Molecular Epidemiology of Clinical Carbapenem-Resistant Acinetobacter baumannii-calcoaceticus complex Isolates in Tertiary Care Hospitals in Java and Sulawesi Islands, Indonesia

Carbapenem-resistant Acinetobacter baumannii (A. baumannii)-calcoaceticus complex (CRAb-cc) is an important pathogen causing nosocomial infections worldwide; however, molecular epidemiology of the A. baumannii-calcoaceticus complex in Indonesian hospitals is scarce. This study aimed to determine the clonal relatedness of CRAb-cc in two tertiary care hospitals in Malang and Manado in Indonesia. The CRAb-cc isolates from routine clinical cultures in two tertiary care hospitals in Malang and Manado were identified using the Vitek2® system (bioMérieux, Lyon, France). Multi-locus variable-number tandem-repeat analysis (MLVA) typing, multi-locus sequence typing (MLST), clonal complex (CC), and phylogenetic tree analysis were conducted for a subset of isolates. Seventy-three CRAb-cc isolates were collected. The CRAb-cc isolates were frequently found among lower-respiratory-tract specimens. We detected the MLVA type (MT) 1, MT3, and MT4 CRAB-cc isolates belonging to the sequence type (ST) 642, and CC1 was the predominant clone in this study. In conclusion, we identified the clonal relatedness of A. baumannii-calcoaceticus complex isolates in two tertiary care hospitals in Malang and Manado in Indonesia. Further study is required to investigate the clinical importance and distribution of ST642 in Indonesian hospitals for developing prevention and control measures.


Introduction
The World Health Organization includes Acinetobacter baumannii (A. baumannii) as one of the six ESKAPE organisms. ESKAPE is the abbreviation of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter spp. [1,2]. The ESKAPE pathogens are potentially resistant to antimicrobial agents and associated with nosocomial infections [3].
A. baumannii is a Gram-negative bacteria that is ubiquitously found in the environment, including soil and water [4]. However, A. baumannii has emerged as the most important healthcare-associated pathogens, which is involved in ventilator-associated pneumonia, urinary tract infections, surgical site infections, and bacteremia [5]. Three related species of the Acinetobacter genus, A. baumannii, Acinetobacter pittii, and Acinetobacter nosocomialis, are often encountered in human specimens and are considered as nosocomial pathogens [4]. They are designated as the A. baumannii-calcoaceticus complex due to the phenotypic similarity [1].

Bacterial Isolates
A. baumannii-calcoaceticus complex isolates were obtained as a part of routine diagnostic procedure and analyzed anonymously. Only one clinical culture with A. baumanniicalcoaceticus complex per patient was included in this study. The strains were isolated and identified from blood, urine, the lower respiratory tract, and wounds based on clinically indicated cultures in each hospital involved in this study.
The identification and antibiotic susceptibility test of CRAB-cc were performed by the VITEK2 ® system (VITEK ® 2 GN ID and VITEK ® 2 AST-GN93; bioMérieux, Lyon, France) [19] and subsequently analyzed according to CLSI 2019 guideline [33]. The CRABcc isolates were stored in trypticase soy broth with 10% glycerin and stored at −80 • C until further characterization.

DNA Extraction and Carbapenemase Genes' Identification
Bacterial DNA was isolated using the GeneAll ® ExgeneTM, Seoul, Republic of Korea. The DNA samples were stored at −20 • C. The detection of carbapenemase genes of seventythree isolates of CRAB-cc were described elsewhere. Briefly, Ambler class A (bla KPC ), Ambler class B (bla NDM ), and Ambler class D (bla OXA-23 ) (Figures 1 and 2) were detected by PCR [23]. The positive control was the CRAB-cc clinical isolates carrying the bla KPC , bla NDM , and bla OXA23 confirmed by PCR in the previous study [19,31], and the negative control was a nuclease free-water. The primer sets and targeted genes are shown in Table 1.

Setting and Study Design
Two tertiary care hospitals were involved in this study: Dr. Saiful Anwar Hospital in Malang (Java Island; 885 beds) and Prof. Dr. R. D. Kandou Hospital in Manado (Sulawesi Island: 1062 beds). The study was performed from March 2019 to August 2019 in Dr. Saiful Anwar Hospital and from June 2019 to November 2019 in Prof. Dr. R. D. Kandou Hospital [32]. The clinical specimens were collected from patients hospitalized in the respective intensive care units (ICU; 37 specimens), neonatal intensive care units (NICU; 8 specimens), cardiovascular intensive care units (CVICU; 3 specimens), medical wards (21 specimens), and surgical wards (4 specimens) in the two tertiary care hospitals. This study was approved by the medical ethics committees of Dr. Saiful Anwar Hospital (Malang) and Prof. Dr. R. D. Kandou Hospital (Manado) (number 400/059/K.3/302/2019 and number 054/EC-KEPK/IV/2019, respectively).

Bacterial Isolates
A. baumannii-calcoaceticus complex isolates were obtained as a part of routine diagnostic procedure and analyzed anonymously. Only one clinical culture with A. baumanniicalcoaceticus complex per patient was included in this study. The strains were isolated and identified from blood, urine, the lower respiratory tract, and wounds based on clinically indicated cultures in each hospital involved in this study.
The identification and antibiotic susceptibility test of CRAB-cc were performed by the VITEK2 ® system (VITEK ® 2 GN ID and VITEK ® 2 AST-GN93; bioMérieux, Lyon, France) [19] and subsequently analyzed according to CLSI 2019 guideline [33]. The CRABcc isolates were stored in trypticase soy broth with 10% glycerin and stored at −80 °C until further characterization.

DNA Extraction and Carbapenemase Genes' Identification
Bacterial DNA was isolated using the GeneAll ® ExgeneTM, Seoul, Republic of Korea. The DNA samples were stored at −20 °C. The detection of carbapenemase genes of seventy-three isolates of CRAB-cc were described elsewhere. Briefly, Ambler class A (blaKPC), Ambler class B (blaNDM), and Ambler class D (blaOXA-23) (Figures 1 and 2) were detected by PCR [23]. The positive control was the CRAB-cc clinical isolates carrying the blaKPC, blaNDM, and blaOXA23 confirmed by PCR in the previous study [19,31], and the negative control was a nuclease free-water. The primer sets and targeted genes are shown in Table 1.

Multi-Locus Variable-Number Tandem Repeat Analysis (MLVA)
MLVA was carried out for 73 CRAB-cc isolates as previously described [34], using eight loci, including 3530, 3002, 2240, 1988, 0826, 0845, 2396, and 3468, as shown in the Table 2. PCR reactions for each locus VNTR program were as follows: initial denaturation of 5 min at 94 °C, 30 cycles of denaturation for 30 s at 94 °C, an annealing step for 30 s, an elongation step at 72 °C, and a final elongation step for 7 min at 72 °C. The number of repetitions for each allele was obtained by subtracting the flanking area and then dividing its value by the length of each repetition. The "failed" designation was given when no amplification or ambiguous amplicon patterns were observed repeatedly at a given locus, and the corresponding allele was indicated with a "-" symbol. Strains were compared using MLVAnet: http://mlva.u-psud.fr/MLVAnet/ (accessed on 1 February 2021) and were assigned into MLVA-8 complexes based on the criteria suggested by Pourcel C et al. [34].

Multi-Locus Variable-Number Tandem Repeat Analysis (MLVA)
MLVA was carried out for 73 CRAB-cc isolates as previously described [34], using eight loci, including 3530, 3002, 2240, 1988, 0826, 0845, 2396, and 3468, as shown in the Table 2. PCR reactions for each locus VNTR program were as follows: initial denaturation of 5 min at 94 • C, 30 cycles of denaturation for 30 s at 94 • C, an annealing step for 30 s, an elongation step at 72 • C, and a final elongation step for 7 min at 72 • C. The number of repetitions for each allele was obtained by subtracting the flanking area and then dividing its value by the length of each repetition. The "failed" designation was given when no amplification or ambiguous amplicon patterns were observed repeatedly at a given locus, and the corresponding allele was indicated with a "-" symbol. Strains were compared using MLVAnet: http://mlva.u-psud.fr/MLVAnet/ (accessed on 1 February 2021) and were assigned into MLVA-8 complexes based on the criteria suggested by Pourcel C et al. [34].

Multilocus Sequence Typing (MLST)
The MLST was carried out based on the sequence analysis of the internal fragments of seven housekeeping genes: cpn60 (60-kDa chaperonin), fusA (elongation factor EF-G), gltA (citrate synthase), pyrG (CTP synthase), recA (homologous recombination factor), rplB (50S ribosomal protein L2), and rpoB (RNA polymerase subunit B) adopted from the Institute Pasteur MLST method (Table 3) [27]. We selected randomly from two to four CRAb-cc isolates from each MLVA type (depends on the MLVA cluster size) for further analysis using MLST. Table 3. Gene primers used for the detection of housekeeping genes by PCR in genes in CRAB-cc isolates in MLST studies.

Clonal Complex (CC) Analysis
The CC analysis was performed using eBURST v.3 (http://eburst.mlst.net) (accessed on 24 December 2021) as previously described [35]. Eleven isolates of CRAB-cc analyzed by MLST were further identified for the CC analysis.

Phylogenetic Analysis
The phylogenetic analysis was generated from the eleven CRAB-cc isolates that were also investigated by MLST and CC analysis. The evolutionary relationship among isolates was built by combining sequences of the seven housekeeping genes, and it was then analyzed using MEGA X (Philadelphia, PA, USA) with the neighbor-joining method [36,37].

Abaum-2396
Abaum-3468    MLST was performed for eleven CRAb-cc isolates, consisting of one isolate from MT1, four isolates from MT2, three isolates from MT3, and one isolate of each of MT4, MT5, and MT6. The MLST analysis identified ST642 as the most frequently found sequence type among CRAb-cc isolates belonging to CC1 (Table 4) and corresponding to MT1, MT3, and MT4. The results of clonal complex analysis of eleven CRAb-cc isolates showed that CC1 and CC2 were predominantly found in Dr. Saiful Anwar Hospital, Malang, and Prof. Dr. R. D. Kandou Hospital, Manado, respectively (Table 5). Table 5   MLST was performed for eleven CRAb-cc isolates, consisting of one isolate from MT1, four isolates from MT2, three isolates from MT3, and one isolate of each of MT4, MT5, and MT6. The MLST analysis identified ST642 as the most frequently found sequence type among CRAb-cc isolates belonging to CC1 (Table 4) and corresponding to MT1, MT3, and MT4. The results of clonal complex analysis of eleven CRAb-cc isolates showed that CC1 and CC2 were predominantly found in Dr. Saiful Anwar Hospital, Malang, and Prof. Dr. R. D. Kandou Hospital, Manado, respectively (Table 5). Table 5 shows MLST and CC analysis of eleven CRAB-cc in Dr. Saiful Anwar Hospital, Malang, and Prof. Dr. R. D. Kandou Hospital, Manado compared to the MLVA types. The phylogenetic analysis detected a close relationship among four isolates from Malang, including MLG-8, MLG-31, MLG-32, and MLG-42 (clade 1); in addition, MLG-30 from Malang was closely related to MDO-9 and MDO-26 from Manado (clade 2). The MDO-2 from Manado showed ST1276, which was independent and singleton; it is suggested that mutation had occurred. We found the MDO-6 strain corresponds to ST642 and CC1; it is suggestive that there are not many mutations in this strain [36]. Clade 1 and clade 2 were in concordance with CC1 and CC2, respectively ( Figure 5). Another molecular epidemiology study on CRAb-cc was performed in a tertiary care hospital in Jakarta, Indonesia, performed by Saharman YR et al.; however, the phylogenetic analysis was not included [19].
The phylogenetic analysis detected a close relationship among four isolates from Malang, including MLG-8, MLG-31, MLG-32, and MLG-42 (clade 1); in addition, MLG-30 from Malang was closely related to MDO-9 and MDO-26 from Manado (clade 2). The MDO-2 from Manado showed ST1276, which was independent and singleton; it is suggested that mutation had occurred. We found the MDO-6 strain corresponds to ST642 and CC1; it is suggestive that there are not many mutations in this strain [36]. Clade 1 and clade 2 were in concordance with CC1 and CC2, respectively ( Figure 5). Another molecular epidemiology study on CRAb-cc was performed in a tertiary care hospital in Jakarta, Indonesia, performed by Saharman YR et al.; however, the phylogenetic analysis was not included [19].

Discussion
To the authors knowledge, the present study was the first clonality study of CRAbcc clinical isolates in Malang and Manado. The present study showed that the CRAb-cc were frequently found in lower-respiratory-tract specimens. Abdulzahra AT et al. reported similar results in the hospital El-Kasr El-Aini (Cairo, Egypt). They found the predominant isolate of the A. baumannii-calcoaceticus complex in lower-respiratory-tract specimens [38].
Multi-locus variable-number tandem-repeat analysis showed two large MLVA types of CRAb-cc isolates in Dr. Saiful Anwar Hospital, Malang, designated MT2 and MT3, whereas one MLVA type (MT2) was predominantly found in Prof. Dr. R. D. Kandou Hospital, Manado. Similarly to other studies, we identified multiple MLVA types, including MT1, MT3, and MT4, within one sequence type (ST642). It is suggested that the three MLVA types are probably related.
The MLST and clonal complex analysis showed that ST642 CC1 corresponds to MT3, the most frequently found clone in this study. The predominance of ST642 CC1 in this study could not be determined to be a short-lived cluster of infections or a prolonged endemic because the baseline data are not available in both hospitals. The ST642 was also reported by another study in Indonesia conducted by Saharman YR et al., but not as the predominant sequence type among CRAb-cc strains in Dr. Cipto Mangunkusumo General Hospital, Jakarta, Indonesia [19], and in another study in Pakistan [39].
This study found five isolates consisting of four isolates from Malang and one isolate from Manado that were assigned to the same clone (ST642 CC1). It is suggested that the cross-transmission of ST642 CC1 may have occurred in Malang; however, the spreading

Discussion
To the authors knowledge, the present study was the first clonality study of CRAb-cc clinical isolates in Malang and Manado. The present study showed that the CRAb-cc were frequently found in lower-respiratory-tract specimens. Abdulzahra AT et al. reported similar results in the hospital El-Kasr El-Aini (Cairo, Egypt). They found the predominant isolate of the A. baumannii-calcoaceticus complex in lower-respiratory-tract specimens [38].
Multi-locus variable-number tandem-repeat analysis showed two large MLVA types of CRAb-cc isolates in Dr. Saiful Anwar Hospital, Malang, designated MT2 and MT3, whereas one MLVA type (MT2) was predominantly found in Prof. Dr. R. D. Kandou Hospital, Manado. Similarly to other studies, we identified multiple MLVA types, including MT1, MT3, and MT4, within one sequence type (ST642). It is suggested that the three MLVA types are probably related.
The MLST and clonal complex analysis showed that ST642 CC1 corresponds to MT3, the most frequently found clone in this study. The predominance of ST642 CC1 in this study could not be determined to be a short-lived cluster of infections or a prolonged endemic because the baseline data are not available in both hospitals. The ST642 was also reported by another study in Indonesia conducted by Saharman YR et al., but not as the predominant sequence type among CRAb-cc strains in Dr. Cipto Mangunkusumo General Hospital, Jakarta, Indonesia [19], and in another study in Pakistan [39].
This study found five isolates consisting of four isolates from Malang and one isolate from Manado that were assigned to the same clone (ST642 CC1). It is suggested that the cross-transmission of ST642 CC1 may have occurred in Malang; however, the spreading of ST642 CC1 in Manado should be investigated with more samples. Further multicenter study involving more samples is required to get more insight into the molecular epidemiology of CRAb-cc strains in Indonesia.
ST2 CC2 was the second sequence type frequently found in this study, including two isolates from Malang and one isolate from Manado. ST2 was also found in Los Angeles [27], which might be associated with human mobility between countries or continents [24]. In addition, we detected three unique sequence types in this study, ST641, ST823, and ST1276, which were also found in Colombia [40], China [41], and Taiwan [42], respectively.
The phylogenetic tree showed the evolutionary relationship among CRAb-cc isolates obtained from Dr. Saiful Anwar Hospital in Malang (clade 1), and that among isolates from Dr. Saiful Anwar Hospital in Malang and Prof. Dr. R. D. Kandou Hospital in Manado (clade 2). A mathematical model study reported that the evolution of multidrug-resistant organisms is more likely to occur when the bacteria are exposed to the antibiotics. The kinds of antibiotics used in the population might be associated with a genome that is ready to evolve [21,[43][44][45]. However, more isolates are required to identify the phylogenetics among CRAB-cc isolates in the two tertiary care hospitals in this study.
The reservoir of CRAB-cc isolates in this study is unknown. The pathogen could survive in the adverse environmental conditions, fostering its persistence and spread in the hospital environment through the hands of the hospital staff, respiratory therapy equipment, tap water, soap dispensers, food (including hospital food), patient beds (mattresses, pillows, bed curtains and blankets), infusion pumps, stainless steel trolleys, door handles, and telephone handles [46,47]. Nevertheless, further investigation is required to discover the source of transmission of CRAB-cc isolates in Dr. Saiful Anwar Hospital and Prof. Dr. R. D. Kandou Hospital.
The present study has certain limitations. First, this study was conducted in two tertiary care hospitals in Indonesia; therefore, it cannot represent national data yet. However, it could be used as a point reference of the molecular epidemiology of CRAB-cc strains in Indonesia. Second, we did not perform the MLST, clonal complex analysis, and phylogenetic analysis for all samples due to limited resources; therefore, the clonal relatedness of all isolates obtained from clinical specimens in Dr. Saiful Anwar Hospital and Prof. Dr. R. D. Kandou Hospital could not be provided.

Conclusions
The ST642-CC1 was detected as the most common clone of CRAb-cc in Dr. Saiful Anwar Hospital in Malang, In addition, this study detected one clade of CRAb-cc isolates designated as clade 2 (ST2-CC2) that consisted of one CRAb-cc isolate from Dr. Saiful Anwar Hospital (Malang) and one CRAb-cc isolate from Prof. Dr. R. D. Kandou Hospital (Manado), representing a clonal relatedness between CRAb-cc in Dr. Saiful Anwar Hospital (Malang) and Prof. Dr. R. D. Kandou Hospital (Manado). Human mobility and antibiotic prescription behavior may be associated with the emerging of a new clade of MDRO in a hospital setting. A national surveillance system is required to have more insight regarding the molecular epidemiology of CRAB-cc in Indonesian hospitals and to develop a strategy to control the spread of CRAB-cc in Indonesian hospitals.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data of this study are available from the corresponding author on request.