Synchronous detection of Burkholderia pseudomallei and its ceftazidime resistance mutation based on RNase-HII hydrolysis combined with lateral flow strip assay

ABSTRACT Melioidosis is a severe and often fatal infectious disease caused by Burkholderia pseudomallei, which poses significant challenges due to its high mortality rate and frequent misdiagnosis. To effectively treat infected patients, especially in epidemic regions like Hainan with a high resistance rate to ceftazidime (CAZ), early diagnosis and resistance testing are critical. Here, we designed a strategy that combines RNase-HII-dependent PCR with lateral flow strip assay (LFSA) for simultaneous detection of B. pseudomallei and its CAZ resistance mutation. In this strategy, we utilized rhPCR technology to design specific primers with a C3-spacer modification. These primers target a 115-base pair region within the ORF2 of the B. pseudomallei type III secretion system gene cluster, as well as the P174L mutation in the penA gene, which is a major mutation associated with CAZ resistance in Hainan. The primers contain complementary RNA bases that can be hydrolyzed only in the presence of thermostable RNase-HII enzymes, facilitating subsequent PCR reactions. Additionally, we introduced two pairs of universal probes carrying fluorescein isothiocyanate (FITC) and biotin, as well as digoxin and biotin. The sequences of these universal probes align with specific regions of the primers, resulting in target products labeled with different tags. The resulting amplification products are then subjected to LFSA, where the presence of specific markers is visually interpreted based on color development. These markers flow to test line 1 (T1) and are captured by a fixed anti-FITC antibody, generating a red line that indicates the presence of B. pseudomallei. Similarly, the occurrence of a red line on test line 2 (T2), which is fixed with anti-digoxin antibodies, indicates the presence of the P174L mutation in B. pseudomallei. This protocol serves as an excellent tool for the rapid diagnosis of melioidosis and provides a basis for selecting appropriate antibiotic medication. It has the potential to revolutionize the diagnosis of melioidosis, particularly in resource-limited settings. Furthermore, this technique holds promise as a versatile tool for disease diagnosis in general. IMPORTANCE This study focused on the development of a reaction system using rhPCR to amplify a specific gene, ORF2, of B. pseudomallei and to identify the P174L mutation associated with increased drug resistance to ceftazidime (CAZ). The system incorporated universal primer probes and a simple temperature cycle reaction. The amplified products were then analyzed using lateral flow strip assay (LFSA) for strain identification and mutation interpretation. The developed system provides a reliable basis for diagnosing melioidosis and selecting appropriate drugs. Its potential impact is particularly significant in resource-limited settings where access to advanced diagnostic techniques is limited. This platform stands out for its simplicity, convenience, sensitivity, specificity, and portability. It shows promise as a point-of-care testing method for detecting single nucleotide polymorphism in genes associated with other diseases. By leveraging the advantages of this platform, researchers and healthcare professionals can potentially expand its use beyond melioidosis and apply it to the rapid detection of genetic variations in other disease-related genes.


IMPORTANCE
This study focused on the development of a reaction system using rhPCR to amplify a specific gene, ORF2, of B. pseudomallei and to identify the P174L mutation associated with increased drug resistance to ceftazidime (CAZ).The system incorporated universal primer probes and a simple temperature cycle reaction.The amplified products were then analyzed using lateral flow strip assay (LFSA) for strain identification and mutation interpretation.The developed system provides a reliable basis for diagnosing melioidosis and selecting appropriate drugs.Its potential impact is particularly significant in resource-limited settings where access to advanced diagnostic techniques is limited.This platform stands out for its simplicity, convenience, sensitiv ity, specificity, and portability.It shows promise as a point-of-care testing method for detecting single nucleotide polymorphism in genes associated with other diseases.By leveraging the advantages of this platform, researchers and healthcare professionals can including notable mutations such as I139M, P145L, T147A, and T264A (18).In addition, it has been confirmed that the P174L mutation in penA can cause enhanced CAZ resistance by expanding the space in a conserved structure called the omega loop, which in turn increased flexibility at the active site (19).The previous study of our group (under review) also showed that this locus is also the main mechanism of CAZ resistance in B. pseudo mallei in Hainan.The presence of these mutation sites severely limits the effectiveness of CAZ, underscoring the importance of timely and accurate identification of these mutations to enable doctors to adjust treatment strategies promptly.
Currently, the gold standard for diagnosing melioidosis relies primarily on the bacterial culture of clinical isolates, followed by serology, mass spectrometry, and other identification methods (20,21).However, these methods often have limitations such as low sensitivity, time-consuming procedures, complexity, and high requirements for technical expertise and equipment (22).Although quantitative real-time PCR technol ogy has reduced identification time significantly, it still involves complex nucleic acid extraction steps, specialized equipment, and strict laboratory safety protocols and has a sensitivity of only around 60%-70% (23,24).Other techniques based on antigen-anti body reactions such as latex agglutination, immunofluorescence analysis (LAA), lateral flow immunoassay (LFI), and indirect hemagglutination test (IHA), while greatly reducing the detection time required (25)(26)(27), also suffer from similar drawbacks such as crossreaction with nontarget bacteria which lead to misdiagnosis and incorrect antibiotic treatment (28,29).Furthermore, these methods do not provide information on pathogen drug resistance, making it challenging to select appropriate antibiotics in a timely and accurate manner, which can lead to disease progression.As a result, there is a need for improved diagnostic methods that overcome these limitations and allow for rapid, sensitive, and accurate detection of melioidosis, including its drug resistance characteris tics.
RNaseH-dependent PCR (rhPCR) amplification, which relies on the cleavage activity of thermostable RNase HII enzymes during hybridization to the complementary target sequence, offers several advantages that can enhance PCR (30).Firstly, the enzyme has minimal activity at low temperatures, allowing for a "heat-start PCR" without the need to modify the DNA polymerase.Secondly, the enzyme's cleavage efficiency decreases in the presence of a mismatch near the RNA residue.Leveraging these properties can be designed for the target sequence, reducing the formation of primer dimers, improving specificity, enabling multiplex PCR with multiple primers, and facilitating the simultane ous detection of SNP (30).Lateral flow strip assay (LFSA) is a technique that combines colloidal gold particles, monoclonal antibody technology, and paper chromatography.LFSA holds great potential for nucleic acid analysis due to its visual detection, rapid response, ease of operation, and compatibility with portable cameras or smartphones.LFSA has already found applications in various fields, including protein analysis, small molecule detection, nucleic acid analysis, and exosome detection (31,32).
In this study, we aimed to achieve rapid identification of B. pseudomallei and POCT for the detection of resistance mutation sites in the penA gene.To accomplish this, we employed rhPCR to amplify the specific house-keeping gene ORF2 of B. pseudomallei and P174L mutation site in penA known to be responsible for altered drug resistance.The amplification process was facilitated by the inclusion of universal primer probes and a simple temperature cycler reaction.Finally, the resulting products were differenti ated based on the bands observed with LFSA.This platform demonstrates simplicity, user-friendliness, high sensitivity, specificity, and adaptability, making it a promising tool for POCT to detect other disease-related genes and SNPs, among other applications.By leveraging the advantages of this platform, it holds the potential for rapid and on-site detection of a wide range of disease-related genetic variations, providing valuable insights for clinical diagnosis and treatment decisions.

Bacteria collection and genomic DNA extraction
B. pseudomallei strain HNBP001, referred to as Bp, was isolated from our laboratory (GenBank accession numbers CP038805 and CP038806).Thirty B. pseudomallei isolates, including 4 strains containing P174L mutation [referred to as Bp (P174L)], and 2 B. cepacia isolates (Bc) were obtained from clinical samples of patients admitted to the Second Affiliated Hospital of Hainan Medical University.Reference strains, including Enterococ cus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, used for specificity testing were obtained from ATCC (Table 1).Unless specified otherwise, bacteria were incubated at 37°C with oxygen in an incubator or shaken at 220 rpm in a shaker.Routine cultures were grown in Luria-Betani (LB) liquid or solid medium.Bacteria for drug susceptibility testing were grown in Mueller-Hinton (MH) liquid medium.All procedures involving live pathogens were conducted in a Biosafety Level 2 facility at the School of Tropical Medicine, Hainan Medical University.For rapid extraction of whole bacterial genomic DNA, the bacterial genome extraction kit (TIANGEN, China) was used following the heat lysis method instructions.The resulting supernatant was collected, and the DNA concentration was determined using a NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific, USA).Adjust the genomic DNA (gDNA) to the required concentration according to the experimental requirements; 1 µL supernatant was served as template for the latter PCR assays and sequencing.

Determination of the growth curves and antibiotic drug susceptibility
Freshly grown single colonies were selected and inoculated into 5 mL of LB liquid medium until reaching the log phase.The bacterial solution was then diluted to OD600 = 0.05.Two hundred microliters of bacterial solution was transferred to a 96-well plate and incubated at 37°C, 180 rpm for 24 hours, with the absorbance at OD 600 being measured continuously at 1-hour intervals.Each well should be repeated three times.These procedures were performed using aseptic techniques and were repeated independently on different dates for a total of three replicates.Strains requiring drug susceptibility testing in LB fresh media were grown in a 37°C incubator until the OD600 = 0.5.The Kirby-Bauer (K-B) test and microbroth dilution susceptibility test were performed according to the experimental method of each kit instruction.
Briefly, for the K-B test, use a sterile cotton swab to dip in bacterial liquid and squeeze out the excess bacterial fluid.Apply the entire MH surface with swab to ensure that the bacterial fluid is evenly applied.Use sterile tweezers to paste the drug sensitivity paper on the surface of the plate after the water was absorbed.The whole process should be completed within 15 minutes after bacterial inoculation.Then, the plate was reversed and inoculated at 37°C for 18-24 hours and the coil diameter was measured with a vernier caliper.
Microbroth dilution susceptibility test was performed according to the instruction with Zhuhai Deer non-fermentation bacteria drug susceptibility testing system (DL Biotech, China).Pure culture individual colonies were picked into 5 mL liquid LB and grown to log phase and adjusted to OD 600 = 0.1 with MH broth medium.Fifty milliliters of the above bacterial suspension was added to the matching MH broth medium tube; after completely mixing, 100 µL was added to each well of a 96-well plate corresponding to different antibiotics.In the process of dropping, pay attention to the suspension or replace the gun head in time to prevent cross-contamination between antibiotics.The reagent board was sealed and incubated at 37°C for 18-24 hours.The results and analysis reports were read using the microbial identification susceptibility analysis system.

Primers, probe designing, and screening
To enable the simultaneous detection of the conserved sequence of B. pseudomallei and the P174L mutation site in penA, two pairs of primers were designed using the NCBI Primer-BLAST website, based on the whole genome of B. pseudomallei strain HNBP001 available in the NCBI database (GenBank accession numbers CP038805 and CP038806).
The primer sequences are listed in Table 2.The product lengths were set in the range of 100 to 200 bp for rhPCR and real-time PCR assays and 400 to 500 bp for sequencing purposes.For LFSA biosensing strategy, primers used for ORF2 region analysis were labeled with biotin and FITC, while the primers used for the P174L mutation site analysis were labeled with digoxin and biotin.All sequences were evaluated using the NUPACK software tool (https://www.nupack.org/) to predicted the likelihood of secondary structure.Each rhPCR mixture consisted of 1 µL of extracted DNA (10 ng) in a total volume of 19 µL, including 2 µL of 10 × ThermoPol Reaction Buffer, 0.5U of Deep Vent (exo-) DNA Polymerase, 400 nM primers in total (with a ratio of blocking primers to universal primer probes of 1:10), 200 µM dNTP Solution, and 100 mU of RNase-HII.For quantitative fluorescence PCR, an additional 1 µL of 20 × EvaGreen was added for amplification and melt curve analysis.PCR was performed with the following parameters: an initial denaturation at 95°C for 5 minutes, followed by 45 cycles of 30 sec at 95°C and 45 sec at 65°C, with fluorescence signal collection at 65°C.For melt curve analysis, an additional warming process from 60°C to 95°C at a rate of 0.5°C per cycle was included, and the fluorescence was collected during this process.No-template controls were included in each run to account for contamination or potential amplifica tion failures.

Lateral flow strip assay
The experiment was performed according to the instructions provided in double-labeled nucleic acid test strip kit from Genenode (Wuhan, China).Briefly, 10 µL of PCRamplified products was absorbed into a new reaction tube and then diluted 10-20 times with the sample solution and fully mixed before testing.Next, 100 µL of diluted amplification products was dropwise added into the colloid gold detection hole.The test results were observed visually by examining the color development of the test strip within 3-10 minutes after the control line (C line) had developed color.The color intensity or presence of a line in the test region provided the basis for interpretation of the results.To document the results, the strip was photographed using a smartphone.After usage, the test strip should be placed in a sealed bag and disposed of appropriately.

Data analysis
In order to determine the suitability of the established method for large-scale applica tion, it is important to assess its accuracy, reproducibility, and stability.To evaluate the accuracy of detecting clinical samples, we tested 36 genomic gDNA samples and bacterial samples using the established method, and the results were compared with the gold standard obtained by sequencing.Each experiment was repeated three times to assess the stability of the experimental protocol.SPSS 20.0 statistical software was used to conduct the analyses.Pairwise comparisons were performed using the t-test and more than three comparisons using one-way ANOVA.P < 0.05 indicates a significant difference.

Ceftazidime-resistant strain identification
In this study, we conducted penA sequencing on a clinically isolated strain of B. pseudomallei that showed resistance to CAZ.The sequencing results revealed a C > T mutation in the conserved sequence of the penA, leading to an amino acid change from proline to leucine at position 174 (P174L), as depicted in Fig. 1A.This result was also consistent with the previous study which showed the same mutation type caused increased resistance to CAZ in B. thailandensis E264 strain reported by Yi, et al. (19).To investigate the impact of the P174L mutation on bacterial growth, we compared the growth curves of the resistant strain Bp(P174L) with the wild-type strain Bp.Turbidity measurements of the growth curves revealed no significant difference in the growth rate between the resistant and wild-type strains, as illustrated in Fig. 1B.Furthermore, we compared the CAZ drug susceptibility between Bp and Bp (P174L) using the K-B test and microbroth dilution susceptibility test.As shown in Fig. 1C and D, the CAZ-sensitive strain Bp exhibited a minimum inhibitory concentration (MIC) of 2 µg/mL, whereas the MIC of CAZ for Bp (P174L) increased to 256 µg/mL, indicating a 128-fold increase in resistance.Notably, this resistance phenotype remained stable during continuous passage culture.These findings demonstrate that the identified P174L mutation in the penA is associated with increased resistance to CAZ in B. pseudomallei, impacting bacterial growth and rendering the strain highly resistant to the antibiotic.

Optimization of the rhPCR system
To optimize the template reaction concentration, a PCR reaction system was established using normal primers (unblocking primers) with the addition of the fluorescent dye EvaGreen.The PCR amplification curve and melt curve analyses were used to evaluate the performance.It was found that a template concentration of 10 ng yielded better Ct values and resulted in a single specific amplification product without any nonspecific amplification (Fig. 3A and B; Fig. S1A).Similarly, the optimal amount of RNase-HII enzyme was determined using specific RNase-HII-dependent primers (blocking primers).The reaction performance was evaluated with varying amounts of RNase-HII, and it was observed that the best results were obtained when 100 mU of RNase-HII was added to the 20 µL reaction system (Fig. 3C and D; Fig. S1B).To determine the optimal annealing temperature for the rhPCR system, different annealing/extension temperatures (63°C, 65°C, and 67°C) were evaluated.The detailed RT-PCR data are presented in Fig. S2.
Based on the results, it was determined that the system exhibited optimal amplification efficiency at an annealing temperature of 65°C.Therefore, this temperature was selected for the subsequent reactions.These optimization steps ensured that the PCR reactions using rhPCR with the designed primers and RNase-HII enzyme were performed under the most favorable conditions, maximizing amplification efficiency and specificity.

Feasibility of the rhPCR-LFSA system
To evaluate the effectiveness of blocking primers in identifying mutant B. pseudomallei, PCR assays were performed using the P174L mutation as an example.It was observed that only the blocking primers produced specific PCR products in the Bp (P174L) group, while the wild-type Bp and blank control groups showed no amplification (Fig. 4A).In contrast, when using regular primers, amplification products were obtained in both the Bp and Bp (P174L) groups, making it difficult to identify the mutation site.Addi tionally, the control group showed nonspecific products, leading to interference in detection.To further analyze the amplification products, a single tube containing the two reactions (ORF2 and P174L) was prepared and universal primers were added.The resulting complex nucleic acid environment was subjected to 12% native PAGE analysis.It was found that the use of unmodified primers produced multiple amplified species, including "primer dimer" products, which were independent of the input target and deviated from the expected amplicon sizes of 189 bp and 155 bp.In contrast, amplifica tion using blocking cleavable primers was dependent on the presence of RNase-HII.The rhPCR format using cleavable primers produced the correct amplicon sizes (189 bp and 155 bp) when the Bp (P174L) target was present and a 155-bp amplicon when the Bp target was present.No amplified products were observed when the target was absent.The use of cleavable primers in the rhPCR system eliminated the formation of primer-dimer artifacts, resulting in more specific and accurate amplification (Fig. 4B).
After PCR amplification, the products were subjected to LFSA.The LFSA results, as well as the typical sequencing results for the genotypes of Bp, Bp (P174L), and negative controls, are shown in Fig. 4 (C and D).LFSA was found to be a simple and easy-to-operate method that did not require expensive instruments, making it suitable for rapid and convenient detection.These results demonstrated the effectiveness of the rhPCR system combined with LFSA for the simultaneous detection of B. pseudomallei and the P174L mutation, highlighting its potential as a reliable and accessible tool for diagnostic applications.

Clinical application of the rhPCR-LFSA system
To evaluate the clinical application of the system, a total of 32 culture isolates derived from hospitalized patients (30 B. pseudomallei isolates and 2 B. cepacia isolates were contained, of which 4 B. pseudomallei isolates carried P174L mutation) and four standard strains from ATCC were analyzed.The results showed that all clinically verified B. pseudomallei strains were detected and showed a positive signal on the T1 line of the LFSA strip.In contrast, B. cepacia strains and the other four reference strains from ATCC only showed bands in the control lines, with negative results in the test lines.Additionally, four strains were positive for the T2 test line, indicating the presence of the P174L mutation in these samples (Fig. 5A).To confirm the accuracy of the method, the P174L locus in these four strains was confirmed by sequencing.The sequencing results showed perfect agreement with the LFSA results, with a coincidence rate of 100% (Fig. 5B).Furthermore, a population genomic analysis of B. pseudomallei in Hainan Island, China, revealed a specific transition from A to G at position 457 nucleotide of penA (T147A) in approximately 60% of the Hainan B. pseudomallei genome (Fig. 5C) (17).To test the universality and transferability of the protocol, specific blocking primers targeting the T147A site carrying the universal probe 1 sequence were designed.The LFSA of the 30 B. pseudomallei isolates was performed after amplification using these primers.The results demonstrated the excellent universality of the protocol, enabling the simultaneous detection of two mutation sites in a single tube, with a coincidence rate of 100% compared with the sequencing results (Fig. 5D; Table 3; Fig. S4).These findings highlight the robustness and reliability of the developed system in clinical settings, providing a promising approach for the rapid and accurate detection of B. pseudomallei and its associated drug resistance mutations.

DISCUSSION
Melioidosis, a severe infectious disease caused by B. pseudomallei, remains a global concern due to its high morbidity and mortality rates.While the disease was initially identified in 1912, its significance surged following the Vietnam War when melioidosis gained attention due to its substantial impact on the US military (4).The disease's endemicity in northern Australia and Southeast Asia, along with sporadic cases in North America, has highlighted the challenges in its diagnosis and management, compounded by its diverse clinical manifestations and high antibiotic resistance (5,(33)(34)(35)(36).The rising incidence in China, particularly since the initiation of the National Health Surveillance Program, underscores the need for improved diagnostics and treatment strategies, especially in the face of emerging drug resistance.In this study, we focused on addressing the diagnostic challenges of melioidosis and the emergence of drug resistance.The current gold standard relies on bacterial cultures, which can be time-consuming.Molecular techniques, such as PCR targeting specific genes like the type III secretion system (TTS1) gene cluster, have shown promise in identifying B. pseudomallei strains.Notably, the resistance of B. pseudomallei strains to key antibiotics like CAZ, a cornerstone of treatment, is becoming a concerning issue.Our study delves into a fatal case involving a penA alteration resulting in high CAZ resistance.Through whole-genome sequencing (WGS) analysis, we identified a P174L substitution in penA associated with a significant increase in minimum inhibitory concentration.This highlights the critical need for a rapid diagnostic method capable of identifying both the presence of B. pseudomallei and specific drug resistance mutations to inform targeted treatment decisions.
Our contribution to this challenge is the development of a PCR protocol utiliz ing thermostable RNase-HII enzymes.This protocol aims to expedite B. pseudomallei identification and improve diagnostic reliability, especially in endemic regions.By incorporating the identification of the P174L mutation, associated with CAZ resistance, we enhance the diagnostic accuracy of our system.Specificity is achieved through blocking primers' design and the cleavage activity of thermostable RNase-HII enzymes.The compatibility of these enzymes with DNA polymerase and their reduced cleavage efficiency in the presence of mismatches near RNA residues allow for multiplex PCR and mutation detection.
Importantly, in resource-limited settings where technical expertise and equipment are scarce, our PCR protocol integrated with lateral flow strip assay presents a practical solution.LFSA, utilizing colloidal gold particles and paper chromatography, offers visual detection and simplicity, making it suitable for point-of-care testing (POCT) (37,38).We incorporated LFSA into our PCR system, introducing universal detection probes consistent with blocking primer sequences.These probes, labeled for LFSA detection, generate red bands on the strip upon flow, enabling visual identification without the need for expensive equipment.This user-friendly approach is well suited for primary healthcare settings.
Our study showcases successful detection of T147A and P174L sites in B. pseudo mallei strains using LFSA, with results matching sequencing outcomes.In summary, the integration of LFSA into our PCR protocol presents a promising avenue for improved diagnosis and management of melioidosis in resource-constrained regions.This approach's simplicity, speed (2.5-3 hours), and adaptability for various strains and drug resistance sites hold significant potential.However, we acknowledge the current need for DNA extraction, a limitation we aim to address in future studies to enhance the protocol's accuracy.Moreover, expanding the sample size will contribute to assessing the protocol's specificity and sensitivity more comprehensively.

FIG 1
FIG 1 Characterization of CAZ-resistant Bp strain.Location of mutations in penA gene in the CAZ-resistant strain Bp (P174L) (A).Comparison of growth curves between Bp and Bp (P174L) (B).Determination of the antibiotic MIC between Bp and Bp (P174L) by K-B test (C) and microbroth dilution susceptibility test (D).

FIG 4
FIG4 Feasibility analysis of the rhPCR-lateral flow system.Amplification curves in common (A, left) and blocked primer (A, right) reaction systems using different templates.Twelve percent native PAGE analysis of amplified products in complex nucleic acid environments under multiplex reaction conditions (B).U represents the unmodified common primers and B represents the blocked primers carrying RNA bases and C3-spacer group.LFSA results (C) and typical sequencing results (D) for the genotypes of Bp, Bp (P174L), and negative controls.

TABLE 1
Strain information involved in this protocol a No subsequent information was traced.

TABLE 2
Nucleotide sequences of primers and probes