinical isolates of Acinetobacter baumannii from Ahvaz, south-west Iran. New Microbes New Infect. 2020;38:100779. doi: 10.1016/j.nmni.2020.100779. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Roca I., Marti S., Espinal P., Martínez P., Gibert I., Vila J. CraA, a major facilitator superfamily efflux pump associated with chloramphenicol resistance in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2009;53:4013–4014. doi: 10.1128/AAC.00584-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Roberts M.C. Update on acquired tetracycline resistance genes. FEMS Microbiol. Lett. 2005;245:195–203. doi: 10.1016/j.femsle.2005.02.034. [DOI] [PubMed] [Google Scholar]
- 46.Sköld O. Resistance to trimethoprim and sulfonamides. Vet. Res. 2001;32:261–273. doi: 10.1051/vetres:2001123. [DOI] [PubMed] [Google Scholar]
- 47.Gordon N.C., Wareham D.W. Multidrug-resistant Acinetobacter baumannii: Mechanisms of virulence and resistance. Int. J. Antimicrob. Agents. 2010;35:219–226. doi: 10.1016/j.ijantimicag.2009.10.024. [DOI] [PubMed] [Google Scholar]
- 48.Coyne S., Courvalin P., Perichon B. Efflux-mediated antibiotic resistance in Acinetobacter spp. Antimicrob. Agents Chemother. 2011;55:947–953. doi: 10.1128/AAC.01388-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Lee Y.T., Chen H.Y., Yang Y.S., Chou Y.C., Chang T.Y., Hsu W.J., Lin I.C., Sun J.R. AdeABC Efflux pump controlled by adeRS two-component system conferring resistance to tigecycline, omadacycline and eravacycline in clinical carbapenem-resistant Acinetobacter nosocomialis. Front. Microbiol. 2020;11:584789. doi: 10.3389/fmicb.2020.584789. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Abbott I., Cerqueira G.M., Bhuiyan S., Peleg A.Y. Carbapenem resistance in Acinetobacter baumannii: Laboratory challenges, mechanistic insights and therapeutic strategies. Expert Rev. Anti-Infect. Ther. 2013;11:395–409. doi: 10.1586/eri.13.21. [DOI] [PubMed] [Google Scholar]
- 51.Ranjbar R., Zayeri S., Afshar D. High frequency of adeA, adeB and adeC genes among Acinetobacter baumannii isolates. Iran. J. Public Health. 2020;49:1539–1545. doi: 10.18502/ijph.v49i8.3898. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.Sharma A., Sharma R., Bhattacharyya T., Bhando T., Pathania R. Fosfomycin resistance in Acinetobacter baumannii is mediated by efflux through a major facilitator superfamily (MFS) transporter-AbaF. J. Antimicrob. Chemother. 2017;72:68–74. doi: 10.1093/jac/dkw382. [DOI] [PubMed] [Google Scholar]
- 53.Carattoli A., Zankari E., García-Fernández A., Voldby Larsen M., Lund O., Villa L., Møller Aarestrup F., Hasman H. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob. Agents Chemother. 2014;58:3895–3903. doi: 10.1128/AAC.02412-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Schwengers O., Barth P., Falgenhauer L., Hain T., Chakraborty T., Goesmann A. Platon: Identification and characterization of bacterial plasmid contigs in short-read draft assemblies exploiting protein sequence-based replicon distribution scores. Microb. Genom. 2020:6. doi: 10.1099/mgen.0.000398. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55.Zankari E., Hasman H., Cosentino S., Vestergaard M., Rasmussen S., Lund O., Aarestrup F.M., Larsen M.V. Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 2012;67:2640–2644. doi: 10.1093/jac/dks261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 56.Salgado-Camargo A.D., Castro-Jaimes S., Gutierrez-Rios R.M., Lozano L.F., Altamirano-Pacheco L., Silva-Sanchez J., Pérez-Oseguera Á., Volkow P., Castillo-Ramírez S., Cevallos M.A. Structure and evolution of Acinetobacter baumannii plasmids. Front. Microbiol. 2020;11:1283. doi: 10.3389/fmicb.2020.01283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 57.Krizova L., Dijkshoorn L., Nemec A. Diversity and evolution of AbaR genomic resistance islands in Acinetobacter baumannii strains of European clone I. Antimicrob. Agents Chemother. 2011;55:3201–3206. doi: 10.1128/AAC.00221-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 58.Lin M.F., Lan C.Y. Antimicrobial resistance in Acinetobacter baumannii: From bench to bedside. World J. Clin. Cases. 2014;2:787–814. doi: 10.12998/wjcc.v2.i12.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 59.Lin M.F., Chang K.C., Yang C.Y., Yang C.M., Xiao C.C., Kuo H.Y., Liou M.L. Role of integrons in antimicrobial susceptibility patterns of Acinetobacter baumannii. Jpn. J. Infect. Dis. 2010;63:440–443. [PubMed] [Google Scholar]
- 60.Huang L.Y., Chen T.L., Lu P.L., Tsai C.A., Cho W.L., Chang F.Y., Fung C.P., Siu L.K. Dissemination of multidrug-resistant, class 1 integron-carrying Acinetobacter baumannii isolates in Taiwan. Clin. Microbiol. Infect. Off. Publ. Eur. Soc. Clin. Microbiol. Infect. Dis. 2008;14:1010–1019. doi: 10.1111/j.1469-0691.2008.02077.x. [DOI] [PubMed] [Google Scholar]
- 61.Rabea R.A., Zaki M.E.S., Fahmy E.M., Fathelbab A. Molecular study of nodulation division genes and integron genes in Acinetobacter baumannii. Clin. Lab. 2020:66. doi: 10.7754/Clin.Lab.2020.200124. [DOI] [PubMed] [Google Scholar]
- 62.Turton J.F., Woodford N., Glover J., Yarde S., Kaufmann M.E., Pitt T.L. Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene intrinsic to this species. J. Clin. Microbiol. 2006;44:2974–2976. doi: 10.1128/JCM.01021-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 63.Linde J., Homeier-Bachmann T., Dangel A., Riehm J.M., Sundell D., Öhrman C., Forsman M., Tomaso H. Genotyping of Francisella tularensis subsp. holarctica from hares in Germany. Microorganisms. 2020;8:1932. doi: 10.3390/microorganisms8121932. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 64.García-Soto S., Abdel-Glil M.Y., Tomaso H., Linde J., Methner U. Emergence of multidrug-resistant Salmonella enterica Subspecies enterica serovar infantis of multilocus sequence type 2283 in German broiler farms. Front. Microbiol. 2020;11:1741. doi: 10.3389/fmicb.2020.01741. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 65.Andrews S. FastQC: A Quality Control Tool for High Throughput Sequence Data. v. 0.11.5. [(accessed on 1 August 2020)]; Available online: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
- 66.Gurevich A., Saveliev V., Vyahhi N., Tesler G. QUAST: Quality assessment tool for genome assemblies. Bioinformatics. 2013;29:1072–1075. doi: 10.1093/bioinformatics/btt086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 67.Wood D.E., Lu J., Langmead B. Improved metagenomic analysis with Kraken 2. Genome. Biol. 2019;20:257. doi: 10.1186/s13059-019-1891-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 68.Diancourt L., Passet V., Nemec A., Dijkshoorn L., Brisse S. The population structure of Acinetobacter baumannii: Expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS ONE. 2010;5:e10034. doi: 10.1371/journal.pone.0010034. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 69.Feldgarden M., Brover V., Haft D.H., Prasad A.B., Slotta D.J., Tolstoy I., Tyson G.H., Zhao S., Hsu C.H., McDermott P.F., et al. Validating the AMRFinder tool and resistance gene database by using antimicrobial resistance genotype-phenotype correlations in a collection of isolates. Antimicrob. Agents Chemother. 2019;63 doi: 10.1128/AAC.00483-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 70.Jia B., Raphenya A.R., Alcock B., Waglechner N., Guo P., Tsang K.K., Lago B.A., Dave B.M., Pereira S., Sharma A.N., et al. CARD 2017: Expansion and model-centric curation of the comprehensive antibiotic resistance database. Nucleic Acids Res. 2017;45:D566–D573. doi: 10.1093/nar/gkw1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 71.Higgins P.G., Prior K., Harmsen D., Seifert H. Development and evaluation of a core genome multilocus typing scheme for whole-genome sequence-based typing of Acinetobacter baumannii. PLoS ONE. 2017;12:e0179228. doi: 10.1371/journal.pone.0179228. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 72.Network S.A.I.D.C.R. Causes and outcomes of sepsis in Southeast Asia: A multinational, multicentre cross-sectional study. Lancet. Glob. Health. 2017;5:e157–e167. doi: 10.1016/s2214-109x(17)30007-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 73.Nga do T.T., Chuc N.T., Hoa N.P., Hoa N.Q., Nguyen N.T., Loan H.T., Toan T.K., Phuc H.D., Horby P., Van Yen N., et al. Antibiotic sales in rural and urban pharmacies in northern Vietnam: An observational study. BMC Pharmacol. Toxicol. 2014;15:6. doi: 10.1186/2050-6511-15-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 74.Kinh N.V., Wertheim H.F.L., Thwaites G.E., Khue L.N., Thai C.H., Khoa N.T., Thi Bich Ha N., Trung N.V., Crook D., van Doorn H.R. Developing an antimicrobial resistance reference laboratory and surveillance programme in Vietnam. Lancet Glob. Health. 2017;5:e1186–e1187. doi: 10.1016/S2214-109X(17)30370-4. [DOI] [PubMed] [Google Scholar]
- 75.Li R., van Doorn H.R., Wertheim H.F.L., Khue L.N., Ha N.T.B., Dat V.Q., Hanh C.T., Nga D.T.T., Trang N.N.M., Nadjm B., et al. Combating antimicrobial resistance: Quality standards for prescribing for respiratory infections in Vietnam. Lancet Glob. Health. 2016;4:e789. doi: 10.1016/S2214-109X(16)30267-4. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
All study data are included in the article and supporting information. The data have also been submitted to the European Nucleotide Archive (ENA). The project accession number is PRJEB43552.