You are viewing the site in preview mode

Skip to main content
Download PDF
Download ePub

Molecular genotyping reveals multiple carbapenemase genes and unique blaOXA-51-like (oxaAb) alleles among clinically isolated Acinetobacter baumannii from a Philippine tertiary hospital

Tropical Medicine and Health volume 52, Article number: 62 (2024) Cite this article

Abstract

Background

Acinetobacter baumannii continued to be an important Gram-negative pathogen of concern in the clinical context. The resistance of this pathogen to carbapenems due to the production of carbapenemases is considered a global threat. Despite the efforts to track carbapenemase synthesis among A. baumannii in the Philippines, local data on its molecular features are very scarce. This study aims to characterize A. baumannii clinical isolates from a Philippine tertiary hospital through genotyping of the pathogen’s carbapenemase genes.

Methods

Antibiotic susceptibility profiling, phenotypic testing of carbapenemase production, and polymerase chain reaction assays to detect the different classes of carbapenemase genes (class A blaKPC, class B blaNDM, blaIMP, blaVIM, and class D blaOXA-23-like, blaOXA-24/40-like, blaOXA-48-like, blaOXA-51-like, ISAba1-blaOXA-51-like, blaOXA-58-like) were performed in all collected A. baumannii, both carbapenem resistant and susceptible (n = 52).

Results

Results showed that the majority of the carbapenem-resistant strains phenotypically produced carbapenemases (up to 84% in carbapenem inactivation methods) and possessed the ISAba1-blaOXA-51-like gene complex (80%). Meanwhile, both carbapenem-resistant and carbapenem-susceptible isolates possessed multi-class carbapenemase genes including blaNDM (1.9%), blaVIM (3.9%), blaOXA-24/40-like (5.8%), blaOXA-58-like (5.8%), blaKPC (11.5%), and blaOXA-23-like (94.2%), which coexist with each other in some strains (17.3%). In terms of the intrinsic blaOXA-51-like (oxaAb) genes, 23 unique alleles were reported (blaOXA-1058 to blaOXA-1080), the majority of which are closely related to blaOXA-66. Isolates possessing these alleles showed varying carbapenem resistance profiles.

Conclusions

In summary, this study highlighted the importance of molecular genotyping in the characterization of A. baumannii by revealing the carbapenemase profiles of the pathogen (which may not be captured accurately in phenotypic tests), in identifying potent carriers of transferrable carbapenemase genes (which may not be expressed straightforwardly in antimicrobial susceptibility testing), and in monitoring unique pathogen epidemiology in the local clinical setting.

Background

Acinetobacter baumannii is a notorious pathogen implicated in serious nosocomial infections worldwide. Among the choice antibiotics for treating severe A. baumannii infections are carbapenems (imipenem and meropenem) [1]. However, global surveillance studies found an increase in the occurrence of carbapenem-resistant A. baumannii (CRAb), particularly among low- to middle-income countries [2]. In Southeast Asia (SEA), a region considered a focal point of global antimicrobial resistance, CRAb emerges as an imminent threat [3]. For instance, the Philippine health authority recently stated that more than 50% of the local A. baumannii isolates are already resistant to imipenem and meropenem [4]. Infections due to CRAb have very limited treatment options and were associated with increased hospitalization, in-patient costs, and mortality [5]. As a result, the World Health Organization (WHO) identified CRAb as a critical priority pathogen requiring new antimicrobials [6].

The mechanisms of carbapenem resistance in A. baumannii include carbapenemase production, efflux pumps, and porin loss. Among these pathways, the synthesis of carbapenemases is the most prevalent [7]. Carbapenemases are types of β-lactamases under molecular classes A (serine β-lactamases), B (metallo-β-lactamases), and D (oxacillinases) with carbapenem-hydrolyzing capabilities. Different acquired carbapenemase genes have been reported for A. baumannii in the last decade such as Ambler class A blaKPC [8], class B blaNDM [9], blaVIM [10], blaIMP [11], class D blaOXA-23-like [12], blaOXA-24/40-like [13], blaOXA-58-like [14], and blaOXA-143-like [15]. The class D blaOXA-51-like (recently referred to as oxaAb) is considered intrinsic β-lactamases in A. baumannii, with significant carbapenemase activity observed in the presence of upstream insertion sequences such as ISAba1 [16].

Clinical isolates of CRAb are profiled for carbapenemase genes given the active spread of transferrable β-lactamases among Gram-negative pathogens [17]. Molecular surveillance of CRAb carbapenemases in SEA has been reported previously [18, 19]. Meanwhile, the Philippine Antimicrobial Resistance Surveillance Program recently released the first comprehensive genomic analysis of 104 CRAb isolates in the country [20]. Despite these efforts, the detection of carbapenemases in the clinical setting remains highly reliant on phenotypic tests. Unfortunately, current tests are not reliable when utilized in A. baumannii [21, 22]. As the clinical management and epidemiology of CRAb are dependent on its mechanism of resistance, molecular genotyping remains to be the gold standard in inferring carbapenemase production through the detection of carbapenemase genes. In this investigation, we revealed the presence of multiple carbapenemase genes in clinically isolated A. baumannii [CRAb and carbapenem-susceptible A. baumannii (CSAb)] and linked them to the pathogen’s antibiotic resistance profiles and carbapenemase production phenotypes. In addition, we described 23 novel blaOXA-51-like gene alleles, indicating the unique molecular epidemiology of Philippine CRAb isolates necessitating further surveillance.

Methods

Collection and characterization of the isolates

Stock cultures of non-duplicate A. baumannii clinical isolates from October 2018 to September 2020 (isolated from different patients with clinically significant infections) were collected from the Section of Clinical Microbiology of The Medical City, Pasig City, Philippines. No identifiable patient-related information was gathered; only microbiological and sample-related data were collected. A total of 52 clinically significant isolates were successfully collected. The original isolation sources include endotracheal aspirate (n = 15), sputum (n = 13), blood (n = 11), urine (n = 6), wound (n = 6), and abscess (n = 1). All isolates were maintained in blood agar medium (Thermo Scientific™, USA) incubated at 35 °C–37 °C. Isolates were identified using Vitek® MS Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF; bioMérieux, France), while the antibiotic susceptibility profiles were generated using Vitek® 2 Compact Analyzer (bioMérieux, France) with Vitek® 2 Advanced Expert System (AES, bioMérieux, France). The antibiotics examined include ceftazidime, ciprofloxacin, ceftriaxone, cefepime, gentamicin, imipenem, meropenem, trimethoprim-sulfamethoxazole, and piperacillin–tazobactam. Carbapenem resistance was determined using the latest recommended minimum inhibitory concentration (MIC) cutoff values from the Clinical and Laboratory Standards Institute (CLSI) [22]. Quality check was done through the routine assay performance testing as conducted by the clinical laboratory (accredited by the local health department) and using CLSI-recommended standard strains for Gram-negative bacteria (E. coli ATCC® 25,922™ and P. aeruginosa ATCC® 27,853™) [22]. The MICs of the isolates to carbapenems are described in Additional File 1.

Detection of carbapenemase production

All isolates underwent phenotypic assessment of carbapenemase production using the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and Tris-modified carbapenem inactivation method (CIM-Tris). For MHT, the protocol of Amjad et al. was used [23]. Briefly, 18–24-h-old isolates were heavily streaked from the edge of 10 µg ertapenem (ERT) disks (Mastdiscs® AST, UK) placed on Mueller–Hinton agar (MHA, Himedia®, India) with a lawn of 0.5 McFarland-standardized Escherichia coli ATCC® 25,922™. The cloverleaf-like zone of inhibition (ZOI) within the streak indicated carbapenemase production. Meanwhile, mCIM was carried out using the procedure of Pierce et al. [24]. Briefly, 1µL loopful of 18–24-h-old isolates was suspended in 2 mL Tryptic Soy Broth (TSB, Himedia®, India) with 10 µg meropenem (MEM) disk (Mastdiscs® AST, UK) and incubated at 35 °C for 4 h. After incubation, the disks were harvested and put on MHA plates with 0.5 McFarland-standardized E. coli ATCC® 25,922™. Isolates with ZOI of 6–10 mm were reported as carbapenemase producers. Finally, CIM-Tris was completed utilizing the methodology of Uechi et al. [25]. In summary, 10µL loopful of overnight isolate cultures was suspended in 400µL of 0.5 M Tris–HCl buffer at pH 7.6 (Trizma®, Germany) with 10 µg MEM disk (Mastdiscs® AST, UK) and incubated at 35 °C for 2 h. The ensuing steps were the same as specified for mCIM. Isolates with ZOI of 6–15 mm were reported as carbapenem producers. All setups were carried out in triplicates and incubated at 35 °C–37 °C for 18–24 h prior to ZOI measurements. For all experiments (including the replicates), the quality control strains recommended by the CLSI were used: Klebsiella pneumoniae ATCC® BAA-1705™ (carbapenemase positive) and ATCC® BAA-1706™ (carbapenemase negative) [22]. Meropenem disk batches used in the assays were also quality-checked through routine disk diffusion in E. coli ATCC® 25,922™, as recommended, prior to usage.

Detection of carbapenemase genes

Genomic DNA from each isolate was extracted using the boil lysis procedure [26] and measured using Nanodrop™ 2000 Spectrophotometer (Thermo Fisher Scientific™, USA). Primers targeting class A (blaKPC), class B (blaNDM, blaIMP, blaVIM), and class D (blaOXA-23-like, blaOXA-24/40-like, blaOXA-48-like, blaOXA-51-like, ISAba1-blaOXA-51-like, blaOXA-58-like) carbapenemase genes were used for the polymerase chain reaction (PCR; Additional File 1). PCR tests were carried out in 25 µL single reactions (with 12.5 µL GoTaq® Green Master Mix (Promega, USA), 1.0 µL of 10 µM primers, 50–100 ng DNA sample, and nuclease-free water) using a GeneExplorer™ thermal cycler (Bioer, China) following the PCR procedures listed in Additional File 1. Gel electrophoresis of the amplicons was performed in 1.8%–2% agarose (1st BASE, Singapore) on a MyGel Instaview® gel electrophoresis system (Accuris, Korea) set at 135 V for 30 min, with 1X Tris–acetate-EDTA running buffer (Vivantis, Malaysia). The final gels were visualized and documented in E-Gel™ Power Snap electrophoresis system (Invitrogen™, USA). All PCR assays were done in two trials using appropriate controls. The expected band size of the amplicons and the controls used for genotyping were outlined in Additional File 1.

Sequence-based clonal typing using bla OXA-51-like genes

Sequence-based typing (SBT) of blaOXA-51-like genes was performed to determine the major clonal lineages of the isolates, conforming with the traditional multilocus sequence typing [27,28,29]. In brief, all blaOXA-51-like amplicons from the isolates, together with an amplicon from A. baumannii ATCC® BAA-1605 as a control, underwent paired-end Sanger sequencing (Macrogen, Korea). Complete consensus DNA from the forward and reverse sequences and their matching amino acid sequences were generated using Bioedit version 7.2.5. Initial identities of the DNA and amino acid sequences were determined using the Basic Local Alignment Search Tool incorporated in the Beta-Lactamase Database-Structure and Function (BLDB; http://www.bldb.eu/; accessed on 10 April 2023). Sequences with 100% matching protein identities with known blaOXA-51-like variants (with or without silent single nucleotide mutations) were reported as the native matching allele. Meanwhile, sequences with at least one mismatch in amino acid sequence compared with all known blaOXA-51-like variants were described as novel alleles following the recent recommendations in β-lactamase reporting and nomenclature [30]. An unrooted maximum likelihood tree of the blaOXA-51-like genes was created in PhyML version 3.1 following the parameters derived from jModelTest version 20,160,303 and using 87 reference blaOXA-51-like gene sequences (Additional File 1) from various subclades of the OXA-51 subfamily tree (http://www.bldb.eu/alignment.php?align=D:OXA-51-like; accessed on 10 April 2023). Changes in amino acids were manually mapped from the multiple sequence alignment of the alleles using Clustal W in Bioedit version 7.2.5, with blaOXA-66 as the leader sequence. All original annotated sequences were submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/; accessed on 15 March 2023) with accession numbers OM617740-OM617792 (Additional File 1). The unique blaOXA-51-like alleles are currently annotated as antimicrobial resistance reference genes in GenBank under Bioproject number PRJNA313047 (reference sequence numbers NG_079900-NG_079922) and are also accessible at the BLDB (http://www.bldb.eu/; accessed on 10 April 2023).

Data analysis and visualization

Phenotypic data including antibiotic susceptibility profiles and carbapenemase production were visualized in a heatmap using Microsoft Office 365 Excel (Microsoft, USA). Genotypic data such as gel electrophoretograms and genotype listing were compiled in Microsoft Office 365 PowerPoint (Microsoft, USA). Amino acid linkage map was made in Microsoft Office 365 PowerPoint (Microsoft, USA) following the model of Evans et al. [31]. Statistical analysis on the sensitivity and specificity of phenotypic tests in detecting carbapenemase production compared to the carbapenem resistance profile of the isolates was computed using the MedCalc version 20.218 (https://www.medcalc.org/calc/diagnostic_test.php; accessed 15 January 2023). Results were reported as 95% confidence intervals whenever appropriate.

Results

A significant number of isolates are carbapenemase-producing CRAb

Of the 52 collected, clinically significant A. baumannii isolates, 25 (48.1%) were considered CRAb based on the Vitek® 2 Compact and Advanced Expert System (AES) results, with their corresponding minimum inhibitory concentrations (MICs) described in Additional File 1. All CRAb has an MIC of ≥ 16 mg/L against meropenem and imipenem, whereas CSAb isolates have MICs ranging from ≤ 0.25 to 1 mg/L. All CRAb isolates were considered multidrug resistant (i.e., resistant to at least two antibiotic classes), with 22 (88.0%) being non-susceptible (resistant and intermediately resistant) to all antibiotics tested. Meanwhile, from the CSAb, three (11.1%) were considered multidrug resistant, with resistance ranging from cephalosporins, aminoglycosides, folate pathway antagonists, and β-lactam combination agents. Only six (11.5%) of the total isolates were susceptible to all antibiotics tested.

Results from several phenotypic tests for carbapenemase production presented variabilities. Among the CRAb, carbapenemase production was detected in 13 (52%), 18 (72%), and 21 (84%) isolates using the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and Tris-modified carbapenem inactivation method (CIM-Tris), respectively. Meanwhile, carbapenemase production was found in 9 (33.3%) and 12 (44.4%) carbapenem-susceptible isolates using mCIM and CIM-Tris, respectively. Carbapenemase production was not reported in any carbapenem-susceptible isolates using MHT. The concordance of the phenotypic assays with the carbapenem resistance profiles showed the highest sensitivity in CIM-Tris [84.0% (63.9%–95.5%)] and the highest specificity in MHT [100% (87.2%–100%)] (Table 1).

Table 1 Comparison among the results of the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and Tris-modified carbapenem inactivation method (CIM-Tris) relative to the carbapenem-resistant (CRAb) and carbapenem-susceptible (CSAb) antibiotic susceptibility testing (AST) of the clinical A. baumannii isolates in Vitek® 2 Compact with Advanced Expert System (bioMérieux, France)
Full size table

A. baumannii possessed multiple carbapenemase genes

All isolates possessed at least one of the examined carbapenemase genes, with Ambler class D genes as the most prevalent (Fig. 1; Additional File 1). All isolates harbored the intrinsic blaOXA-51-like genes, with 25 (48.1%) also possessing the upstream ISAba1. The majority [20/25 (80.0%)] of the isolates with ISAba1-blaOXA-51-like genes were classified as CRAb.

Fig. 1
figure 1

Summary of A. baumannii isolate characteristics based on their antibiotic susceptibility profiles in Vitek® 2 Compact with Advanced Expert System (antibiotics used: CAZ, ceftazidime; CIP, ciprofloxacin; CRO, ceftriaxone; FEP, cefepime; GEN, gentamicin; IPM, imipenem; MEM, meropenem; SXT, trimethoprim-sulfamethoxazole; TZP, piperacillin–tazobactam), carbapenemase production (MHT, modified Hodge test; mCIM, modified carbapenem inactivation method; CIM-Tris, Tris-modified carbapenem inactivation method), type of blaOXA-51-like alleles detected, presence of ISAba1 upstream the blaOXA-51-like genes, and other carbapenemase genes detected through PCR (KPC- Klebsiella pneumoniae carbapenemase, NDM- New Delhi Metallo-β-lactamase, VIM- Verona Integron-associated Metallo-β-lactamase, OXA- Oxacillinase)

Full size image

Among the Ambler class A carbapenemase genes, blaKPC was reported in six (11.5%) isolates. Meanwhile, blaNDM [1 (1.92%)] and blaVIM [2 (3.85%)] were the class B carbapenemase genes found. Among the class D carbapenemase genes (except blaOXA-51-like), blaOXA-23-like dominated [49 (94.2%)]. Only three each of blaOXA-24/40-like and blaOXA-58-like (5.8%) genes were found, all of which were from CSAb. The blaIMP and blaOXA-48-like genes were not detected in our isolates even after repeat PCR assays using appropriate controls.

Multi-class carbapenemase genes (i.e., class D genes in combination with either blaKPC, blaNDM, or blaVIM) were detected in nine (17.3%) isolates, whereas blaOXA-23-like in combination with blaOXA-24/40-like or blaOXA-58-like were detected in five (9.62%) isolates. Only three of the 25 CRAb isolates (12.0%) possessed multi-class carbapenemase genes.

A. baumannii clinical isolates harbored unique bla OXA-51-like alleles

Sequencing and phylogenetic analysis of the blaOXA-51-like genes from the clinical isolates indicated that 25 (48.1%) had known blaOXA-51-like alleles, with blaOXA-508 [10 (19.2%)] and blaOXA-66 [8 (15.4%)] as the most common (Fig. 2). One each of other known alleles such as blaOXA-68, blaOXA-70, blaOXA-90, blaOXA-98, blaOXA-100, blaOXA-120, and blaOXA-342 was also detected. The reference isolate, A. baumannii ATCC BAA-1605™, had the confirmed blaOXA-69 (GenBank accession number of OM617776). The rest of the isolates (n = 28; 53.8%) possessed unique blaOXA-51-like alleles based on their amino acid sequences (Additional File 1). The majority [37 (71.2%)] of the discovered blaOXA-51-like alleles clustered with the blaOXA-66 subclade [international clonal complex 2 in sequence-based typing (SBT)], while only three (5.8%), four (7.7%), and eight (15.4%) alleles clustered with blaOXA-120, blaOXA-51, and blaOXA-68/69 subclades (international clonal complex 1 in SBT), respectively (Fig. 2).

Fig. 2
figure 2

Unrooted maximum likelihood tree based on the 825 nucleotides of the blaOXA-51-like genes of the study isolates and known references (Additional File 1) using the three-parameter (TPM1—unequal frequencies) with gamma distribution model of DNA substitution. Only the general tree topology was shown. Genes were clustered based on the subclades shown in the blaOXA-51-like gene tree generated in the Beta-Lactamase Database-Structure and Function (http://www.bldb.eu/; accessed on 13 March 2023). Samples marked with red circles are alleles matching with known blaOXA-51-like variants, while those with black triangles are the unique alleles. Most of the rare alleles clustered under the blaOXA-66 subclade under the international clonal complex 2 in sequence-based typing

Full size image

The 23 new alleles reported were assigned as blaOXA-1058 to blaOXA-1080, with blaOXA-1067 and blaOXA-1066 detected in two (3.8%) and three (5.8%) isolates, respectively. Amino acid differences from the leader protein (blaOXA-66) range from one to nine (Fig. 3). The predicted conserved active site residues (S80, K83, S127, W166, K217, R260) [32] were unchanged for the new alleles, except for blaOXA-1064, and blaOXA-1075 with K217Q and K217E mutations, respectively (Additional File 1). Among the novel alleles, only five (21.7%) were found to have the upstream ISAba1 (Fig. 1).

Fig. 3
figure 3

Amino acid linkage map of the unique blaOXA-51-like alleles from the study. The amino acid substitution nomenclature indicates the original amino acid followed by the position and the replacement. The substitutions are relative to the reference blaOXA-66 sequence. Boxes colored yellow indicate alleles with amino acid changes in a predicted active site residue (K217)

Full size image

Discussion

In the Philippines, the occurrence of CRAb has been independently reported [4]. Additionally, the genomic features of local CRAb isolates were recently characterized [20]. Our study contributed to the growing molecular investigations on A. baumannii by providing evidence on the presence of multiple and multi-class carbapenemase genes and unique blaOXA-51-like alleles among the local clinical isolates. Specifically, we reported the occurrence of cryptic Ambler class A, B, and D carbapenemase genes in some CRAb and CSAb isolates and discovered 23 novel blaOXA-51-like alleles mostly clustering with blaOXA-66 (international clonal complex 2).

Our findings demonstrated that most of the CRAb isolates phenotypically produced carbapenemases as evident in their meropenem and imipenem resistance profiles and their capacity to degrade carbapenems in disk diffusion-based assays. Although the chosen phenotypic tests (MHT, mCIM, CIM-Tris) produced inconsistent results, all assays confirmed that carbapenemase production is more common among CRAb isolates than CSAb. There is currently no gold standard for the phenotypic detection of carbapenemase production in A. baumannii due to the inherently low specificity and reproducibility of the available disk diffusion-based assays [21, 22, 33, 34]. Hence, it is not surprising to see non-concordant results when comparing the phenotypic assay result to the AST profile. Another potential explanation for this non-concordance is the involvement of porin loss or efflux pump overexpression in the carbapenem-resistant phenotype [7] which is not detected in the selected disk diffusion-based assays. At least ten other potentially more accurate and rapid phenotypic tests for carbapenemase production in A. baumannii have been described in the last decade, but none has been accepted globally for use in the clinical setting [33]. Hence, phenotypic testing alone may not be reliable in determining actual carbapenemase production (or the propensity to produce the enzyme) in A. baumannii. Molecular genotyping remains to be useful in inferring carbapenemase production and activity among A. baumannii clinical isolates [35].

Ambler class D carbapenemases predominate in CRAb globally. As expected, all our A. baumannii clinical isolates possessed blaOXA-51-like which is known to be an intrinsic carbapenemase gene in the pathogen [36]. However, recent studies revealed that blaOXA-51-like genes can also be discovered in non-baumannii species of Acinetobacter [37], in some Enterobacteriaceae species [38], and in Pseudomonas aeruginosa [39] due to the transfer activity of the gene’s frequently linked insertion sequence, ISAba1. Aside from facilitating gene transfer, ISAba1 is also associated with carbapenem resistance due to its ability to induce overexpression of the intrinsic blaOXA-51-like genes [40]. Our findings supported this association by demonstrating that most of our CRAb isolates had the ISAba1 upstream their blaOXA-51-like genes. However, we also detected the ISAba1-blaOXA-51-like complex in a few CSAb isolates, indicating that the presence of this gene complex alone is not a guaranteed predictor of carbapenem-resistant phenotype in A. baumannii, as also described in previous studies [41]. Exploring the genetic environment of the ISAba1-blaOXA-51-like genes through whole genome analysis of the carbapenem-susceptible isolates could potentially explain this observed phenomenon.

Our research showed that blaOXA-23-like genes dominated in our A. baumannii clinical isolates. This result corroborated with the findings of the recent genomic surveillance of A. baumannii in the Philippines [20] and the SENTRY study indicating blaOXA-23-like as the most prevalent carbapenemase genes among A. baumannii in the Asia–Pacific region [18]. While most reports of this gene in A. baumannii were correlated with carbapenem resistance, recent research revealed that it can also be associated with CSAb strains as demonstrated by our findings [42]. This phenomenon can be considered a silent threat as the carbapenem-susceptible strains could serve as reservoirs of transferrable carbapenemase genes such as blaOXA-23-like [43]. This case demonstrates the role of molecular epidemiologic research in the effective management and control of A. baumannii, wherein the phenotypic carbapenem susceptibility of the isolate may conceal the isolate’s potential to be a source of significant drug-resistant genes. The same situation could be true for other transferable carbapenemase genes such as blaOXA-24/40-like and blaOXA-58-like as these genes were also detected in our CSAb isolates, albeit only in a few strains. Notably, all our isolates harboring the blaOXA-58-like genes have slightly higher MIC in meropenem (0.5 mg/L) compared to those without (≤ 0.25 mg/mL). This seemingly insignificant increase in MIC may be easily dismissed in the clinical laboratory but may contribute to the undetectable spread of blaOXA-58-like genes [44]. Overall, the high occurrence of the class D ISAba1-blaOXA-51-like and blaOXA-23-like genes in the local isolates could be driven by high antibiotic pressure (i.e., from extensive third-generation cephalosporin and carbapenem usage) which promotes the interchange of oxacillinases between strains through mobile insertion sequences [45].

Class A blaKPC and class B blaNDM and blaVIM were just recently found in A. baumannii and were associated with significant carbapenem resistance [8,9,10]. In the Philippines, blaKPC-2 was reported from one CRAb isolate through genomic surveillance [20]. In the literature, blaKPC gene is only found in CRAb, but our findings suggested that some CSAb isolates may also harbor the gene. Similar to our observations in the blaOXA-58-like genes, three out of five of our CSAb isolates harboring blaKPC gene have slightly higher MIC in meropenem (0.5 mg/L). We speculate that the genetic environment of this gene in the individual A. baumannii strains influences the expression of the carbapenem-resistant phenotype in this species as recently discovered in genome studies [46]. Similarly, the presence of blaVIM did not directly translate to carbapenem-resistant phenotype in some of our isolates. These findings suggest that CSAb in the clinical context may also be potent carriers of serine carbapenemases and metallo-β-lactamases that could be expressed later on as carbapenem-resistant phenotype or transferred to other Gram-negative bacteria. This phenomenon of cryptic carbapenemase genes, wherein carbapenem-susceptible pathogens do not immediately express the carbapenemase genes but may do so upon exposure to high levels of carbapenems, has been described in other recent reports [47, 48]. Similar high occurrence of cryptic carbapenemase genes in CSAb has been recently described in Indonesia [49], potentially indicating an underexplored yet rampant presence of this phenomenon in Southeast Asia. Finally, our only isolate with detected blaNDM was reported to be CRAb, although the gene’s contribution to the carbapenem-resistant phenotype is not clear as other carbapenemase genes (ISAba1-blaOXA-51-like, blaOXA-23-like) coexist within the strain.

In general, modern strains of Gram-negative clinical isolates have multiple β-lactamase (i.e., carbapenemase) genes due to the extensive genetic transfer between species [50]. Our results showed the co-occurrence of multiple class D or class D in combination with class A or B carbapenemase genes in a few CRAb and CSAb isolates. Our study added evidence on the co-occurrence of multiple and multi-class carbapenemase genes in A. baumannii clinical isolates, making them potent reservoirs of various transferrable β-lactamase genes. This discovery has significant implications in A. baumannii infection management and control as the choice of antibiotics and outbreak investigations depend on the types of carbapenemase genes present in the pathogen. Evidently, recent studies showed that specific carbapenemase genes (i.e., ISAba1-blaOXA-51-like, blaOXA-23-like) are significant determinants of patient mortality and hospital outbreaks [51].

In addition to the occurrence of carbapenemase genes, we also investigated the diversity of the intrinsic blaOXA-51-like genes among our isolates to determine locally occurring alleles and infer pathogen clonality. Since its discovery in 2005, there are already more than 370 alleles of blaOXA-51-like genes, with the list still growing (http://www.bldb.eu/alignment.php?align=D:OXA-51-like, accessed on 5 April 2023). Our study added 23 new blaOXA-51-like alleles from our clinical isolates, potentially indicating the unique molecular characteristics of A. baumannii in the locality. In addition, our SBT analysis revealed that the majority of the isolates clustered under the international clonal complexes 2 and 1 due to the presence of blaOXA-66 and blaOXA-69 alleles, respectively. This finding is supported by previous studies correlating SBT with MLST clones [15, 52, 53].

Among the new alleles reported from our study, two genes (blaOXA-1064 and blaOXA1075) showed mutations in one predicted active carbapenemase site (K217). Particularly, the lysine moiety at amino acid position 217 was hypothesized to be involved in connecting the two clefts of the active site through hydrogen bonding with serine residue in one of the protein’s loops [32]. Hence, mutations in this site could lessen the ability of the OXA-51 enzyme to hydrolyze carbapenems. This finding could potentially explain the carbapenem-susceptible phenotype of the strains where the alleles were discovered. Further enzymatic expression and kinetics study on these alleles will be needed to confirm the effect of the observed mutations on the carbapenemase activity of the enzymes. None of the new alleles had the I129L or L167V mutations predicted to increase the carbapenemase activity of OXA-51 [54].

Overall, our study presented several limitations. For instance, only a limited number of clinically significant A. baumannii isolates were collected and successfully maintained from the clinical laboratory (due to missed and unmaintained cultures). Despite this, we gathered representatives from both CRAb and CSAb isolates, limiting the bias of molecularly characterizing only the resistant isolates. This isolate collection strategy helped us reveal that some CSAb strains may possess cryptic carbapenemase genes which could be expressed later on (as CRAb phenotype) or transferred to other species. Meanwhile, in terms of the choice of the genes, we only used the targets commonly reported in A. baumannii [7] and those with available appropriate controls to minimize reporting false positives. Meanwhile, we only deduced the clonality of our strains from SBT, not MLST. Despite this, SBT using blaOXA-51-like has been used previously to correctly infer the major clones of A. baumannii, corroborating with MLST findings [27,28,29]. Finally, this study was limited to using single gene-based analyses and genotyping experiments, not whole genome sequencing. Despite this, we ensured the validity of our results by incorporating appropriate quality check measures.

Conclusions

Our research emphasized the value of molecular genotyping for identifying and monitoring carbapenem resistance in A. baumannii. The rampant spread of transferable carbapenemase genes and rapid clonal expansion of CRAb necessitate reporting of the molecular characteristics of these isolates, especially in the Philippine clinical setting which only utilizes unreliable phenotypic tests for carbapenemase detection in A. baumannii. In conclusion, our investigation reported the dominance of ISAba1-blaOXA-51-like and blaOXA-23-like genes among the clinical isolates, confirming earlier local studies. Meanwhile, we also reported the occurrence of multiple carbapenemase genes even in CSAb strains, indicating the presence of cryptic carbapenemases that could be expressed into resistant phenotype or transferred to other pathogens. Finally, our investigation discovered new blaOXA-51-like alleles, suggesting a potentially distinct molecular epidemiology of the local isolates that calls for more surveillance. Future studies may delve into the whole genome characteristics of the A. baumannii isolates with interesting genotypic profiles to reveal the genetic environment of the important carbapenemase genes. Enzymatic investigations on the rare alleles may also aid in understanding the actual impact of single amino acid mutations in the activity of the intrinsic blaOXA-51-like genes in A. baumannii.

Availability of data and materials

All original DNA sequence data were deposited in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) with accession numbers OM617740-OM617792. All other relevant data were supplied within the paper and the supplementary data.

References

  1. Michalopoulos A, Falagas ME. Treatment of Acinetobacter infections. Expert Opin Pharmacother. 2010;11(5):779–88. https://doiorg.publicaciones.saludcastillayleon.es/10.1517/14656561003596350.

    Article  CAS  PubMed  Google Scholar 

  2. Oldenkamp R, Schultsz C, Mancini E, et al. Filling the gaps in the global prevalence map of clinical antimicrobial resistance. Proc Natl Acad Sci USA. 2021;118(1):e2013515118. https://doiorg.publicaciones.saludcastillayleon.es/10.1073/pnas.2013515118.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Chua AQ, Verma M, Hsu LY, et al. An analysis of national action plans on antimicrobial resistance in Southeast Asia using a governance framework approach. Lancet Reg Health West Pac. 2021;7:100084. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.lanwpc.2020.100084.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Department of Health [Internet]. Antimicrobial Resistance Surveillance Program Annual Report 2022 [cited 2023 March 13]. Available from: https://arsp.com.ph/download/1864/?tmstv=1696147644

  5. Pogue JM, Zhou Y, Kanakamedala H, et al. Burden of illness in carbapenem-resistant Acinetobacter baumannii infections in US hospitals between 2014 and 2019. BMC Infect Dis. 2022;22(1):36. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12879-021-07024-4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Tacconelli E, Carrara E, Savoldi A, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis. 2018;18(3):318–27. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/S1473-3099(17)30753-3.

    Article  PubMed  Google Scholar 

  7. Lee CR, Lee JH, Park M, et al. Biology of Acinetobacter baumannii: pathogenesis, antibiotic resistance mechanisms, and prospective treatment options. Front Cell Infect Microbiol. 2017;7:55. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fcimb.2017.00055.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Robledo IE, Aquino EE, Santé MI, et al. Detection of KPC in Acinetobacter spp. Puerto Rico Antimicrob Agents Chemother. 2010;54(3):1354–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/AAC.00899-09.

    Article  CAS  PubMed  Google Scholar 

  9. Revathi G, Siu LK, Lu PL, et al. First report of NDM-1-producing Acinetobacter baumannii in East Africa. Int J Infect Dis. 2013;17(12):e1255–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ijid.2013.07.016.

    Article  CAS  PubMed  Google Scholar 

  10. Perilli M, Pellegrini C, Celenza G, et al. First report from Italy of blaVIM-1 and blaTEM-1 genes in Pseudomonas putida and Acinetobacter baumannii isolated from wastewater. J Chemother. 2011;23(3):181–2. https://doiorg.publicaciones.saludcastillayleon.es/10.1179/joc.2011.23.3.181.

    Article  CAS  PubMed  Google Scholar 

  11. Da Silva GJ, Correia M, Vital C, et al. Molecular characterization of blaIMP-5, a new integron-borne metallo-beta-lactamase gene from an Acinetobacter baumannii nosocomial isolate in Portugal. FEMS Microbiol Lett. 2002;215(1):33–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1574-6968.2002.tb11366.x.

    Article  PubMed  Google Scholar 

  12. Fouad M, Attia AS, Tawakkol WM, et al. Emergence of carbapenem-resistant Acinetobacter baumannii harboring the OXA-23 carbapenemase in intensive care units of Egyptian hospitals. Int J Infect Dis. 2013;17(12):e1252–4. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ijid.2013.07.012.

    Article  CAS  PubMed  Google Scholar 

  13. Sarı AN, Biçmen M, Gülay Z. The first report on the outbreak of OXA-24/40-like carbapenemase-producing Acinetobacter baumannii in Turkey. Jpn J Infect Dis. 2013;66(5):439–42.

    Article  PubMed  Google Scholar 

  14. de Souza GC, Bertholdo LM, Otton LM, et al. First occurrence of blaOXA-58 in Acinetobacter baumannii isolated from a clinical sample in Southern Brazil. Braz J Microbiol. 2012;43(1):243–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1590/S1517-838220120001000027.

    Article  Google Scholar 

  15. Rafei R, Hamze M, Pailhoriès H, et al. Extrahuman epidemiology of Acinetobacter baumannii in Lebanon. Appl Environ Microbiol. 2015;81(7):2359–67. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/AEM.03824-14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nigro SJ, Hall RM. Does the intrinsic oxaAb (blaOXA-51-like) gene of Acinetobacter baumannii confer resistance to carbapenems when activated by ISAba1? J Antimicrob Chemother. 2018;73(12):3518–20. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/jac/dky334.

    Article  CAS  PubMed  Google Scholar 

  17. Bonomo RA, Burd EM, Conly J, et al. Carbapenemase-producing organisms: a global scourge. Clin Infect Dis. 2018;66(8):1290–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/cid/cix893.

    Article  CAS  PubMed  Google Scholar 

  18. Mendes RE, Bell JM, Turnidge JD, et al. Emergence and widespread dissemination of OXA-23, -24/40 and -58 carbapenemases among Acinetobacter spp. in Asia-Pacific nations: report from the SENTRY Surveillance Program. J Antimicrob Chemother. 2009;63(1):55–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/jac/dkn434.

    Article  CAS  PubMed  Google Scholar 

  19. Wareth G, Linde J, Nguyen NH, et al. WGS-based analysis of carbapenem-resistant Acinetobacter baumannii in Vietnam and molecular characterization of antimicrobial determinants and MLST in Southeast Asia. Antibiotics (Basel). 2021;10(5):563. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/antibiotics10050563.

    Article  CAS  PubMed  Google Scholar 

  20. Chilam J, Argimón S, Limas MT, et al. Genomic surveillance of Acinetobacter baumannii in the Philippines, 2013–2014. Western Pac Surveill Response J. 2021;12(4):1–15. https://doiorg.publicaciones.saludcastillayleon.es/10.5365/wpsar.2021.12.4.863.

    Article  PubMed  PubMed Central  Google Scholar 

  21. The European Committee on Antimicrobial Susceptibility Testing [Internet]. EUCAST guideline for the detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance version 2.0 [cited 2023 March 13]. Available from: https://www.eucast.org/resistance_mechanisms

  22. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: M100. 33rd ed. Wayne, PA, USA: CLSI; 2023.

  23. Amjad A, Mirza IA, Abbasi S, et al. Modified Hodge test: a simple and effective test for detection of carbapenemase production. Iran J Microbiol. 2011;3(4):189–93.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Pierce VM, Simner PJ, Lonsway DR, et al. Modified carbapenem inactivation method for phenotypic detection of carbapenemase production among Enterobacteriaceae. J Clin Microbiol. 2017;55(8):2321–33. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/JCM.00193-17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Uechi K, Tada T, Shimada K, et al. A modified carbapenem inactivation method, CIMTris, for carbapenemase production in Acinetobacter and Pseudomonas species. J Clin Microbiol. 2017;55(12):3405–10. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/JCM.00893-17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Mu X, Nakano R, Nakano A, et al. Loop-mediated isothermal amplification: rapid and sensitive detection of the antibiotic resistance gene ISAba1-blaOXA-51-like in Acinetobacter baumannii. J Microbiol Methods. 2016;121:36–40. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.mimet.2015.12.011.

    Article  CAS  PubMed  Google Scholar 

  27. Pournaras S, Gogou V, Giannouli M, et al. Single-locus-sequence-based typing of blaOXA-51-like genes for rapid assignment of Acinetobacter baumannii clinical isolates to international clonal lineages. J Clin Microbiol. 2014;52(5):1653–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/JCM.03565-13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zander E, Nemec A, Seifert H, Higgins PG. Association between β-lactamase-encoding bla(OXA-51) variants and DiversiLab rep-PCR-based typing of Acinetobacter baumannii isolates. J Clin Microbiol. 2012;50(6):1900–4. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/JCM.06462-11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hamouda A, Evans BA, Towner KJ, Amyes SG. Characterization of epidemiologically unrelated Acinetobacter baumannii isolates from four continents by use of multilocus sequence typing, pulsed-field gel electrophoresis, and sequence-based typing of bla(OXA-51-like) genes. J Clin Microbiol. 2010;48(7):2476–83. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/JCM.02431-09.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Bradford PA, Bonomo RA, Bush K, et al. Consensus on β-lactamase nomenclature. Antimicrob Agents Chemother. 2022;66(4):e0033322. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/aac.00333-22.

    Article  CAS  PubMed  Google Scholar 

  31. Evans BA, Hamouda A, Towner KJ, et al. OXA-51-like beta-lactamases and their association with particular epidemic lineages of Acinetobacter baumannii. Clin Microbiol Infect. 2008;14(3):268–75. https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1469-0691.2007.01919.x.

    Article  CAS  PubMed  Google Scholar 

  32. Smith CA, Antunes NT, Stewart NK, et al. Structural basis for enhancement of carbapenemase activity in the OXA-51 family of class D β-lactamases. ACS Chem Biol. 2015;10(8):1791–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1021/acschembio.5b00090.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mitteregger D, Wessely J, Barišić I, Bedenić B, Kosak D, Kundi M. A variant carbapenem inactivation method (CIM) for Acinetobacter baumannii group with shortened time-to-result: rCIM-A. Pathogens. 2022;11(4):482. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/pathogens11040482.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Khuntayaporn P, Thirapanmethee K, Kanathum P, Chitsombat K, Chomnawang MT. Comparative study of phenotypic-based detection assays for carbapenemase-producing Acinetobacter baumannii with a proposed algorithm in resource-limited settings. PLoS ONE. 2021;16(11):e0259686. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0259686.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Jin X, Zhang H, Wu S, et al. Multicenter evaluation of Xpert Carba-R assay for detection and identification of the carbapenemase genes in rectal swabs and clinical isolates. J Mol Diagn. 2021;23(1):111–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jmoldx.2020.10.017.

    Article  CAS  PubMed  Google Scholar 

  36. Turton JF, Woodford N, Glover J, et al. Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene intrinsic to this species. J Clin Microbiol. 2006;44(8):2974–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/JCM.01021-06.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lee YT, Kuo SC, Chiang MC, et al. Emergence of carbapenem-resistant non-baumannii species of Acinetobacter harboring a blaOXA-51-like gene that is intrinsic to A. baumannii. Antimicrob Agents Chemother. 2012;56(2):1124–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/AAC.00622-11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Leski TA, Bangura U, Jimmy DH, et al. Identification of blaOXA-51-like, blaOXA-58, blaNDM-1, and blaVIM carbapenemase genes in hospital Enterobacteriaceae isolates from Sierra Leone. J Clin Microbiol. 2013;51(7):2435–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/JCM.00832-13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chaudhary M, Pavasi A. Prevalence, genotyping of Escherichia coli and Pseudomonas aeruginosa clinical isolates for oxacillinase resistance and mapping susceptibility behaviour. J Microb Biochem Technol. 2014;6:2. https://doiorg.publicaciones.saludcastillayleon.es/10.4172/1948-5948.1000123.

    Article  CAS  Google Scholar 

  40. Evans BA, Hamouda A, Amyes SG. The rise of carbapenem-resistant Acinetobacter baumannii. Curr Pharm Des. 2013;19(2):223–38.

    Article  CAS  PubMed  Google Scholar 

  41. Pagano M, Martins AF, Machado AB, et al. Carbapenem-susceptible Acinetobacter baumannii carrying the ISAba1 upstream blaOXA-51-like gene in Porto Alegre, southern Brazil. Epidemiol Infect. 2013;141(2):330–3. https://doiorg.publicaciones.saludcastillayleon.es/10.1017/S095026881200074X.

    Article  CAS  PubMed  Google Scholar 

  42. Zhang Y, Fan B, Luo Y, et al. Comparative analysis of carbapenemases, RND family efflux pumps and biofilm formation potential among Acinetobacter baumannii strains with different carbapenem susceptibility. BMC Infect Dis. 2021;21(1):841. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12879-021-06529-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Carvalho KR, Carvalho-Assef APD, dos Santos LG, et al. Occurrence of blaOXA-23 gene in imipenem susceptible Acinetobacter baumannii. Mem Inst Oswaldo Cruz Rio de Janeiro. 2011;106(4):505–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1590/S0074-02762011000400020.

    Article  Google Scholar 

  44. Wu W, He Y, Lu J, et al. Transition of blaOXA-58-like to blaOXA-23-like in Acinetobacter baumannii clinical isolates in Southern China: An 8-Year Study. PLoS ONE. 2015;10(9):e0137174. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0137174.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hashemizadeh Z, Hatam G, Fathi J, et al. The spread of insertion sequences element and transposons in carbapenem resistant Acinetobacter baumannii in a hospital setting in Southwestern Iran. Infect Chemother. 2022;54(2):275–86. https://doiorg.publicaciones.saludcastillayleon.es/10.3947/ic.2022.0022.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Martinez T, Martinez I, Vazquez GJ, et al. Genetic environment of the KPC gene in Acinetobacter baumannii ST2 clone from Puerto Rico and genomic insights into its drug resistance. J Med Microbiol. 2016;65(8):784–92. https://doiorg.publicaciones.saludcastillayleon.es/10.1099/jmm.0.000289.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Abordo AMS, Carascal MB, Remenyi R, Dalisay DS, Saludes JP. Clinically isolated β-lactam-resistant Gram-negative bacilli in a Philippine tertiary care hospital harbor multi-class β-lactamase genes. Pathogens. 2023;12(8):1019. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/pathogens12081019.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Stasiak M, Maćkiw E, Kowalska J, Kucharek K, Postupolski J. Silent genes: antimicrobial resistance and antibiotic production. Pol J Microbiol. 2021;70(4):421–9. https://doiorg.publicaciones.saludcastillayleon.es/10.33073/pjm-2021-040.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Anggraini D, Kemal RA, Hadi U, Kuntaman K. The susceptibility pattern and distribution of blaOXA-23 genes of clinical isolate Acinetobacter baumannii in a tertiary hospital. Indonesia J Infect Dev Ctries. 2022;16(5):821–6. https://doiorg.publicaciones.saludcastillayleon.es/10.3855/jidc.15902.

    Article  CAS  PubMed  Google Scholar 

  50. Bush K, Bradford PA. Epidemiology of β-lactamase-producing pathogens. Clin Microbiol Rev. 2020;33(2):e00047-e119. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/CMR.00047-19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Kim MH, Jeong H, Sim YM, et al. Using comparative genomics to understand molecular features of carbapenem-resistant Acinetobacter baumannii from South Korea causing invasive infections and their clinical implications. PLoS ONE. 2020;15(2):e0229416. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0229416.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Rafei R, Pailhoriès H, Hamze M, et al. Molecular epidemiology of Acinetobacter baumannii in different hospitals in Tripoli, Lebanon using bla(OXA-51-like) sequence based typing. BMC Microbiol. 2015;15:103. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-015-0441-5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Rafei R, Dabboussi F, Hamze M, et al. Molecular analysis of Acinetobacter baumannii strains isolated in Lebanon using four different typing methods. PLoS ONE. 2014;9(12):e115969. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0115969.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Chan KW, Liu CY, Wong HY, Chan WC, Wong KY, Chen S. Specific amino acid substitutions in OXA-51-type β-lactamase enhance catalytic activity to a level comparable to carbapenemase OXA-23 and OXA-24/40. Int J Mol Sci. 2022;23(9):4496. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/ijms23094496.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank Ms. Ma. Sheila Monda and the medical technologists of the Section of Clinical Microbiology and Molecular Diagnostics (Department of Laboratory Medicine and Pathology), Mr. Alecks Megxel Abordo and Dr. Roland Gilbert Remenyi of the Biomedical Research Laboratory (Clinical and Translational Research Institute) of The Medical City, and the Pathogen-Host-Environment Interaction Research Laboratory of the University of the Philippines Diliman for the technical assistance and provision of materials for the experiments. They also acknowledge Dr. Esperanza Cabrera and Engr. Marvin Belen for the insights and support in manuscript preparation.

Funding

This study was partially funded by The Medical City Intramural Grant Fund (Grant No. 2020-001) awarded to MBC and the Emerging Inter-Disciplinary Research (EIDR) Grant of the Office of the Vice President for Academic Affairs, University of the Philippines System (Grant No. C04-003) awarded to WLR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Pathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of Science, University of the Philippines Diliman, 1101, Quezon City, Philippines

    Mark B. Carascal & Windell L. Rivera

  2. Clinical and Translational Research Institute, The Medical City, Ortigas Avenue, 1605, Pasig City, Philippines

    Mark B. Carascal & Raul V. Destura

  3. Institute of Molecular Biology and Biotechnology, National Institutes of Health, University of the Philippines, 1159, Manila, Philippines

    Raul V. Destura

Authors
  1. Mark B. Carascal
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Raul V. Destura
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Windell L. Rivera
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

MBC contributed to conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing—original draft, and writing—review and editing; RVD contributed to conceptualization, investigation, methodology, project administration, resources, supervision, validation, and writing—review and editing; WLR contributed to conceptualization, investigation, funding acquisition, project administration, resources, supervision, validation, visualization, writing—original draft, and writing—review and editing.

Corresponding author

Correspondence to Windell L. Rivera.

Ethics declarations

Ethics approval and consent to participate

The protocol for this study was examined and acknowledged by The Medical City Institutional Review Board (GCS-2018-123) on October 2, 2018. The protocol was exempted from full ethics review since there is no human intervention or data used in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

41182_2024_629_MOESM1_ESM.docx

Additional file 1: Supplementary data on the methods and experimental outcomes. This additional file contains tables of the detailed characteristics of the isolates, Polymerase Chain Reaction protocols, GenBank accession numbers of the reference and original sequences used in the analysis, raw gel profiles of PCR amplicons, and complete multiple sequence alignment of the genes investigated

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Carascal, M.B., Destura, R.V. & Rivera, W.L. Molecular genotyping reveals multiple carbapenemase genes and unique blaOXA-51-like (oxaAb) alleles among clinically isolated Acinetobacter baumannii from a Philippine tertiary hospital. Trop Med Health 52, 62 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s41182-024-00629-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s41182-024-00629-w

Keywords

Tropical Medicine and Health

ISSN: 1349-4147

Contact us