The emergence of NDM-1, IMP-7, IMP-26, IMP-62, IMP-76, VIM-4 and VIM-5 producing Pseudomonas aeruginosa strains, identification of a novel sequence type (ST3891) and analysis of virulence genes from a Malaysian tertiary hospital

Kalaivani Kalai Chelvam; Yue Min Lim; Cindy Shuan Ju Teh; Sasheela Ponnampalavanar; Jamuna Vadivelu; Vanitha Mariappan; Kumutha Malar Vellasamy

doi:10.1371/journal.pone.0350200

Abstract

Multidrug-resistant (MDR) Pseudomonas aeruginosa is a medically crucial nosocomial pathogen causing significant mortality in patients with weakened immune systems. It is sensitive to carbapenems such as meropenem and imipenem. However, recent research indicates the rise and rapid transmission of carbapenem-resistant P. aeruginosa (CRPA). This study analysed the phenotypic and genotypic traits linked with CRPA strains obtained from the University Malaya Medical Centre (UMMC), a Malaysian tertiary teaching hospital. MDR P. aeruginosa (n = 215) were isolated from the Medical Microbiology Diagnostic Laboratory (MMDL). The VITEK-2 automated system was used for antimicrobial susceptibility and data for susceptibility to various antimicrobial agents were retrieved from the MMDL database at the UMMC. The minimum inhibitory concentration (MIC) of the carbapenems (meropenem and imipenem) was analysed using the broth microdilution method. Pyocyanin, protease and biofilm assays were conducted to assess the phenotypic traits. Furthermore, polymerase chain reaction (PCR) was used to detect six virulence genes and three carbapenemase genes. P. aeruginosa strains that displayed high-level resistance to meropenem and imipenem showed an association with 140 strains (65.1%) that were strong biofilm producers (OD_570nm ≥ 3.0). The majority of the strains (73.5%) exhibited high to moderate pyocyanin production, while 95.3% were capable of secreting protease. Among the six virulence genes tested, most of the strains harboured the toxR (109/215, 50.7%) and algD (108/215, 50.2%) genes. The identification of carbapenem genes showed bla_IMP (n = 24, 11.2%) followed by bla_NDM (n = 16, 7.4%) and bla_VIM (n = 4, 1.86%). A novel sequence type, ST3891 was discovered using multilocus sequence typing (MLST). The rising incidence of carbapenem-resistant strains is concerning, as carbapenems with antipseudomonal activity play a vital role in treating P. aeruginosa infections. Additionally, this study found a positive correlation between increased biofilm production and resistance to carbapenem in P. aeruginosa. This emphasises the importance of stringent infection control strategies, particularly considering the clonal dissemination of the high-risk ST235 clone and the identification of the novel ST3891 strain in Malaysia.

Citation: Kalai Chelvam K, Lim YM, Teh CSJ, Ponnampalavanar S, Vadivelu J, Mariappan V, et al. (2026) The emergence of NDM-1, IMP-7, IMP-26, IMP-62, IMP-76, VIM-4 and VIM-5 producing Pseudomonas aeruginosa strains, identification of a novel sequence type (ST3891) and analysis of virulence genes from a Malaysian tertiary hospital. PLoS One 21(6): e0350200. https://doi.org/10.1371/journal.pone.0350200

Editor: Dwij Raj Bhatta, Tribhuvan University, NEPAL

Received: March 18, 2025; Accepted: May 11, 2026; Published: June 10, 2026

Copyright: © 2026 Kalai Chelvam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: the Universiti Malaya Specialist Centre (UMSC) C.A.R.E Research Grant (Grant Number: PV024-2017).

Competing interests: The authors have declared that no competing interests exist.

Introduction

Pseudomonas aeruginosa related to multidrug resistance (MDR) is a medically crucial nosocomial pathogen causing significant mortality in patients with weakened immune systems. It is also one of the most frequently occurring bacterial co-infections in COVID-19 patients [1], with severe infections complicating their clinical management [1,2].

The Southeast Asian region recorded the highest history of P. aeruginosa prevalence [3]. It frequently affects patients with compromised immune systems [4]. Various studies have reported increasing isolation of MDR P. aeruginosa from hospitalised patients, especially in critically ill patients [5,6]. P. aeruginosa infections are generally treated with antibiotics. However, treatment is becoming more difficult due to increasing resistance to antibiotics.

In Malaysia, the National Surveillance of Antibiotic Resistance (NSAR) report indicated that both meropenem and imipenem showed an increasing trend in resistance until around 2014–2015, after which the resistance gradually declined up to 2018 [7]. By 2023, the resistance rates for meropenem and imipenem further decreased from 6.8% to 6.5% and 7.1% to 6.7%, respectively [8]. In University Malaya Medical Centre (UMMC), carbapenem-resistant Enterobacteriaceae (CRE), particularly Klebsiella pneumoniae was first reported in 2013, then peaked from 2014 to 2015 [9]. It continued to cause different infections among the patients from 2016 to 2017, leading to an increase in carbapenem-resistant K. pneumoniae (CRKP) infections [10]. Carbapenem-resistant Acinetobacter baumannii (CRAB) strains at UMMC exhibited resistance to most antibiotics, with colistin being the only effective option, highlighting limited treatment alternatives [11,12]. Data regarding the burden and underlying mechanisms of carbapenem-resistant P. aeruginosa (CRPA) in Malaysia is scarce, particularly when compared to information on CRKP and CRAB. Therefore, this study investigated phenotypic and genotypic characteristics associated with CRPA in UMMC. The potential benefits of this study include improved treatment strategies, better infection control measures and contributions to the broader scientific knowledge based on antibiotic resistance. This may positively influence patient care, public health and worldwide efforts against antibiotic resistance.

Materials and methods

Data collection

A retrospective analysis of archived bacterial cultures from clinical specimens were conducted. The data were accessed on 21 August 2020 for research purposes. Data involving patients’ demographics, including ethnicity, as well as the source of the specimen, location of the ward and antibiotic susceptibility were retrieved from the Medical Microbiology Diagnostic Laboratory (MMDL) database at University Malaya Medical Centre (UMMC), following institutional guidelines and ethical approval.

Ethical considerations

The UMMC Medical Research Ethics Committee approved the ethics with the following reference number (MRECID.NO: 2019815−7748). Informed consent was not obtained. During data collection, the authors had access to identifiable information by using the patient web portal and written requests were required to access the patient data. After data extraction, all records were anonymised to protect patient confidentiality, in compliance with ethical and regulatory requirements.

Bacterial strains

A total of 215 MDR P. aeruginosa strains from the MMDL collection from patients in UMMC from January 2015 to December 2018 were used in this study. MDR P. aeruginosa was defined as non-susceptible to at least one agent in ≥3 antimicrobial categories [13]. Non-MDR data were excluded from this study to focus on clinical relevance and resistance burden. The bacterial cultures were identified and confirmed via a polymerase chain reaction (PCR) assay (Table 1). P. aeruginosa ATCC 27853 was used as the positive control [14]. Strains PA153 and PACTR were isolated from the same patient three years apart. PA153 was recovered from bone in 2015 and PACTR was obtained from tissue in 2018.

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Table 1. 16S rDNA-based primer set [14].

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Antimicrobial susceptibility test (AST)

The antimicrobial susceptibility of the strains was evaluated using the AST-N314 card on the automated VITEK-2 system (BioMérieux, Marcy L’Etoile, France) by MMDL. Minimum inhibitory concentration (MIC) values of meropenem and imipenem (Oxoid, Hampshire, United Kingdom) were determined for each isolate using the broth microdilution method with concentrations ranging between 0 and 256 µg/mL, according to Clinical and Laboratory Standards Institute (CLSI) guidelines [15]. MIC breakpoints for meropenem and imipenem were established as ≤2 μg/mL for susceptible, 4 μg/mL for intermediate, and ≥8 μg/mL for resistant. P. aeruginosa ATCC 27853 was used as a quality control strain.

Biofilm assay

Biofilm adherence assay was conducted using the crystal violet staining method as formerly outlined by Kalai Chelvam et al. [16] with slight modifications. The planktonic cultures (OD_600nm 1.0) were diluted in fresh Luria Bertani (LB) broth (Oxoid, UK) to 1:100 (~OD_600nm 0.01). Briefly, 100 μl of the culture was added into each well of a sterile 96-well plate and incubated at 37 °C for 24 hours. For the control wells, bacterial cultures were substituted with fresh LB. After incubation, the culture supernatant was aseptically transferred to a new sterile 96-well plate, and the absorbance at OD_600nm was measured to assess bacterial growth under biofilm conditions. The empty wells containing biofilm were stained with 150 μl of 1% crystal violet (w/v) and further incubated for 30 mins at room temperature. Excess stain was removed by washing the wells twice with 175 μl of sterile distilled water. Subsequently, 175 μl of dimethyl sulfoxide (DMSO) was added to each well, and after a 10-minute incubation at room temperature, the absorbance was measured at OD_570nm. The quantification of biofilm formed (based on the OD_570nm of crystal violet stain) ranged between 0.01 and 5.71. The strains were divided into low (OD_570nm ≤ 1.5), moderate (OD_570nm 1.5 to 3) and high biofilm (OD_570nm ≥ 3) producers as suggested by published criteria [16]. The experiment was conducted in triplicate, with the mean OD_600nm value and its standard deviation (SD) reported.

Protease assay

According to a protocol by García-Reyes, Moustafa [17], a skimmed milk agar was used to qualitatively determine the protease secretion. The inoculated plate was incubated for 48 hours at 37 °C. A clear zone around the colony becomes readily visible. The results were scored as no protease production – 0 (0 mm); low – 1 (≤ 1.5 mm; medium – 2 & 3 (1.5–3.0 mm); high – 4 (≥ 3 mm) based on the halo diameter [18]. The experiment was repeated twice to check for reproducibility. P. aeruginosa ATCC 27853 was used as the control strain.

Pyocyanin assay

King A medium (comprising 20.0 g/L tryptone, 3.3 g/L MgCl₂·6H₂O, 20.0 g/L KOH, 5.5 mL H₂SO₄, and 10.0 g/L glycerol, adjusted to pH 7.2) [19] was inoculated with an overnight culture to an OD_600nm of 0.2 and incubated at 37 °C with constant shaking at 230 rpm. Following 24 hours of incubation, the medium was used to assess pyocyanin production by visual assessment [20]. After centrifuging the bacterial culture at 8000 rpm for 10 minutes, the absorbance of the supernatant was measured at OD_695nm [21]. All strains were divided into low (OD_695nm ≤ 1.5), moderate (OD_695nm of 1.5 to 3), and high (OD_695nm ≥ 3) pyocyanin producers. P. aeruginosa ATCC 27853 was used as a positive control. King A medium without any bacterial culture was used as a negative control.

Genomic DNA Extraction

The template DNA for PCR was obtained from all P. aeruginosa strains using the boiling method [22]. A single colony from an overnight P. aeruginosa culture grown on nutrient agar was suspended in 100 μl of sterile deionised water and boiled for 10 minutes in a water bath. The suspension was then centrifuged at 13,800 rpm for 5 minutes, after which the supernatant was collected to be stored at −80 °C.

Molecular identification of virulence-related genes

PCR was conducted to identify six virulence-associated markers: adhesion (algD), motility (fliC), T3SS (exoS), toxins (plcH, toxR), and quorum sensing or regulation (lasR) by using previously reported primers (Table 2) with minor modifications to the reaction parameters [23,24]. A Bio-Rad thermocycler (CFX96) was used for PCR amplifications using these cycling conditions: an initial denaturation at 95 °C for 15 minutes, followed by 35 cycles comprising 1 minute at 95 °C, 45 seconds at 60 °C, and 1 minute at 72 °C, with a final extension at 72 °C for 7 minutes. The PCR products were subjected to agarose (1.5%) gel electrophoresis and stained with SYBR Safe (Invitrogen) for visualisation. The gels were visualised using a UV trans-illumination system. P. aeruginosa ATCC 27853 was used as the positive control. A gene was considered present if a specific amplicon of expected size was visible on the agarose gel under UV illumination.

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Table 2. Primers used in this study.

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PCR identification of carbapenem-resistant genes

Three carbapenemase genes (bla_IMP, bla_VIM and bla_NDM) (Table 2) were selected based on their prevalence in Malaysia. Identification of these genes was performed using PCR on a CFX96 Bio-Rad thermocycler as described by Nordmann et al. [25]. PCR products were analysed using 1.5% agarose gel electrophoresis and stained with SYBR Safe (Invitrogen) for identification. The gels were visualised using a UV transillumination imaging system. Amplified products were sent for DNA sequencing by Sanger Sequencing and BLAST analysis was performed using the NCBI nucleotide database using Standard Nucleotide BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to validate the identity.

Molecular typing based on MLST

Out of 215 MDR P. aeruginosa strains, nine strains were chosen for MLST. The selection criteria were: (i) different biofilm-forming abilities (strong, moderate and non-biofilm) and (ii) variable carbapenem-resistance levels (high, > 256 µg/mL; medium, ≥ 32 μg/mL and borderline ≥ 8 μg/mL). According to a protocol by Curran et al. [26], genes acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE (Table 3) were selected and PCR assay was performed using a Bio-Rad thermocycler (CFX96). Single-plex PCRs were performed for each gene, starting with an initial denaturation at 96 °C for 1 minute. This was followed by 30 cycles of denaturation at 96 °C for 1 minute, primer annealing at 55 °C for 1 minute, and extension at 72 °C for 1 minute. A final extension step was conducted at 72 °C for 10 minutes. The QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used to purify the amplicon for each target gene. DNA sequencing was carried out on an automated DNA sequencing system (Applied Biosystems, Foster City, CA). The results were compared with the data available in the PubMLST database (https://pubmlst.org/organisms/pseudomonas-aeruginosa) to determine allelic profiles and sequence types (STs). Newly identified alleles and STs were submitted to the PubMLST database for validation.

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Table 3. Primers used for MLST [26].

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Statistical analysis

Statistical analysis was carried out using IBM Statistical Package for Social Science (SPSS) software version 27.0 (https://www.ibm.com). The relationship between phenotypic characteristics and carbapenem resistance was assessed using the Pearson’s coefficient. For all analyses, a p-value below 0.05 was regarded as statistically significant.

Results and discussion

Bacterial identification and strain distribution

Multidrug-resistant P. aeruginosa strains (n = 215) were collected over the three-year period from 2015 to 2018. The identity of all the strains were further verified using the PCR assay. PCR amplification targeting the 16S rDNA gene showed amplification of products with an amplicon size of 956 bp (Fig 1).

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Fig 1. PCR-based identification of P. aeruginosa strains using 16S rDNA amplification.

Lane M: 100 bp markers; lane 1: positive control (P. aeruginosa ATCC27853); lanes 2-4: clinical isolates (PACTR: lane 3: PA153; lane 4: PA205); lane 5: negative control (deionised water). The expected amplicon size is 956 bp.

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The 215 MDR P. aeruginosa strains were obtained from various sources across different UMMC wards. Malays were the majority race (n = 74, 34%) followed by Indians (n = 66, 31%), Chinese (n = 64, 30%) and others (n = 11, 5%). The strains were obtained from respiratory secretions (43/215, 20%), sputum (39/215, 18.1%), tissue (38/215, 17.7%) and other sources (95/215, 44.2%). Most strains were obtained from patients in the urology ward (39/215, 18.1%), intensive care unit (36/215, 16.7%), orthopaedic ward (33/215, 15.3%), neuro-intensive care unit (21/215, 9.8%), and various other wards (86/215, 40.1%). P. aeruginosa is a significant nosocomial pathogen, which creates major challenges due to its high rates of mortality, morbidity and the resulting healthcare expenses. While its prevalence has remained stable over the last two decades, MDR strains have increased dramatically worldwide. In Malaysia, the NSAR reported rising carbapenem resistance rates compared to 2018, with 77% of strains in this study resistant to both meropenem and imipenem [8].

Antimicrobial susceptibility features

The antibiotic susceptibility results based on the VITEK-2 System (BioMérieux, France) showed the highest resistance rates against ceftazidime (87.4%), meropenem (80.9%) and imipenem (85.6%) (Table 4). Overall, the MDR rate was 98.6% (212/215) and 85.6% (184/215) were carbapenem-resistant strains. The MIC values for imipenem and meropenem (8 to ≥ 256 µg/mL) confirmed that 165 (76.7%) and 166 (77.2%) of the strains showed high-level resistance, respectively.

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Table 4. Antibiotic susceptibility rates of MDR P. aeruginosa as determined using the VITEK-2 system.

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Phenotypic characterisation

In this study, 140 (65.1%) were identified as high biofilm producers. Additionally, 47 strains (21.9%) demonstrated moderate biofilm production and 28 strains (13.0%) exhibited low biofilm production. Protease secretion was detected in 95.3% of the strains (205/215), while 4.7% (10 out of 215) could not secrete protease (Table 5). Protease secretion was detected through the transparent halo that resulted from the degradation of milk protein by protease (Fig 2). The diameter of the hydrolysis zone indicated the intensity of protease produced by the strains. P. aeruginosa ATCC 27853 served as the positive control.

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Table 5. Protease production levels in MDR P. aeruginosa strains (n = 215) based on zone diameter on skim milk agar.

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Fig 2. Protease activity of MDR P. aeruginosa strains on skim milk agar.

Transparent halos indicate protease-mediated casein hydrolysis. Plates were incubated for 48 hours at 37°C. Positive control: P. aeruginosa ATCC 27853.

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A total of 57 strains representing 26.5% showed low pyocyanin production ability. Additionally, 45 strains (20.9%) were categorised as medium producers of pyocyanin, while 113 strains (52.6%) were classified as high producers of pyocyanin (Table 6).

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Table 6. Categorisation of MDR P. aeruginosa strains (n = 215) by pyocyanin production level (absorbance at OD_695nm).

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Biofilm production was observed in 92% of strains, predominantly high to moderate producers. Biofilms are critical for antibiotic resistance and persistent infections, as evidenced by prior studies [27,28]. The correlation between MBL presence and biofilm strength highlights the role of these enzymes in promoting bacterial survival under antibiotic stress, especially in hospital settings [29]. AST of the strains obtained from UMMC revealed that all strains were resistant to most of the antibiotics applied for treatment in UMMC. This test, supported by Saha et al. [30], showed that all biofilm-producing strains were highly resistant to antibiotics in comparison to the non-biofilm producers. While colistin can be effective against P. aeruginosa, its penetration into biofilms may be limited. Biofilm-associated infections often require a combination of antimicrobial agents and may involve the use of additional strategies, such as the use of biofilm-disrupting agents or surgical intervention [31,32]. Production of protease and biofilm were correlated, with 43% of high protease producers demonstrating moderate to high biofilm formation, consistent with findings by Zaki et al. [33]. Pyocyanin, detected in 82% of strains, further supports biofilm formation and virulence. Its interaction with extracellular DNA (eDNA) promotes bacterial aggregation, a key factor in infection persistence [34]. Phenotypic characteristics of known virulence determinants such as biofilm formation and biofilm-associated phenotypes (secretion of protease and pyocyanin) are important to understand the antibiotic resistance mechanisms that hamper treatment and control of the infection.

Virulence gene identification and association with phenotypic traits

Among the six virulence-related genes tested, the majority of the strains harboured toxR (109/215, 50.7%) and algD (108/215, 50.2%) genes (Fig 3). The lowest identification frequencies were observed for the plcH (17/215, 7.9%) and lasR (43/215, 20.0%) genes, while all four genes fliC, exoS, toxR and algD were present in 12 of the strains. Among the total strains, 48, constituting 22%, displayed no virulence genes.

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Fig 3. Prevalence of virulence-associated genes in the 215 MDR P. aeruginosa strains.

Genes assessed include fliC, exoS, toxR, algD, plcH and lasR.

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In this study, 51% of the strains carried the toxR gene, while 50% harboured the algD gene. The toxR in P. aeruginosa regulates surface-associated behaviours like swarming motility, ability to form biofilm and virulence-related factors by binding to the second messenger c-di-GMP [35]. During infection, bacteria transition from nonmucoid to mucoid phenotypes, producing alginate is essential for biofilm development [36]. Mucoid-producing bacteria become dominant in the advanced stage of infection, causing deterioration and a high mortality rate [37]. In this study, the algD gene was present in 50% of the strains. Several studies have investigated how virulence and antibiotic susceptibility interact, revealing an antagonistic relationship. Resistance mechanisms generate a cost to bacterial virulence [38–40]. However, other researchers proposed that the toxR and algD virulotypes are linked to carbapenem resistance, particularly in high antibiotic pressure environments, such as ICUs [41,42].

Carbapenemase Genes: Clinical and Epidemiological Implications

Using PCR, 44 out of 215 MDR P. aeruginosa strains were confirmed to harbour carbapenem-resistant genes. The carbapenemase genes identified among the strains were bla_IMP (n = 24, 11.2%), followed by bla_NDM (n = 16, 7.4%) and bla_VIM (n = 4, 1.86%). Four allelic variants, bla_IMP-7 (1 strain), bla_IMP-26 (21 strains), bla_IMP-62 (1 strain) and bla_IMP-76 (1 strain), were identified. Of the 215 strains, no carbapenemase genes were detected in 190 (88.4%) strains. PA153 and PACTR showed the presence of both bla_IMP-26 and bla_VIM-4 genes. None of the strains had all three carbapenemase genes present. Eight, four, 14 and 18 strains from the years 2015, 2016, 2017 and 2018, respectively were positive for the carbapenemase gene (Table 7).

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Table 7. Distribution of carbapenemase gene variants (bla_IMP, bla_NDM, bla_VIM) in CRPA strains by year of isolation (n = 184).

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There was 98−100% DNA sequence similarity for bla_NDM-1 gene for 16 P. aeruginosa strains studied and a P. aeruginosa isolate from Singapore with a GenBank accession of KT364224.1. The bla_VIM nucleotide sequence for the strains PA153 and PACTR had 100% identity to the bla_VIM-4 gene with a GenBank accession of NG_050367.1 whereas PA005 and PA187 showed 99−100% identity to the bla_VIM-5 gene with a GenBank accession of AY456196.1. Fourteen P. aeruginosa strains showed 98−100% homology to bla_IMP-26 gene with a GenBank accession of NG049190.1. The GenBank accession numbers for the CRPA strains studied are shown in Table 8.

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Table 8. GenBank accession numbers or sequenced carbapenemase genes in selected P. aeruginosa strains.

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In this study, P. aeruginosa isolates harboured bla_IMP, bla_NDM and bla_VIM carbapenemase genes, with bla_IMP-26 being the most prevalent (21 out of 24 IMP-positive strains). This is consistent with prior reports from Malaysia, where bla_IMP-26 was associated with endemic strains in hospital settings [43]. The study also detected strains carrying carbapenemase genes (bla_IMP-7, bla_IMP-62, bla_IMP-76), albeit at low prevalence. This finding aligns with earlier reports from UMMC [44]. IMP- and VIM-type metallo-β-lactamases (MBLs) are widely reported across Asia, although prevalence varies by country and institution. In a global cohort of 972 CRPA isolates, 22% harboured carbapenemase genes [45]. In Japan, genome surveillance of 382 meropenem‐resistant isolates found a wide range of sequence types and relatively low carbapenemase prevalence [46]. In Thailand, P. aeruginosa isolates carrying bla_IMP and bla_VIM remain common, with ST235 reported as a major high-risk clone in national collections and outbreaks [47,48]. In China, whole-genome sequencing of CRPA strains (2018–2020) has revealed high-risk clones circulating in hospitals, highlighting the potential for horizontal spread of resistance determinants, including MBLs [49]. In India, recent work shows widespread expression of IMP and VIM MBL genes among biofilm-forming, clinical P. aeruginosa, suggesting a more endemic distribution than previously appreciated [50]. In the Middle East, studies from Iraq and Bahrain (2020s) report bla_VIM (especially VIM-2) and bla_IMP-1 in carbapenem-resistant isolates [51]. Beyond epidemiology, there is growing concern over novel IMP variants that may evade inhibition by newer drugs (e.g., xeruborbactam) [52]. Therapeutically, promising preclinical work shows that combining imipenem with meso-dimercaptosuccinic acid (DMSA) can restore activity against MBL producers (including IMP-13 and VIM-2) in a sepsis model [53]. Together, these data indicate that while IMP- and VIM-type carbapenemases are historically well-established in Asia, especially in high-risk clones like ST235, their variant diversity is increasing, with implications for both surveillance and treatment.

The detection of rare variants like bla_IMP-62 and bla_IMP-76 also suggests ongoing evolution of resistance mechanisms. Other carbapenemase genes, such as bla_NDM-1, linked to international transmission, highlight the role of travel in spreading resistant strains [54]. Globally, bla_NDM-1, initially reported in K. pneumoniae, has become widespread in P. aeruginosa, often linked to horizontal gene transfer via plasmids [55]. Its presence in Malaysian isolates further supports the hypothesis of interspecies gene dissemination and regional mobility, possibly influenced by medical tourism and population movement [54]. The identification of bla_VIM-4 and bla_VIM-5, albeit in fewer isolates, remains significant due to their association with outbreaks and high-risk clones, such as ST235. These enzymes, particularly NDM and VIM types, are part of MBLs, a subclass capable of hydrolysing all β-lactams except aztreonam. They are concerning not only for their extensive resistance profiles but also for their role in promoting biofilm production, as shown by positive correlations in this study. Such dual mechanisms intensify the difficulty in treating infections and necessitate urgent containment strategies. Deeper studies are important to find out about other carbapenem resistance mechanisms.

Novel Sequence Type ST3891: A Signpost of Genetic Diversification

MLST analysis was conducted on nine CRPA strains and identified four different STs presented in descending order by the number of strains in which the ST was found: ST235 (n = 6), ST357 (n = 1), ST882 (n = 1) and ST3891 (n = 1). PA208 was deposited in the PubMLST database on 17 February 2022, and ST3891, a novel ST, was assigned with an ID: 8202 (Table 9).

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Table 9. Multilocus sequence typing (MLST) profiles of nine CRPA isolates based on seven housekeeping genes.

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ST235 and ST357 are high-risk clones due to their MDR phenotypes [56]. In this study, ST235 was prevalent in the UMMC. A similar predominance of ST235 and ST357 in MDR P. aeruginosa was observed in Malaysia and other countries [54,57,58]. It is ubiquitous worldwide and linked with various resistance traits, including carbapenemases [59]. The ST235 subtype is frequently associated with poor clinical outcomes. Meanwhile, ST882 was reported in other countries but not in Malaysia [60]. The PubMLST database showed another three P. aeruginosa strains with ST882 were present in different locations: two strains in Australia and one strain in Russia. Three strains from different countries, including one from this study, were isolated from sputum specimens. This suggests the global distribution of this specific sequence type. A novel sequence type, ST3891, was also identified, underscoring a local evolutionary adaptation or an undetected transmission pathway within Malaysian healthcare settings. Although ST3891 was found in only one isolate, its presence alongside carbapenem resistance and virulence traits like biofilm development indicates the importance of genomic surveillance. Comparative genomics with other novel or emerging STs (e.g., ST882 found in other countries but first reported here in Malaysia) may reveal shared resistance islands or mobile genetic elements driving resistance and virulence. These findings highlight the need for continuous genetic monitoring to track the dissemination of high-risk clones.

Correlations of phenotypic characteristics and carbapenem resistance

The results indicated that strains with high resistance to both meropenem and imipenem were also strong biofilm producers (n = 132, 61.4%). Only three carbapenem-resistant strains produced low biofilm (1.4%). The sensitivity profile of P. aeruginosa strains to carbapenem showed that 18 strains (8.4%) were classified as weak biofilm formers and 21 strains (9.8%) were classified as moderate biofilm formers. Significant positive correlations were observed between biofilm and protease activity (r = 0.142, p = 0.038), biofilm and pyocyanin production (r = 0.405, p < 0.001), biofilm and meropenem resistance (r = 0.729, p < 0.001), biofilm and imipenem resistance (r = 0.595, p < 0.001) and biofilm and carbapenem-resistance (r = 0.721, p < 0.001). This suggested that as the biofilm development increases, the levels of protease, pyocyanin and carbapenem-resistance tend to increase (Table 10). P. aeruginosa strains producing MBLs, including those with bla_IMP-26 variants, demonstrated increased biofilm formation capabilities.

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Table 10. Pearson correlation coefficients between biofilm and phenotypic characteristics of P. aeruginosa (n = 215).

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Biofilm and virulence-related genes

Of the 215 strains categorised as high biofilm formers, 65 strains (30.2%) were found to carry the algD gene (biofilm associated). In contrast, low biofilm producers comprised 14/215 strains (6.5%) that harbour the algD gene. Among the moderate biofilm formers, 29/215 strains (13.5%) were identified to carry the algD gene. Most strains harboured the toxR (109/215, 51%) and algD gene (108/215, 50%). There were 83 (39%), 62 (29%) and 43 (20%) strains that exhibited fliC, exoS and lasR genes, respectively. The lowest identification frequencies were observed for the plcH gene (17/215, 8%).

Limitations of the study

There are several limitations in this study. Firstly, it is unknown if the infections originated from environmental sources, as environmental samples were not included. This crucial information may reduce infection control implications for implementing targeted interventions that may help to prevent the spread of resistant organisms within the hospital. The current study focused on class B metallo-β-lactamases (MBLs: bla_IMP, bla_VIM and bla_NDM) because surveillance reports in Malaysia (NSAR 2018–2023) indicated these as the predominant carbapenemase classes. Screening for GES and KPC was not performed due to resource limitations and their historically low prevalence in local P. aeruginosa isolates. Knowledge on the functional relevance of these genes is also missing due to the lack of expression-level data such as quantitative PCR or transcriptomics. This limits the understanding of whether the identified genes are actively expressed and contributing to the phenotypic resistance observed. In addition, clinical correlation could not be performed due to the retrospective nature of the study and the lack of clinical metadata, such as the comorbidities and treatment outcomes. Finally, multivariate analyses were also not performed due to limitations in the dataset, including the absence of comprehensive covariate information and insufficient sample size. This restricts the ability to control for potential confounding variables and evaluate the independent contribution of each factor. Future studies with larger, well-annotated datasets, broader genomic coverage and functional validation will be crucial to improve the interpretation and clinical relevance and enable more robust interpretations.

Conclusion

A positive correlation was found between high biofilm producers and P. aeruginosa strains that are resistant to carbapenem. Biofilm development may cause persistent infections and is less effective in antibiotic treatment. Based on the AST and MIC results, it can be concluded that high resistance to carbapenem may be due to the ability to form biofilm. The high biofilm formers were also supported by the result of biofilm-associated phenotype analysis, such as protease and pyocyanin assay. The clonal dissemination of ST235 in Malaysia necessitates continued close monitoring. This study presents the first report of the novel ST3891 CRPA in Malaysia. The rise of CRPA strains is alarming, highlighting the need for strict infection control policies to reduce the spread of carbapenemase-encoding genes among strains. Studies incorporating clinical and environmental strains would provide more valuable insights into the dissemination of P. aeruginosa.

Supporting information

S1 File. Uncropped gel and plate images of carbapenem-resistant Pseudomonas aeruginosa isolates.

This file contains all the uncropped gel and plate images obtained in this study.

https://doi.org/10.1371/journal.pone.0350200.s001

(DOCX)

S1 Table. Complete dataset of 215 carbapenem-resistant Pseudomonas aeruginosa isolates.

The dataset contains antimicrobial susceptibility, virulence profiling, biofilm formation and molecular characterisation data for all clinical isolates analysed in this study.

https://doi.org/10.1371/journal.pone.0350200.s002

(XLSX)

Acknowledgments

We thank Norazilawati Mohd Isa, a lab technician at the Medical Laboratory, Department of Infectious Disease at the UMMC, for her support during strain collection data. Kalaivani Kalai Chelvam was supported by the Universiti Malaya Faculty of Medicine Postgraduate Scholarship Fund (Account Number: UM.0000092/KWD.BH).

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Figures

Abstract

Introduction

Materials and methods

Data collection

Ethical considerations

Bacterial strains

Antimicrobial susceptibility test (AST)

Biofilm assay

Protease assay

Pyocyanin assay

Genomic DNA Extraction

Molecular identification of virulence-related genes

PCR identification of carbapenem-resistant genes

Molecular typing based on MLST

Statistical analysis

Results and discussion

Bacterial identification and strain distribution

Antimicrobial susceptibility features

Phenotypic characterisation

Virulence gene identification and association with phenotypic traits

Carbapenemase Genes: Clinical and Epidemiological Implications

Novel Sequence Type ST3891: A Signpost of Genetic Diversification

Correlations of phenotypic characteristics and carbapenem resistance

Biofilm and virulence-related genes

Limitations of the study

Conclusion

Supporting information

S1 File. Uncropped gel and plate images of carbapenem-resistant Pseudomonas aeruginosa isolates.

S1 Table. Complete dataset of 215 carbapenem-resistant Pseudomonas aeruginosa isolates.

Acknowledgments

References