Loop-mediated isothermal amplification assay detects multiple alleles of blaOXA-51-like genes in Acinetobacter baumannii and other Gram-negative bacteria despite primer-template mismatches

Mark B Carascal; Lawrence S Macalalad; Joy Ann Petronio-Santos; Raul V Destura; Windell L Rivera

doi:10.1016/j.heliyon.2024.e35653

. 2024 Aug 5;10(15):e35653. doi: 10.1016/j.heliyon.2024.e35653

Loop-mediated isothermal amplification assay detects multiple alleles of bla_OXA-51-like genes in Acinetobacter baumannii and other Gram-negative bacteria despite primer-template mismatches

Mark B Carascal ^a,^b, Lawrence S Macalalad ^b, Joy Ann Petronio-Santos ^a,^c, Raul V Destura ^b,^d, Windell L Rivera ^a,^⁎

PMCID: PMC11337129 PMID: 39170108

Abstract

The known intrinsic and polymorphic bla_OXA-51-like genes of Acinetobacter baumannii were recently reported in other non-A. baumannii Gram-negative pathogens. Accurate detection of this potentially transferrable carbapenemase gene in the clinical setting is critical. This study developed a loop-mediated isothermal amplification (LAMP) assay targetting multiple alleles of bla_OXA-51-like genes. Specifically, an alignment-based primer design, in silico primer screening, and in vitro assay confirmation were conducted. Both in silico and in vitro results revealed the tolerance of the LAMP assay to up to five primer-template mismatches outside the 3′-end primer regions. Within 90 min, the LAMP assay also detected the gene targets in other Gram-negative bacteria with known and novel bla_OXA-51-like genes. Finally, it showed a superior limit of detection (as low as 10¹ CFU/mL) compared with polymerase chain reaction, and high specificity against non-targets. This study developed a highly adaptable LAMP assay to monitor bla_OXA-51-like genes in the clinical setting and provided important insights into LAMP primer design and screening.

Keywords: Acinetobacter baumannii, Carbapenemase, Loop-mediated isothermal amplification, OXA-51

1. Introduction

The World Health Organization identified carbapenem-resistant Gram-negative bacteria as critical priority pathogens that pose the greatest threat to humans [1]. Infections caused by these bacteria have limited treatment options, resulting in high hospitalization and mortality rates [2]. The mechanisms of resistance among these pathogens are highly diverse, with the production of carbapenemases as the most prevalent in carbapenem-resistant Acinetobacter baumannii (CRAb). CRAb is now a growing concern in the global clinical setting, with up to 90 % prevalence in certain parts of Europe, Asia, and Africa [3]. The global increase in CRAb incidence may be due to the rapid spread of carbapenemase genes across Gram-negative species.

The bla_OXA-51-like is a subfamily of chromosomal class D β-lactamase genes in A. baumannii that confers significant hydrolyzing activities against carbapenems [4]. As an intrinsic gene, it has been used as a target for confirming species identity and investigating CRAb in hospital outbreaks [5,6]. However, in the past years, there have been reports of bla_OXA-51-like in non-A. baumannii Gram-negative bacterial species [[7], [8], [9]]. The spread of these genes is due to their association with insertion sequences, which makes them transferrable to other pathogenic Gram-negative species through transposition [10]. Therefore, bla_OXA-51-like may also be used to indicate the spread of antibiotic resistance genes in the hospital and environment.

Within the class D β-lactamase genes, the bla_OXA-51-like subfamily is the biggest, with >370 alleles (http://bldb.eu/accessed on August 1, 2023). The diversity of sequences under this subfamily is due to single-nucleotide polymorphisms (SNPs) dispersed throughout the gene. Hence, when designing molecular probes for nucleic acid amplification tests (NAATs), the high variations among the alleles should be considered. The loop-mediated isothermal amplification (LAMP) assay, a type of NAAT, is dependent on at least six different primers that form loop structures facilitating the subsequent amplification of the target DNA region [11,12]. It is performed using Bst polymerase, whose activity depends on the high fidelity of the 3′-ends of the primers serving as the initial point of DNA elongation [13]. Hence, any mismatch on the 3′-end of the primers was believed to result in low amplification efficiency.

The dynamics of the molecular interactions between the LAMP primers and templates are still understudied, with mismatches considered as a culprit for suboptimal assay [14,15]. Unfortunately, the extent of the effect of the mismatches was not thoroughly investigated, especially those outside the 3′-end primer region. In this study, we developed a LAMP assay that detects multiple variants of the bla_OXA-51-like alleles from A. baumannii and other Gram-negative species by fine-tuning the parameters of primer design and screening. We also explored the effects of different outer and inner primer mismatches in the performance of the LAMP assay using both in silico and in vitro experimental models.

2. Materials and methods

2.1. Collection of reference DNA samples

The protocol for this study was acknowledged by The Medical City–Institutional Review Board (GCS-2020-050). All isolates were acquired from the microbial biobank of The Medical City, Philippines. Only microbiological data were collected from the isolates. Genomic DNA samples were isolated using the boil lysis method [16] from clinical A. baumannii strains with sequence-confirmed bla_OXA-51-like variants and standard strains of the American Type Culture Collection (ATCC) (A. baumannii ATCC BAA-1605 and ATCC 19606). Additional DNA from ATCC and the National Collection of Type Cultures (NCTC) standard multidrug-resistant Gram-negative strains were also obtained. Finally, DNA from three Klebsiella pneumoniae and five Pseudomonas aeruginosa clinical isolates with previous polymerase chain reaction (PCR)-confirmed bla_OXA-51-like genes were also collected (Supplementary File 1). All DNA samples were checked for quality and quantity using Spectrostar Nano UV–Vis Spectrophotometer (BMG Labtech, Germany).

2.2. Analysis of the bla_OXA-51-like genes in non-A. baumannii samples

All non-A. baumannii DNA samples underwent bla_OXA-51-like PCR with A. baumannii ATCC 19606 and Staphylococcus aureus ATCC 29213 as positive and negative controls, respectively. PCR assays were conducted in a 25 μL reaction with 12.5 μL of a 2 × GoTaq Green PCR master mix (Promega, Wisconsin, USA), 10 μM of primers [17], at least 25 ng/μL template DNA, and nuclease-free water. The reaction was performed in a GeneExplorer thermal cycler (Bioer, Hangzhou, China) following a published protocol [17]. All PCR assays were conducted in two independent trials. Amplicons were visualized in 2 % agarose gel using MyGel Instaview® gel electrophoresis (Accuris, Kyonggi, Korea). PCR was confirmed by observing amplicons with a size of 825 bp. Gel profiles were presented as raw images and compiled using Microsoft PowerPoint (Microsoft, Washington, USA). The bla_OXA-51-like amplicons generated from the non-A. baumannii samples were bidirectionally sequenced using Sanger sequencing (Macrogen, Seoul, Korea) to determine the allelic profiles. Amplicons from A. baumannii ATCC BAA-1605 and ATCC 19606 were sequenced as controls. The complete DNA and amino acid sequences were compared with all known bla_OXA-51-like variants using the β-lactamase database-embedded Basic Local Alignment Search Tool (BLDB BLAST; http://www.bldb.eu:4567/, accessed on August 1, 2023) [18]. The recent consensus for β-lactamase nomenclature was followed in naming alleles [19]. All original sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on September 5, 2023) with accession numbers OR449063–OR449072.

2.3. LAMP primer design for bla_OXA-51-like genes

The primer design workflow suggested by Shirshikov et al. was followed with slight modifications [20]. Briefly, the sequences of 34 unique bla_OXA-51-like alleles from our available isolates, together with the native bla_OXA-51 gene, were gathered from the GenBank database (Supplementary File 2). The chosen alleles correspond to those that have counterpart samples for the in vitro experiments, ensuring accurate confirmation of our in silico hypothesis on primer-template mismatch tolerance later on. The sequences were aligned in Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on January 14, 2023) using default parameters. The multiple sequence alignment file was then uploaded to MorphoCatcher (http://morphocatcher.ru/, accessed on January 20, 2023) to detect and mark sites with high variations. The generated file was uploaded to PrimerExplorer v.5 (http://primerexplorer.jp/lampv5e/index.html, accessed on January 20, 2023) using AT-rich sequence parameters. The resulting primer sets were checked for the absence of primer secondary structures using the default parameters of Oligo Calculator v.3.27 (http://biotools.nubic.northwestern.edu/OligoCalc.html, accessed on January 25, 2023). The top primer set (TMC6: F3, B3, Forward Inner Primer (FIP) = F2 + F1c, and Backward Inner Primer (BIP) = B2 + B1c) was chosen based on the set parameters. The final primer sequences are TMC6–F3 (5′-GCCAAACTCAACAAAGCTATGG-3′), TMC6–B3 (5′-CGGAATAGCGGAAGCTTTCA-3′), TMC6–FIP (5′-GCTCAAGGCCGATCAAAGCATTAAG–TCGTGCTTCGACCGAGTA-3′), and TMC6–BIP (5′-ACAGAAGTATTTAAGTGGGACGGGC–CCTAGGGTCATGTCCTTTTCC-3′).

2.4. In silico analysis of the LAMP primers

To determine the mismatches of the designed LAMP primer set on the target genes, the primers were aligned to the available bla_OXA-51-like alleles from our reference sequences using Clustal W in Bioedit v.7.2.5. The alignment was visualized as conservation plots. Meanwhile, to determine the initial performance of the chosen primer set, an in silico simulation was performed using the electric LAMP program (eLAMP) with modified parameters [14]. The TMC6 primer set and comparable primers from the literature (Primer Sets 1 and 2) [21,22] were compiled into one comma-separated values file. Meanwhile, sequences from 373 known bla_OXA-51-like alleles were compiled into one FASTA file and used as the template for the in silico simulation. The simulation was performed using combinations of different primer mismatch parameters (Supplementary File 3). Amplification results were scored from 0 to 3 (0 for no amplification and 1–3 for amplification with one to three matching 3′-end sequences). Scores were visualized as a heatmap using Microsoft Excel (Microsoft, Washington, USA). The in silico performance of the TMC6 primer set was compared with Primer Sets 1 and 2 using analysis of variance with Tukey's test in R v.4.3.1.

2.5. LAMP assay development

The LAMP assay was performed in a 25 μL reaction with 12.5 μL of a 2 × DNA Polymerase Buffer B (LGC, Biosearch Technologies, London, UK), 1 μL 100 mM of MgSO₄ (New England Biolabs, Massachusetts, USA), 10 mM of a deoxynucleotide triphosphate mix (Vivantis, Selangor, Malaysia), 5 μM of F3 and B3 primers, 12 μM of FIP and BIP, 20–25 ng of template DNA, 8 units of Bst DNA Polymerase Exonuclease Minus (LGC, Biosearch Technologies, London, UK), and nuclease-free water. All reference DNA from the isolates with sequence-confirmed bla_OXA-51-like alleles underwent the LAMP assay. DNA from S. aureus ATCC 29213 was used as the negative control. All assays were performed in at least two independent trials using a GeneExplorer thermal cycler (Bioer, Hangzhou, China) set at 64 °C for at least 60 min and terminated at 80 °C for 2 min. Amplification products were visualized by adding a 1000 × SYBR Green I dye (Invitrogen, California, USA) to the completed reactions and by 2 % gel electrophoresis, as described earlier. Colorimetric and gel profiles were presented as raw images and compiled using Microsoft PowerPoint (Microsoft, Washington, USA).

2.6. Analytical performance testing of the LAMP assay

The limit of detection (LOD) of the LAMP assay was assessed using different dilutions of A. baumannii TMC36 (bla_OXA-1071 with no primer–template mismatch) and A. baumannii ATCC 1605 (bla_OXA-69 with multiple primer–template mismatches) to achieve cell concentrations of 10⁰–10⁶ colony forming units (CFU)/mL. Serial dilutions were performed from 0.5 McFarland standardized cultures in nuclease-free water, and colony counts were confirmed through plating. DNA samples from each dilution were extracted as described previously and used as templates for both LAMP and PCR assays. The LOD was defined as the lowest tested sample concentration that yielded a positive assay result. Meanwhile, to determine the specificity of the LAMP assay, at least 20–25 ng of DNA from the 15 bla_OXA-51-like-negative standard multidrug-resistant Gram-negative strains was used as the templates. LAMP assays were performed as with the previous experiments.

3. Results

3.1. PCR and sequencing confirmed bla_OXA-51-like genes in other Gram-negative clinical isolates

The bla_OXA-51-like PCR analysis revealed the presence of faint amplicon bands in the three K. pneumoniae and four P. aeruginosa clinical isolates (Supplementary Files 4-5). Sequence analyses of the resulting amplicons confirmed the genes to be known (bla_OXA-66, bla_OXA-769, and bla_OXA-1070) and novel (bla_OXA-1217, bla_OXA-1218, bla_OXA-1219, and bla_OXA-1220) alleles (GenBank: OR449065–OR449072). Conversely, all other reference multidrug-resistant, non-A. baumannii Gram-negative strains tested negative for the gene (Supplementary Files 4-5). Our controls A. baumannii ATCC BAA-1605 and A. baumannii ATCC 19606 had the confirmed bla_OXA-69 and bla_OXA-98 alleles, respectively (GenBank: OR449063–OR449064).

3.2. Primer design workflow generated primers with ideal characteristics

The initial multiple sequence alignment of the 34 reference alleles showed SNPs dispersed throughout the bla_OXA-51-like genes (Supplementary File 6). The MorphoCatcher program determined sites with low average mutational index, chose the best template (Supplementary File 7), and marked the template sequence with informative sites for the primer design. The output from the MorphoCatcher was used in PrimerExplorer v.5, which generated a total of 13 primer designs within the target gene positions (160–700 bp). The top five primers with higher dimerization energies were chosen and screened using the set parameters. The TMC6 primer set was finally selected on the basis of its ideal characteristics (Supplementary File 8).

3.3. In silico LAMP showed high amplification success rates

The in silico LAMP analysis showed that the TMC6 primer set had high mismatch tolerance in detecting various bla_OXA-51-like alleles (Fig. 1). The primer set amplified 334 of the 373 alleles with as low as 80 % overall primer matching and exact 3′-end fidelity (Supplementary File 9). The TMC6 primer set was found to have comparable in silico performance with that of Primer Set 2 [21] and superior sensitivity compared with that of Primer Set 1 [22] (Fig. 1). These results were confirmed by the statistically insignificant differences (p-values >0.05) between TMC6 primers and Primer Set 2 in all set conditions and a significant difference (p-value <0.001) between Primer Set 1 and the TMC6 primer set at 80 % primer–template matching (Supplementary File 10).

3.4. LAMP assay confirmed the mismatch tolerance of the designed primers

The reference DNA samples for the in vitro experiments were grouped based on the characteristics of their bla_OXA-51-like alleles (Fig. 2). Primer–template mismatches can be found in all primer positions (outer, middle, and inner) and directions (forward and backward), with a higher occurrence of mismatches in the backward primers. None of the outer and middle primers have a 3′-end mismatch with the template, and none of the inner primers have a 5′-end mismatch. The LAMP assay showed positive amplification reactions for all the tested bla_OXA-51-like alleles regardless of their primer–template mismatch groupings (Fig. 3; Supplementary File 11). The efficiency of the LAMP reaction is not dependent on the number of mismatches as most of the representatives have close band intensity profiles regardless of the grouping and at almost similar starting DNA template concentrations (20–25 ng/μL).

Fig. 3 — The LAMP assay showed positive detection (as a green fluorescence and ladder-like gel profile) of the *bla*_OXA-51-like genes in all reference A. *baumannii* samples regardless of the target allele. Groups 1 (lanes 1–3: *bla*_OXA-1070, *bla*_OXA-1071, and *bla*_OXA-1072), 2 (lane 4: *bla*_OXA-1074), 3 (lanes 5–7: *bla*_OXA-68 and two *bla*_OXA-98), 4 (lanes 8–10: *bla*_OXA-70, *bla*_OXA-100, and *bla*_OXA-1078), 5 (lanes 11–13: *bla*_OXA-120, *bla*_OXA-1060, and *bla*_OXA-1079), 6 (lane 14: *bla*_OXA-1069), 7 (lanes 15–19: *bla*_OXA-66, *bla*_OXA-90, *bla*_OXA-342, *bla*_OXA-508, and *bla*_OXA-1080), and 8 (lane 20: *bla*_OXA-69) alleles with different levels of primer–template mismatch showed almost similar levels of amplification in the LAMP assay. N, negative control (S. *aureus* ATCC 29213); NTC, no-template control (nuclease-free water). Raw images are provided as Supplementary File 11.

3.5. LAMP assay detected bla_OXA-51-like alleles in other Gram-negative bacteria

Using the grouping in Fig. 2, the bla_OXA-51-like alleles of the clinical K. pneumoniae and P. aeruginosa isolates were identified to be under Groups 1 (bla_OXA769, bla_OXA-1070, and bla_OXA-1217), 2 (bla_OXA-1220), 3 (bla_OXA-1219), and 7 (bla_OXA-66 and bla_OXA-1218). These alleles were successfully detected in the LAMP assay as well. However, the LAMP products from the K. pneumoniae and P. aeruginosa DNA templates were significantly detected only after at least 90 min of reaction time (compared with only 60 min in the initial assays, Fig. 4A and B; Supplementary File 12). The intensity and sizes of the LAMP products are not dependent on the number of primer–template mismatches.

Fig. 4 — The LAMP assay showed detectable positive results (as a green fluorescence and ladder-like gel profile) for *bla*_OXA-51-like genes in non-A. *baumannii* clinical samples after a 90-min reaction time regardless of the target allele. (A) The standard 60-min LAMP assays showed minimal to no positivity for all samples in colorimetric and gel electrophoresis profiles. (B) The positivity was enhanced when the assays were extended to 90 min. Lanes 1–3: K. *pneumoniae* TMCK01, TMCK03, and TMCK04 with *bla*_OXA-1217, *bla*_OXA-769, and *bla*_OXA-66, respectively. Lanes 4–8: P. *aeruginosa* TMCP02, TMCP03, TMCP04, TMCP12, and TMCP14 with *bla*_OXA-1070, *bla*_OXA-1218, *bla*_OXA-1219, *bla*_OXA-1220, and *bla*_OXA-66, respectively. N, negative control (S. *aureus* ATCC 29213); NTC, no-template control (nuclease-free water). Raw images are provided as Supplementary File 12.

3.6. The developed LAMP assay has a superior LOD than PCR and high specificity

The LAMP assay showed robust detection of the bla_OXA-51-like genes in A. baumannii in as low as 10¹ CFU/mL (Fig. 5A; Supplementary File 13). This observation is true whether there is no primer–template mismatch (bla_OXA-1071) or in templates with up to five mismatches (bla_OXA-69; Supplementary Files 14-15). Conversely, PCR was only able to detect the targets at 10⁶ CFU/mL cell concentration. Regarding specificity, the LAMP assay did not produce any spurious LAMP products when tested against the bla_OXA-51-like-negative standard Gram-negative strains (Fig. 5B).

4. Discussion

NAATs play an important role in detecting and monitoring antibiotic resistance in healthcare and community settings. LAMP is considered among the promising NAATs due to its high adaptability and accuracy [23]. However, it can be challenging to design primers that will ensure high sensitivity and specificity, especially for highly polymorphic targets like most antibiotic resistance genes. The conventional LAMP primer design promised versatility in terms of targeting common sites in different sequences [12]. However, limitations exist in using large datasets as target references and in setting limits on the number and positions of allowable mismatches. As multiple variants of the bla_OXA-51-like genes exist, we developed a LAMP assay that could detect different alleles of the genes even in the presence of up to five mismatches in the primer binding regions. The developed assay was also found to detect the presence of the gene when acquired by other Gram-negative bacteria.

The presence of bla_OXA-51-like genes from non-A. baumannii species was reported in Acinetobacter nosocomialis [24], Enterobacterales [8,25], and P. aeruginosa [9] in the last 15 years. Our study showed the presence of the same genes in clinical K. pneumoniae and P. aeruginosa isolates. More importantly, we showed that four of the detected genes correspond to new alleles (bla_OXA-1217 to bla_OXA-1220). Our results provide preliminary evidence on the potential cross-species transfer of the intrinsic bla_OXA-51-like carbapenemase genes among non-A. baumannii Gram-negative clinical isolates in a Philippine hospital setting. The presence of these genes in other species as a transferrable element may further spread the carbapenem resistance phenotype, eventually rendering β-lactam antibiotics ineffective in several Gram-negative pathogens [26]. Although only four of the non-A. baumannii isolates reported are carbapenem-resistant, the presence of the carbapenemase genes gives the pathogens the potential to become resistant later. Herein, we propose bla_OXA-51-like genes to be important markers in monitoring the spread of carbapenem resistance in Gram-negative pathogens.

LAMP assay development starts by understanding the peculiarities of primer design. Although targeting conserved gene regions is straightforward in PrimerExplorer v.5, using multiple representative sequences for highly polymorphic targets can be challenging. In the original design manual, uploading multiple alignment sequences is possible, but only limited to up to 2000 bp of input sequence. The workflow used in our study addressed this limitation using Clustal Omega, which allows for unlimited sequence length input [20]. Additionally, the MorphoCatcher program allowed for the creation of an annotated input file that can be interpreted by PrimerExplorer v.5 [20]. This combination provided an efficient method of representing multiple variations of polymorphic genes as input sequences for primer design.

Technologists only rely on empirical testing of multiple primer sets to decide which primers will work [27]. The eLAMP program solved this limitation by allowing the simulation of different primer–template conditions and predicting LAMP amplification with allowance for mismatches [14]. The program could be a crude way to test the initial sensitivity of the primers against variants of polymorphic targets. Our results indicate that the assay could tolerate up to 20 % mismatch (up to 4-bp mismatch per primer, assuming an average primer length of 20 bp) in silico. We hypothesize that these in silico conditions could provide good estimates of the allowable number of mismatches for LAMP primer design, as also supported by another study [14]. Given its potential predictive power, the eLAMP results can be used to screen for the best primer to synthesize, eventually saving both time and resources for technologists developing LAMP assays.

The eLAMP analysis revealed a high sensitivity of the TMC6 primer set to at least 89.5 % of all known bla_OXA-51-like alleles. This performance is comparable with that of Primer Set 2 [21]. The difference lies in considering multiple sequence alignment data in our primer design workflow, resulting in an additional 18 in silico amplified targets with exact primer–template matches and 3′-end fidelity. Conversely, Primer Set 1 [22] showed a more selective in silico amplification compared with the TMC6 primer set. A quick evaluation of their primers showed nonideal primer characteristics such as low GC rates of F3 and BIP (35%–38 %) and the presence of potential primer secondary structures. This observation highlights the importance of evaluating individual primer characteristics before choosing the best set for the assay.

To determine if the eLAMP results complement the actual LAMP assay, DNA templates with different variants of the bla_OXA-51-like alleles were used. Our results showed that the developed LAMP was able to tolerate up to a total of five primer mismatches if the 3′-ends of the individual primers were exactly matching. Previous studies hypothesized that general LAMP primer mismatches may result in nonamplification of the target [14,28]. However, this hypothesis was not supported by in vitro data. Our results add to the limited experimental evidence showing mismatch tolerance of 2–3 bp in middle primers and at the 5′-ends [15,29]. Our study provided evidence that mismatches at the 5′-ends of the outer primers (F3 and B3) and middle positions within BIP (B1c and B2) are tolerated under the standard assay. Therefore, technologists may add these considerations in primer design workflows, making allowance for mismatches, especially for highly polymorphic antibiotic resistance gene targets. Our in vitro results supported the in silico results, showing up to approximately 20 % primer–template mismatch tolerance (outside the 3′-end) of the LAMP assay.

Previous LAMP assays targeting bla_OXA-51-like genes were used for rapid A. baumannii detection and investigations of hospital outbreaks [21,22]. However, none of these assays has been tested in detecting the transferrable gene in non-A. baumannii isolates or in novel bla_OXA-51-like alleles. Our developed LAMP assay addressed these gaps by detecting the target genes in K. pneumoniae and P. aeruginosa, and four novel bla_OXA-51-like alleles (bla_OXA-1217 to bla_OXA-1220). However, longer assay times are necessary for properly detecting the genes in non-A. baumannii samples. One possible explanation is the potentially low initial quantity of the mobile genetic elements carrying the bla_OXA-51-like genes in non-A. baumannii isolates. Our PCR results support this hypothesis by showing only faint target amplicons for the tested K. pneumoniae and P. aeruginosa. Increasing the LAMP reaction time to 90 min provided an advantage in preventing false-negative or inconclusive results. A 90-min LAMP assay has been described in the literature for testing targets in mobile genetic elements from crude DNA samples [30].

Our developed LAMP assay showed superior LODs compared with PCR, making it more sensitive in detecting the target antibiotic resistance genes even at low concentrations [31]. Additionally, no spurious amplification was detected in DNA samples from other multidrug-resistant Gram-negative pathogens without the target gene, suggesting high specificity [32]. The rapid nature of the reaction (relative to its PCR counterpart), coupled with high sensitivity and specificity, makes the developed LAMP assay ideal in fast-paced epidemiological and clinical investigations adaptable to low-resource clinical settings.

Overall, this study presented limitations. First, we only used selected reference sequences for the primer design. The chosen alleles correspond to those that have counterpart samples for the in vitro experiments, ensuring accurate confirmation of our in silico hypothesis on primer-template mismatch tolerance. Second, only a few non-A. baumannii strains with bla_OXA-51-like genes were used for additional in vitro testing. Despite this low sample availability, our study provided initial evidence of the potential transferability of the bla_OXA-51-like genes in non-A. baumannii Gram-negative pathogens, which could be further studied in future investigations. Finally, only limited DNA samples possessing different alleles with primer-template mismatches were used in the in vitro experiments. The meticulous primer design workflow, extensive complementary in silico analysis, and confirmatory in vitro experimental design helped supplement this limitation.

5. Conclusions

Our results imply the need for more efforts to monitor transferrable resistance genes. Poor resistance detection and control may ultimately result in increased resistance rates and improper patient management. The developed LAMP assay is useful in detecting multiple alleles of the bla_OXA-51-like genes, which are emerging to be important markers for the spread of antibiotic resistance. Meanwhile, the LAMP assay development workflow described herein also provided important insights into mismatch considerations, which can be used in designing assays against other polymorphic resistance gene targets. Future studies may delve into designing closed-tube (minimizing potential cross-contamination of products), or allele-specific LAMP assays for other applications.

Funding

This study was partially funded by The Medical City Intramural Grant Fund (Grant No. 2020-001) awarded to Mark B. Carascal. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability statement

All data are provided within the manuscript and its supplementary files. All original annotated sequences have been deposited in the GenBank database under the accession numbers OR449063–OR449072.

Ethics declaration statement

This study was reviewed and acknowledged by The Medical City Institutional Review Board, with a research registry number: GCS-2020-050. Full review and/or approval by the ethics committee was not needed for this study because only samples stored in the hospital biobank, without any patient-related data, were used for the experiments and analyses.

CRediT authorship contribution statement

Mark B. Carascal: Writing – review & editing, Writing – original draft, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Lawrence S. Macalalad: Methodology, Investigation, Formal analysis, Data curation. Joy Ann Petronio-Santos: Validation, Supervision, Resources, Formal analysis, Conceptualization. Raul V. Destura: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Conceptualization. Windell L. Rivera: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors would like to acknowledge Dr. Roland Gilbert Remenyi and the rest of The BRU Labs staff of the Clinical and Translational Research Institute for providing the facilities for the experiments, and Dr. Esperanza Cabrera and Engr. Marvin Belen for the support in manuscript preparation.

Footnotes

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e35653.

Appendix A. Supplementary data

The following are the Supplementary data to this article.

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References

1.Tacconelli E., Carrara E., Savoldi A., Harbarth S., Mendelson M., Monnet D.L., 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):318–327. doi: 10.1016/s1473-3099(17)30753-3. [DOI] [PubMed] [Google Scholar]
2.Zhou R., Fang X., Zhang J., Zheng X., Shangguan S., Chen S., et al. Impact of carbapenem resistance on mortality in patients infected with Enterobacteriaceae: a systematic review and meta-analysis. BMJ Open. 2021;11(12) doi: 10.1136/bmjopen-2021-054971. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Ma C., McClean S. Mapping global prevalence of Acinetobacter baumannii and recent vaccine development to tackle it. Vaccines (Basel) 2021;9(6):570. doi: 10.3390/vaccines9060570. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Takebayashi Y., Findlay J., Heesom K.J., Warburton P.J., Avison M.B., Evans B.A. Variability in carbapenemase activity of intrinsic OxaAb (OXA-51-like) β-lactamase enzymes in Acinetobacter baumannii. J. Antimicrob. Chemother. 2021;76(3):587–595. doi: 10.1093/jac/dkaa502. [DOI] [PubMed] [Google Scholar]
5.Hassan R.M., Salem S.T., Hassan S.I.M., Hegab A.S., Elkholy Y.S. Molecular characterization of carbapenem-resistant Acinetobacter baumannii clinical isolates from Egyptian patients. PLoS One. 2021;16(6) doi: 10.1371/journal.pone.0251508. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Zhao Y., Hu K., Zhang J., Guo Y., Fan X., Wang Y., et al. Outbreak of carbapenem-resistant Acinetobacter baumannii carrying the carbapenemase OXA-23 in ICU of the eastern Heilongjiang Province, China. BMC Infect. Dis. 2019;19(1):452. doi: 10.1186/s12879-019-4073-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Lee Y.T., Kuo S.C., Chiang M.C., Yang S.P., Chen C.P., Chen T.L., 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):1124–1127. doi: 10.1128/aac.00622-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Leski T.A., Bangura U., Jimmy D.H., Ansumana R., Lizewski S.E., Li R.W., et al. Identification of blaOXA-₅₁-like, blaOXA-₅₈, blaDIM-₁, and blaVIM carbapenemase genes in hospital Enterobacteriaceae isolates from Sierra Leone. J. Clin. Microbiol. 2013;51(7):2435–2438. doi: 10.1128/jcm.00832-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Chaudhary M., Payasi A. Prevalence, genotyping of Escherichia coli and Pseudomonas aeruginosa clinical isolates for oxacillinase resistance and mapping susceptibility behavior. J. Microb. Biochem. Technol. 2014;6(2):63–67. [Google Scholar]
10.Chen T.L., Lee Y.T., Kuo S.C., Hsueh P.R., Chang F.Y., Siu L.K., et al. Emergence and distribution of plasmids bearing the blaOXA-51-like gene with an upstream ISAba1 in carbapenem-resistant Acinetobacter baumannii isolates in Taiwan. Antimicrob. Agents Chemother. 2010;54(11):4575–4581. doi: 10.1128/aac.00764-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12) doi: 10.1093/nar/28.12.e63. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Tomita N., Mori Y., Kanda H., Notomi T. Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat. Protoc. 2008;3(5):877–882. doi: 10.1038/nprot.2008.57. [DOI] [PubMed] [Google Scholar]
13.Zhou Y., Wan Z., Yang S., Li Y., Li M., Wang B., et al. A Mismatch-tolerant reverse transcription loop-mediated isothermal amplification method and its application on simultaneous detection of all four serotype of dengue viruses. Front. Microbiol. 2019;10:1056. doi: 10.3389/fmicb.2019.01056. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Salinas N.R., Little D.P. Electric LAMP: virtual loop-mediated isothermal AMPlification. ISRN Bioinform. 2012;2012 doi: 10.5402/2012/696758. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Wang D. Effect of internal primer–template mismatches on loop-mediated isothermal amplification. Biotech Biotechnol Equip. 2016;30(2):314–318. doi: 10.1080/13102818.2015.1125765. [DOI] [Google Scholar]
16.Mu X., Nakano R., Nakano A., Ubagai T., Kikuchi-Ueda T., Tansho-Nagakawa S., 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. doi: 10.1016/j.mimet.2015.12.011. [DOI] [PubMed] [Google Scholar]
17.Hu W.S., Yao S.M., Fung C.P., Hsieh Y.P., Liu C.P., Lin J.F. An OXA-66/OXA-51-like carbapenemase and possibly an efflux pump are associated with resistance to imipenem in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2007;51(11):3844–3852. doi: 10.1128/aac.01512-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Naas T., Oueslati S., Bonnin R.A., Dabos M.L., Zavala A., Dortet L., et al. Beta-lactamase database (BLDB) - structure and function. J. Enzym. Inhib. Med. Chem. 2017;32(1):917–919. doi: 10.1080/14756366.2017.1344235. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Bradford P.A., Bonomo R.A., Bush K., Carattoli A., Feldgarden M., Haft D.H., et al. Consensus on β-lactamase nomenclature. Antimicrob. Agents Chemother. 2022;66(4) doi: 10.1128/aac.00333-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Shirshikov F.V., Pekov Y.A., Miroshnikov K.A. MorphoCatcher: a multiple-alignment based web tool for target selection and designing taxon-specific primers in the loop-mediated isothermal amplification method. PeerJ. 2019;7 doi: 10.7717/peerj.6801. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Li P., Niu W., Li H., Lei H., Liu W., Zhao X., et al. Rapid detection of Acinetobacter baumannii and molecular epidemiology of carbapenem-resistant A. baumannii in two comprehensive hospitals of Beijing, China. Front. Microbiol. 2015;6:997. doi: 10.3389/fmicb.2015.00997. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Garciglia Mercado C., Gaxiola Robles R., Ascencio F., Silva-Sanchez J., Estrada-Garcia M.T., Gomez-Anduro G. Development of a LAMP method for detection of carbapenem-resistant Acinetobacter baumannii during a hospital outbreak. J Infect Dev Ctries. 2020;14(5):494–501. doi: 10.3855/jidc.11692. [DOI] [PubMed] [Google Scholar]
23.Garg N., Ahmad F.J., Kar S. Recent advances in loop-mediated isothermal amplification (LAMP) for rapid and efficient detection of pathogens. Curr Res Microb Sci. 2022;3 doi: 10.1016/j.crmicr.2022.100120. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Lee Y.T., Turton J.F., Chen T.L., Wu R.C., Chang W.C., Fung C.P., et al. First identification of blaOXA-51-like in non-baumannii Acinetobacter spp. J. Chemother. 2009;21(5):514–520. doi: 10.1179/joc.2009.21.5.514. [DOI] [PubMed] [Google Scholar]
25.Budak S., Aktas Z., Oncul O., Acar A., Ozruyt M., Turhan V., et al. Detection of Acinetobacter baumannii derived oxa-51 and oxa-58 carbapenemase genes in enteric bacteria: a new dimension on carbapenemase resistance. Clin. Microbiol. Infect. 2012;18(Suppl):311. [Google Scholar]
26.Abordo A.M.S., Carascal M.B., Remenyi R., Dalisay D.S., Saludes J.P. 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. doi: 10.3390/pathogens12081019. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Zhang Y., Tanner N.A. Improving RT-LAMP detection of SARS-CoV-2 RNA through primer set selection and combination. PLoS One. 2022;17(4) doi: 10.1371/journal.pone.0254324. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Subramaniam K., Shariff M., Omar A.R., Hair-Bejo M., Ong B.L. Use of acridine orange to visually improve the loop-mediated isothermal amplification for detection of infectious spleen and kidney necrosis virus. Fish Pathol. 2014;49(4):173–180. [Google Scholar]
29.Peyrefitte C.N., Boubis L., Coudrier D., Bouloy M., Grandadam M., Tolou H.J., et al. Real-time reverse-transcription loop-mediated isothermal amplification for rapid detection of rift valley Fever virus. J. Clin. Microbiol. 2008;46(11):3653–3659. doi: 10.1128/jcm.01188-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Yu G., Chen L., Lin C.W., Li B., Cui H., Chen S., et al. Loop-mediated isothermal amplification assays for screening of bacterial integrons. Biol. Res. 2014;47(1):53. doi: 10.1186/0717-6287-47-53. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Hassan M.M., van Vliet A.H.M., Higgins O., Burke L.P., Chueiri A., O'Connor L., et al. Rapid culture-independent loop-mediated isothermal amplification detection of antimicrobial resistance markers from environmental water samples. Microb. Biotechnol. 2023;16(5):977–989. doi: 10.1111/1751-7915.14227. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Kim E.J., Lee J., Yoon Y., Lee D., Baek Y., Takano C., et al. Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria. Front. Cell. Infect. Microbiol. 2023;12 doi: 10.3389/fcimb.2022.1000445. [DOI] [PMC free article] [PubMed] [Google Scholar]

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