Background Candida species cause superficial, deep, and systemic infections, and non-Candida albicans have become a pathogenic species, affecting individuals with normal immunity or immunosuppression. Candida haemulonii complex is a rare pathogen. The complex was reclassified into three species, (Candida haemulonii sense stricto (C. haemulonii sense stricto) and Candida duobushaemulonii (C. duobushaemulonii)) and a variety (Candida haemulonii var. vulnera (C. haemulonii var. vulnera)). Case presentation A 78-year-old woman presented with a diabetic foot ulcer due to poor diabetes self-management for over 20 years. The strain was identified as C. duobushaemulonii in the hospital laboratory using a right foot trauma secretion culture, and drug sensitivity testing showed sensitivity to 5-flucytosine and resistance to amphotericin B. Symptomatic treatment, wound cleaning, and dressing changes were administered; systemic anti-infective treatment was administered when necessary. After treatment, the yellow discharge from the patient's second, third, and fourth toes along with the dorsum of the right foot and the lower third segment of the lower extremity of the embryonic bone in front of the skin wound was significantly reduced. Blood tests suggested that the indicators improved, and nutritional support was continued. We proposed surgical treatment; however, the patient and her family members took the initiative to request discharge and were advised to continue the treatment outside the hospital. Conclusion Early detection of fungal infections and the initiation of appropriate treatment for diabetic foot ulcers may lead to better healing and amputation avoidance. Therefore, timely and accurate etiological diagnosis is particularly important for the treatment and prognosis of diabetic foot infections.

Severe Fever with Thrombocytopenia Syndrome (SFTS), caused by SFTS virus (SFTSV), is a widely distributed infection with significant mortality. Diagnosis in resource-limited settings remains challenging. For rapid and convenient diagnosis, we developed a portable rapid diagnosis method that combines Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a technology with immunochromatographic test strips. The SFTSV RNA was amplified by reverse transcription loop-mediated isothermal amplification (RT-LAMP). The homologous target sequence and single stranded DNA (ssDNA) reporter gene were cleaved by CRISPR/Cas12a in parallel, and ssDNA probes labeled with FAM fluorescein and biotin were captured by an immunochromatographic strip. Finally, the signal on the immunochromatographic strips became visible to the naked eye. Based on CRISPR/Cas12a, a rapid SFTSV detection method was developed, featuring simplicity, rapidity, low cost, and ease of use. The method was applied for the nucleic acid detection of SFTSV in 40 clinical serum samples and compared with RT-polymerase chain reaction (PCR). The new method showed 100% sensitivity and 100% specificity with a detection agreement rate of 100%. The minimum detection limit of the method was 2.5 copies/μL, and no cross-reactivity with nucleic acids from other common pathogens was observed. Detection can be completed within 80 min, and results are observable with the naked eye. For the analysis of clinical samples, the method exhibits good detection performance and thus provides an attractive option for the nucleic acid detection of SFTSV in point-of-care and resource-limited medical settings.

IntroductionSevere fever with thrombocytopenia syndrome virus (SFTSV), a novel bunyavirus associated with febrile illness, thrombocytopenia, and leukopenia, represents a considerable public health threat.MethodsIn this study, we developed a visual detection platform for SFTSV by integrating reverse transcription-recombinase polymerase amplification (RT-RPA) with lateral flow nucleic acid chromatography assay (LFNA). The method operates under isothermal conditions, and delivers results within 40 minutes. Using a conserved region of the S segment as the amplification target.ResultsThe assay demonstrated a detection limit of 101 copies/μL and showed no cross-reactivity with 12 other common clinical pathogens or 6 less common clinical pathogens. An evaluation of 80 SFTSV-positive clinical samples and 16 SFTSV-negative clinical samples showed that, compared with quantitative PCR (qPCR), the method demonstrated excellent sensitivity and specificity.DiscussionThe RT-RPA-LFNA platform is easy to operate, provides rapid results and is cost-effective, and therefore has potential for use in point-of-care testing (POCT), particularly in resource-limited primary healthcare settings. This approach not only provides a reliable tool for SFTSV diagnosis but also holds potential for the detection of other pathogens.

Background and aimsSevere fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease characterized by high mortality and rapid clinical progression. This longitudinal study aimed to investigate the temporal changes in laboratory parameters and evaluate the prognostic value of hematology-derived systemic inflammatory indices in patients with SFTS.MethodsAltogether, 235 hospitalized patients with SFTS were prospectively enrolled between 2022 and 2024. Demographic, clinical, treatment, and outcome data were collected and reassessed. Hematological, liver, and kidney function parameters, and myocardial parameters were routinely measured on days 0, 3, 7, and 10 after admission. Time-points stepwise Cox proportional hazards regression and receiver operating characteristic (ROC) curve analyses were performed at each time point to evaluate predictive performance.ResultsOf the 235 patients, 61 (26.0%) died of SFTS. The median age was 64.0 (IQR 54.0-71.0) years, and 93 (39.6%) were male. After adjusting for confounding factors, serum creatinine (HR 1.017 [95% CI 1.006-1.029], p = 0.003), urea (HR 1.102 [95% CI 1.047-1.161], p < 0.001), platelet counts (HR 0.987 [95% CI 0.977-0.997], p = 0.010), neutrophil-to-lymphocyte-platelet ratio (NLPR) (HR 1.021 [95% CI 1.007-1.034], p = 0.002), and neutrophil percentage-to-albumin ratio (NPAR) (HR 1.689 [95% CI 1.028-2.776], p = 0.039) were identified as independently associated with prognosis at each time point. Among them, NLPR presented consistently high predictive performance, with an area under the curve (AUC) of 0.802 (95% CI 0.736-0.868) on day 0, 0.833 (95% CI 0.774-0.892) on day 3, 0.861 (95% CI 0.784-0.938) on day 7, and 0.840 (95% CI 0.706-0.975) on day 10. Patients with high NLPR (> 3.76) had significantly shorter survival times (HR 6.584 [95% CI 3.840-11.290], p < 0.001 by the log-rank test). In addition, albumin treatment was associated with a reduced risk of mortality in the high-NLPR group (HR 0.423 [95% CI 0.195-0.919], p = 0.030).ConclusionsNLPR may serve as a simple and effective prognostic marker for SFTS. Albumin treatment may be associated with a lower risk of mortality in patients with a high NLPR.

This study aims to establish a platform and to achieve rapid, simultaneous detection of Helicobacter pylori along with its key virulence genes, cytotoxin-associated gene A (cagA) and vacuolating cytotoxin A (vacA). We developed a multiplex recombinase polymerase amplification combined with lateral flow strip (mRPA-LFS) assay for the rapid, simultaneous detection of H. pylori (targeting the 16 S rRNA gene) and its key virulence genes, cagA and vacA. The assay employs a nucleic acid tag (NAT)-bridging strategy for multiplex visualization without instrumentation. Performance was evaluated using recombinant plasmids and clinical saliva samples (n = 28), with 16 S rRNA detection compared against the urea breath test and genotyping results against quantitative PCR (qPCR). The mRPA-LFS assay completes detection within 30 min at a constant temperature of 39 °C. It demonstrated high sensitivity with limits of detection of 10¹, 10³, and 10² copies/µL for the 16 S rRNA, cagA, and vacA genes, respectively, and showed no cross-reactivity with nine common non-H. pylori pathogens. The detection results of the mRPA-LFS for H. pylori were highly consistent with those of qPCR (complete agreement for 16 S rRNA and cagA genes; sensitivity of 94.4

BACKGROUND:The adaptive evolution of Klebsiella pneumoniae carbapenemase (KPC) is the primary mechanism mediating the development of ceftazidime-avibactam (CZA) resistance, and the continuous emergence of novel KPC variants has posed a critical challenge to contemporary CZA-based therapeutic strategies. OBJECTIVES:This study aimed to investigate the mechanisms underlying the development of CZA resistance in an ST11-KL64 K. pneumoniae strain Kpn_54H carrying the blaKPC-176 gene. METHODS:Whole-genomic sequencing (WGS), gene cloning, antimicrobial susceptibility testing (AST), agar well diffusion assay, molecular docking, and molecular dynamics simulations were conducted to explore CZA resistance mechanisms. Conjugation experiments, plasmid stability testing, and growth assays were performed to assess fitness costs. RESULTS:The carbapenem-resistant Klebsiella pneumoniae (CRKP) strain Kpn_54H (ST11-KL64) harbours KPC-176, a variant derived from a T508C mutation in blaKPC-2, resulting in an S171P substitution within the its Omega loop (residues 164-179). CZA resistance emerged post-drug exposure, and the variant reverted to wild-type KPC-2 after drug withdrawal. Molecular dynamics simulations indicated this mutation may enhance hydrogen bonds and residue rigidity during ceftazidime (CAZ) binding, increasing enzyme affinity for CAZ while reducing avibactam (AVI) affinity. Consequently, KPC-176 exhibits markedly elevated CAZ hydrolysis. blaKPC-176 is embedded in a core △ISKpn6-blaKPC-176-ISKpn27 structure, flanked by IS26 elements, and resides on a horizontally transmissible IncFII (pHN7A8) plasmid. KPC-176 conferred lower competitive fitness, but enhanced tolerance to sub-inhibitory CZA. CONCLUSIONS:KPC-176 mediates K. pneumoniae resistance to CZA by enhancing ceftazidime affinity while impairing avibactam inhibition. It poses a significant transmission threat due to the conjugative pKpn_54H-KPC plasmid and IS26 flanked genetic environment.

Vaginitis frequently poses serious threats to women's health. If left untreated, it can increase the risk of infection by other pathogens such as HPV, leading to cervical dysplasia. This study developed a rapid detection method based on multiplex recombinase polymerase amplification (mRPA) combined with lateral flow strips (LFS) for the simultaneous identification of five common vaginitis pathogens: Neisseria gonorrhoeae, Gardnerella vaginalis, Candida albicans, Ureaplasma urealyticum, and Trichomonas vaginalis. The novelty of this technology lies in the fact that, the 5' end of the RPA primer was modified by a nucleic acid tagged (NAT) sequence to complementarily pair with the LFS capture probe. The entire testing process can be completed within 50-55 min and provides visual results. Furthermore, the lowest detection limits of the platform for Neisseria gonorrhoeae, Gardnerella vaginalis, Candida albicans, Ureaplasma urealyticum, and Trichomonas vaginalis respectively were 1.5 × 10 CFU/mL, 1.5 × 102 CFU/mL, 1.5 × 10 CFU/mL, 1.35 × 102 copies/μL, and 1.02 × 102 copies/μL, and there was no nucleic acid cross-reactivity with other pathogens. Clinical validation using 122 samples showed a sensitivity of 97.1%, specificity of 90%, and accuracy of 95.9% compared to conventional culture and microscopy. This mRPA-LFS platform is rapid, specific, and sensitive, showing promise as an effective point-of-care testing (POCT) tool.

Since Candida albicans, a type of fungus, causes severe infections that pose a significant threat to human health, its rapid detection is critical in clinical antifungal therapy. Traditional fungal diagnostic approaches are largely based on the culture method. This method is time-consuming and laborious, taking about 48-72 h, and cannot identify emerging species, making it unsuitable for critically ill patients with bloodstream infections, sepsis, and so on. Other antigen or nucleic acid amplification-based methods were also found to be unsuitable for Point-ofCare Testing (POCT) diagnosis due to various limitations. Therefore, establishing a new approach for the rapid diagnosis of Candida spp is imperative. Herein, we proposed a novel diagnostic method for invasive fungi detection. Specifically, we created a new CRISPR diagnostic platform for Candida albicans-specific Internal Transcriptional Spacer 2 (ITS2) gene by combining the DNase cleavage activity of Cas12a with Recombinase Polymerase Amplification (RPA). Furthermore, to achieve rapid on-site detection under low-resource conditions, we used a transverse lateral flow strip with a single target to visualize the Cas12a single enzyme digestion product. We designated the platform as a rapid molecular detection tool that integrates RPA and the CRISPRCas12a technology. The entire platform can accurately identify Candida albicans within 50 minwhile remaining unaffected by other fungi or bacteria. Furthermore, the detection limit of the platform could reach 102 CFU/ ml. Moreover, this approach offers additional benefits, including easy operation, low set-up cost, and broad applicability for Candida albicans detection across medical institutions at all levels, especially in township health centers in resource-poor regions.

Aim:To identify the causative bacteria of healthcare-associated sepsis in preterm infants and analyze their antibiotic resistance trends over ten years, providing evidence for infection prevention strategies. Materials and Methods:We retrospectively analyzed blood culture data from preterm infants (<37 weeks) with healthcare-associated sepsis (onset >72 hours after birth) admitted between January 2014 and December 2023. Pathogen distribution and antibiotic resistance patterns were compared between two periods (2014-2018 vs 2019-2023). Results:Among 9928 preterm infants, 3.3% (332 cases) had positive blood cultures, with incidence increasing from 1.4% (2014-2018) to 2.7% (2019-2023). Gram-negative bacteria remained predominant (48.00% to 61.07%), led by Klebsiella pneumoniae. Gram-positive bacteria increased significantly (5.33% to 31.30%), primarily coagulase-negative staphylococci, while fungal infections decreased (46.67% to 7.63%). Resistance to third-generation cephalosporins persisted in K. pneumoniae (~80%) and increased in Enterobacter cloacae (60% to 90%). Emerging carbapenem resistance was observed in E. coli (0% to 33.33%) and K. pneumoniae (5.25% to 4.08%), with Enterobacter cloacae showing a significant rise (0% to 60%). ESBL-producing strains rose from 13.33% to 30.53%. All Gram-positive isolates remained susceptible to linezolid, except one vancomycin-resistant Staphylococcus capsulatus. Conclusion:The incidence of healthcare-associated sepsis in preterm infants increased significantly, with rising carbapenem resistance in Gram-negative bacteria and a marked increase in coagulase-negative staphylococci. These trends underscore the need for enhanced infection control and judicious antibiotic use guided by blood culture results.

Whooping cough (pertussis) is an acute respiratory infectious disease caused by Bordetella pertussis (BP). It poses a risk to infants and young children. This investigation aimed to construct a simple, rapid, and accurate diagnostic protocol for BP detection that does not depend on complex equipment or large-scale instruments. This study combines Recombinase Polymerase Amplification (RPA) technology with the CRISPR/Cas12a system, utilizing immunochromatographic lateral flow strips (ILFS) test and fluorescence curves to observe data. This diagnostic strategy does not require complex equipment used in traditional diagnostic approaches (such as bacterial culture, pathogen detection, and molecular biology techniques), which has increased its accessibility and ease of use. The validation data indicate that the RPA-CRISPR/Cas12a-ILFS and RPA-CRISPR/Cas12a fluorescence detection analyses had a lower detection threshold of 102 copies/µL and did not cross-react with other prevalent infections. Furthermore, 40 clinical samples were evaluated and compared via qPCR, which revealed that the RPA-CRISPR/Cas12a method has 100

IntroductionZinc exhibits potent antimicrobial properties due to its ability to compromise bacterial structure and protein functionality, effectively inhibiting and eradicating bacteria. However, bacteria have evolved mechanisms to expel excess zinc ions from their cells, enabling them to thrive in environments rich in metal ions at high concentrations. This evolutionary advancement limits the clinical application of metal ions as antimicrobial agents. In this study, we aimed to investigate the potential of zinc ionophores to overcome bacterial resistance by elevating intracellular zinc ion levels.MethodsWe employed the zinc ionophore PBT2 to elevate intracellular zinc ion levels in Klebsiella pneumoniae, a bacterium known for its resistance to various antibiotics. By treating K. pneumoniae with PBT2, we aimed to assess its impact on bacterial resistance to tigecycline, an antibiotic commonly used in clinical settings. The changes in intracellular zinc ion levels, superoxide dismutase activity, reactive oxygen species concentration, and cell wall synthesis pathway were monitored to evaluate the mechanism of action of PBT2.ResultsOur results revealed that PBT2 successfully reversed the resistance of K. pneumoniae to tigecycline. Specifically, PBT2 increased the concentration of intracellular zinc ions in K. pneumoniae, leading to a suppression of superoxide dismutase activity within the cell and an elevation of reactive oxygen species concentration. These changes impaired the oxidative stress response of the bacteria. Additionally, the disruption of zinc homeostasis significantly inhibited the cell wall synthesis pathway in K. pneumoniae, potentially restricting the efflux pump mechanism that predominantly drives tigecycline resistance.DiscussionThe findings of this study pave the way for innovative strategies and approaches in the clinical development of novel antimicrobial agents. By using zinc ionophores such as PBT2 to elevate intracellular zinc ion levels, we can overcome bacterial resistance to antibiotics like tigecycline. The suppression of superoxide dismutase activity and elevation of reactive oxygen species concentration suggest that PBT2 impairs the oxidative stress response of K. pneumoniae, further enhancing its susceptibility to antibiotics. Furthermore, the inhibition of the cell wall synthesis pathway and restriction of the efflux pump mechanism provide additional mechanisms by which PBT2 reverses antibiotic resistance. These results highlight the potential of zinc ionophores as a novel class of antimicrobial agents and warrant further investigation into their clinical applications.

Currently, the molecular mechanisms of azole resistance in C. glabrata are unresolved. This study aims to detect azole resistance of C. glabrata after exposure to fluconazole (Diflucan) in vitro. After 50 days of induction, the five susceptible isolates of C. glabrata demonstrated cross-resistance to azoles (fluconazole (Diflucan), voriconazole and itraconazole). Mutations in PDR1 or ERG11 genes are key nodes in azole resistance of C. glabrata. DNA-Sequencing revealed three(3/5) fluconazole (Diflucan)-resistant isolates had undergone missense mutations (R376Q, R772K, E1083K in PDR1 and F135L in ERG11), all of which were newly discovered and previously unreported. mRNA expression of resistant genes in five resistant isolated was elevated, with CDR1 being the most prominent. Analysis using flow cytometry revealed that resistant strains showed decreased R6G uptake and increased efflux efficiency, but no obvious significance difference in biofilm production. C. glabrata acquires azole cross-resistance upon continuous exposed to fluconazole (Diflucan) and could remain resistant without antifungal agents. The development of azole resistance in C. glabrata has been linked to genes associated with efflux pump transporters and the ergosterol synthesis pathway. However, the relationship between resistance and newly discovered missense mutation sites requires further investigation.

Vancomycin-resistant Enterococcus (VRE) has demonstrated increasing global prevalence in recent years. Clinical detection currently relies on phenotypic methods including agar screening, minimum inhibitory concentration (MIC) testing, Kirby-Bauer disk diffusion, and Etest. In addition, molecular approaches such as polymerase chain reaction (PCR) and quantitative PCR (qPCR) can be applied for VRE identification. Nevertheless, these methods cannot achieve point-of-care detection (POCT). Thus, novel rapid diagnostic platforms have become urgently needed for curbing VRE transmission and containing nosocomial outbreaks. Recombinase polymerase amplification (RPA) and lateral flow strips (LFS) are effective tools for achieving rapid POCT. In this study, RPA was combined with LFS to establish a fast, sensitive, and specific detection method. This study established a multiplex RPA-LFS (mRPA-LFS) that delivers results within 30-40 min, with detection limits of 102 copies/μl for vanA, vanB, and vanM. Notably, the assay demonstrated high specificity without cross-reactivity to common bacterial/fungal pathogens, and showed 100% concordance with conventional PCR in 30 clinical samples. In this study, a rapid detection assay for vanA, vanB, and vanM genes in VRE was developed using mRPA-LFS technology. Characterized by high sensitivity, specificity, operational simplicity, and cost-effectiveness, this method is suitable for on-site detection.

Carbapenem-resistant Enterobacterales (CRE) infections are posing a critical global health threat as their prevalence, multidrug resistance, and mortality rates are all increasing.To address this challenge, we developed a multiplex recombinase polymerase amplification (mRPA) coupled with lateral flow immunoassay strip (LFS) to simultaneously detect five carbapenemase genes (blaKPC, blaNDM, blaOXA-48, blaVIM, and blaIMP) via molecular hybridization. This platform enables rapid, minimal equipments across diverse clinical and community settings.Validation demonstrated no cross-reactivity with non-target clinical strains and a detection limit of 102 CFU/mL (n = 66 clinical samples). Compared to qPCR, the assay achieved 98.2 % sensitivity, 100 % specificity, and 98.5 % concordance. The platform's portability and minimal technical requirements make it suitable for decentralized healthcare settings, including primary hospitals and point-of-care facilities.This multiplex, field-deployable assay provides a critical tool for timely clinical decision-making and precision antimicrobial stewardship in CRE management, particularly in resource-limited regions where conventional molecular diagnostics remain inaccessible.

Mycoplasma pneumoniae can cause respiratory infections and pneumonia, posing a serious threat to the health of children and adolescents. Early diagnosis of Mycoplasma pneumoniae infection is crucial for clinical treatment. Currently, diagnostic methods for Mycoplasma pneumoniae infection include pathogen detection, molecular biology techniques, and bacterial culture, all of which have certain limitations. Here, we developed a rapid, simple, and accurate detection method for Mycoplasma pneumoniae that does not rely on large equipment or complex operations. This technology combines the CRISPR-Cas12a system with recombinase polymerase amplification (RPA), allowing the detection results to be observed through fluorescence curves and immunochromatographic lateral flow strips.It has been validated that RPA-CRISPR/Cas12a fluorescence analysis and RPA-CRISPR/Cas12-immunochromatographic exhibit no cross-reactivity with other common pathogens, and The established detection limit was ascertained to be as low as 102 copies/µL.Additionally, 49 clinical samples were tested and compared with fluorescence quantitative polymerase chain reaction, demonstrating a sensitivity and specificity of 100%. This platform exhibits promising clinical performance and holds significant potential for clinical application, particularly in settings with limited resources, such as clinical care points or resource-constrained areas.

Norovirus is highly infectious and rapidly transmissible and represents a major pathogen of sporadic cases and outbreaks of acute gastroenteritis worldwide, causing a substantial disease burden. Recent years have witnessed a dramatic increase in norovirus outbreaks in China, significantly higher than in previous years, among which GII norovirus is the predominant prevalent strain. Therefore, rapid norovirus diagnosis is critical for clinical treatment and transmission control. Hence, we developed a molecular assay based on RPA combined with the CRISPER-CAS12a technique targeting the conserved region of the GII norovirus genome, the results of which could be displayed by fluorescence curves and immunochromatographic lateral-flow test strips. The reaction only required approximately 50 min, and the results were visible by the naked eye with a sensitivity reaching 102 copies/μl. Also, our method does not cross-react with other common pathogens that cause intestinal diarrhea. Furthermore, this assay was easy to perform and inexpensive, which could be widely applied for detecting norovirus in settings including medical institutions at all levels, particularly township health centers in low-resource areas.

BACKGROUND:Simple, rapid, and accurate diagnosis of tuberculous pleural effusion (TPE) remains challenging. This study aimed to determine the accuracy of IFN-γ in diagnosing TPE. METHODS:We quantified the expression of interferon-gamma (IFN-γ) in blood (B), adenosine deaminase (ADA), and IFN-γ in pleural effusions (PE) from 25 TPE patients and 31 non-TPE patients using a combination of immunological assays and flow cytometric analysis. The diagnostic performance of these three biomarkers was evaluated using receiver operating characteristic (ROC) curves. RESULTS:We found that IFN-γ levels in blood and pleural fluid were higher in the TPE group than in the non-TPE group. The mean concentration of IFN-γ in pleural fluid of the TPE group was 3140.90 (1817.94, 6611.05) pg/mL, while that of the non-TPE group was 4.91 (0.69, 8.6) pg/mL), and the difference was statistically significant (z = 6.39, P < 0.001). The mean blood IFN-γ was 40.19 (16.45, 59.08) pg/mL in the TPE group and 2.76 (1.96, 6.02) pg/mL in the non-TPE group, which was statistically different (z = 5.12, P < 0.001). The area under the ROC curve (AUC) for pleural fluid IFN-γ, blood IFN-γ, and ADA were 0.999 (95 % CI: 0.994-1.00), 0.901 (95 % CI: 0.798-1.00) and 0.996 (95 % CI: 0.987-1.00), respectively. CONCLUSION:This study confirms that IFN-γ has high diagnostic validity in patients with TPE and can potentially be an excellent biomarker.

Tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) is increasing and has emerged as a global public health issue. However, the mechanism of tigecycline resistance remains unclear. The objective of this study was to investigate the potential role of efflux pump system in tigecycline resistance. 29 tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains were collected and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The ramR, acrR, rpsJ, tet(A), and tet(X) were amplified by polymerase chain reaction (PCR). The mRNA expression of different efflux pump genes and regulator genes were analyzed by real-time PCR. Additionally, KP14 was selected for genome sequencing. KP14 genes without acrB, oqxB, and TetA were modified using suicide plasmids and MIC of tigecycline of KP14 with target genes knocked out was investigated. It was found that MIC of tigecycline of 20 out of the 29 TNSKP strains decreased by over four folds once combined with phenyl-arginine-β-naphthylamide dihydrochloride (PaβN). Most strains exhibited upregulation of AcrAB and oqxAB efflux pumps. The strains with acrB, oqxB, and tetA genes knocked out were constructed, wherein the MIC of tigecycline of KP14∆acrB and KP14∆tetA was observed to be 2 µg/mL (decreased by 16 folds), the MIC of tigecycline of KP14ΔacrBΔTetA was 0.25 µg/mL (decreased by 128 folds), but the MIC of tigecycline of KP14∆oqxB remained unchanged at 32 µg/mL. The majority of TNSKP strains demonstrated increased expression of AcrAB-TolC and oqxAB, while certain strains showed mutations in other genes associated with tigecycline resistance. In KP14, both overexpression of AcrAB-TolC and tet(A) gene mutation contributed to the mechanism of tigecycline resistance.

In this study, we devised a diagnostic platform harnessing a combination of recombinase polymerase amplification (RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. Notably, this platform obviates the need for intricate equipment and finds utility in diverse settings. Two result display methods were incorporated in this investigation: the RPA-Cas12a-fluorescence method and the RPA-Cas12a-LFS (lateral flow strip). Upon validation, both display platforms exhibited no instances of cross-reactivity, with seven additional types of fungal pathogens responsible for respiratory infections. The established detection limit was ascertained to be as low as 10 2 copies/µL. In comparison to fluorescence quantitative PCR, the platform demonstrated a sensitivity of 96.7%, a specificity of 100%, and a consistency rate of 98.0%.This platform provides expeditious, precise, and on-site detection capabilities, thereby rendering it a pivotal diagnostic instrument amenable for deployment in primary healthcare facilities and point-of-care settings.

The global proliferation of carbapenemase-producing bacteria (CPB) has garnered significant attention worldwide. Early diagnosis of CPB and accurate identification of carbapenemases are crucial for preventing the spread of CPB and ensuring targeted antibiotic therapy. Therefore, efficient and accurate identification of carbapenemases is paramount in clinically treating diseases associated with CPB. In this study, 58 CPB strains were collected and detected using the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) method, a rapid detection platform based on CRISPR-Cas12a gene editing and isothermal amplification. Additionally, four conventional methods (the APB/EDTA method, PCR, NG-test Carba 5, and GeneXpert Carba-R) were employed and compared against whole genome sequencing (WGS) results, considered the gold standard, to evaluate their efficacy in detecting carbapenemases. Detection by the APB/EDTA method revealed that 29 strains were positive for Class A serine endopeptidases, while 29 strains were positive for Class B metalloenzymes. The classification of these zymotypes was consistent with the sequencing result. All target carbapenemases for KPC were identified with 100% sensitivity using NG-test Carba 5, PCR, DETECTR, and GeneXpert Carba-R. In the case of NDM, both Xpert Carba-R and DETECTR showed a sensitivity of 100%. In contrast, NG-test Carba 5 and PCR had a slightly lower sensitivity of 96.7%, each missing one target carbapenemase. n this study, the APB/EDTA method is capable of identifying the zymotype classification but not the specific resistant genes, while Xpert Carba-R and DETECTR are able to detect all target carbapenemases.