Transcriptional and translational regulators ('switches') function as biosensors by controlling how gene circuits respond to specific stimuli. However, contemporary switches suffer from spontaneous activation or off-target responses, limiting sensitivity and specificity. Here we introduce base gap switches, a class of programmable transcriptional regulators that are selectively activated by DNA. Base gap switches are simple to design and install, provide tight control of gene expression and exhibit high specificity and orthogonality. They can discriminate single-nucleotide mismatches and integrate with nucleic acid amplification methods. Using base gap switches, we developed an assay for Trichomonas vaginalis with a limit of detection of 0.6 copies μl[-1] and validated its performance using clinical samples. We further demonstrate a one-pot assay for differentiating Neisseria gonorrhoeae and Chlamydia trachomatis, and an assay for detecting single-nucleotide variants of EGFR-associated cell-free DNA. These results establish base gap switches as a versatile tool for gene circuit-based biosensing.

Mosaic penA alleles encoding highly mutated variants of penicillin-binding protein 2 (PBP2) are the principal determinants of ceftriaxone resistance in Neisseria gonorrhoeae . Resistance-associated mutations in PBP2 from the ceftriaxone-resistant strain H041 restrict formation of the inward conformation of the β3-β4 loop associated with efficient acylation, but how β-lactam recognition is coupled to this conformational switch is unknown. Because the conserved active-site residue Tyr422 interacts with the R 1 substituent of β-lactams, we investigated its role in coupling ligand recognition and acylation activity. Mutation of Tyr422 to Ala lowered acylation rates by up to 120-fold for cefoperazone and piperacillin, whereas acylation rates of ceftriaxone increased 4-fold. Unexpectedly, the crystal structure of the Y422A mutant acylated by ceftriaxone revealed that the β3-β4 loop had adopted the inward, high-activity conformation, despite position 422 being spatially distant from the loop. Transformation experiments showed that cell viability requires a tyrosine at position 422, indicating the residue is essential for transpeptidase function. Together, these findings reveal an energetic coupling between an active-site residue in PBP2 and a conformational switch whose equilibrium is altered by resistance mutations. The previously observed higher activity of β-lactams containing extended R 1 groups is consistent with stronger interactions with Tyr422 that favor the conformational switch. Molecular modeling suggests that such groups enhance activity by mimicking the iso -Glu region of the pentapeptide substrate. Overall, we propose that access to the high-activity state of PBP2 where the β3-β4 loop is inward is regulated by interactions between Tyr422 and β-lactam R 1 groups, and that resistance mutations function by tilting the balance toward a lower activity state.

Vaginal infections are commonly treated with antibiotics but increasing antimicrobial resistance and side effects of commercial intimate wash have encouraged the search for safer herbal alternatives. This study aimed to develop and evaluate an intimate wash formulated with Andrographis paniculata extract against selected pathogens; Candida albicans, Neisseria gonorrhoeae, Escherichia coli and Streptococcus agalactiae. The antimicrobial activity of A. paniculata intimate wash was assessed by using the agar well diffusion method to observe inhibition zones. Toxicity evaluations were conducted, including heavy metal detection for the intimate wash and the brine shrimp lethality assay for the extract. Stability and sterility testing of intimate wash were also conducted to determine the product's shelf-life. The results showed that the intimate wash exhibited optimum inhibition at 3.125 mg/mL. Heavy metals were below the permissible limits, and the extract also demonstrated non-cytotoxic effect towards brine shrimp. The intimate wash showed good stability and remained free from contamination throughout 24 months of storage. In conclusion, A. paniculata intimate wash is acceptable for preliminary safety findings and has potential as herbal alternative for the management of vaginal infections. However, further in vivo and clinical studies are required to confirm its safety and efficacy.

There is an urgent need for novel antimicrobials to treat multidrug-resistant strains of Neisseria gonorrhoeae. In a phase III trial (EAGLE-1), gepotidacin, a first-in-class triazaacenaphthylene antibacterial, demonstrated non-inferiority to ceftriaxone plus azithromycin as measured by microbiological success for the treatment of uncomplicated urogenital gonorrhea. Here, we assessed the in vitro activity of gepotidacin against key N. gonorrhoeae drug-resistant phenotypes and genotypes from EAGLE-1. There were 408 baseline urogenital isolates recovered from 406 patients during the EAGLE-1 study. Agar dilution minimum inhibitory concentration (MIC) susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines, and all isolates were molecularly characterized. All of the isolates were susceptible to ceftriaxone. All isolates had gepotidacin MICs of ≤2 µg/mL, with values ranging from ≤0.06 to 2 µg/mL; MIC50, 0.25 µg/mL, and MIC90, 0.5 µg/mL. As defined by the Food and Drug Administration (FDA) breakpoints and interpretive criteria (MIC breakpoints of ≤1 µg/mL [susceptible]; 2 µg/mL [intermediate]; and ≥4 µg/mL [resistant]), 99% of the study isolates were susceptible to gepotidacin, 1% were intermediate, and no isolates were resistant. Susceptibility percentages remained high across resistance phenotypes and genotypes. These findings support the in vitro activity of gepotidacin against the N. gonorrhoeae isolates, including drug-resistant phenotypes and genotypes, recovered in the EAGLE-1 clinical study.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT04010539.

CLSI M100, 36th edition (2026), lowered the ceftriaxone susceptible MIC breakpoint for Neisseria gonorrhoeae from ≤0.25 mg/L to ≤0.12 mg/L, classified 0.25 mg/L as intermediate, and removed ceftriaxone disk diffusion interpretive criteria. We conducted a single-center, retrospective method-comparison study of 111 consecutive, nonduplicate, culture-confirmed isolates with paired ceftriaxone gradient strip MICs, agar dilution MICs, and archived 30-µg disk diffusion zone diameters. Gradient strip readings were prespecified to be rounded upward to the next nominal twofold dilution for comparison with agar dilution. Standardized essential agreement within ±1 dilution was 111/111 (100.0%); because upward rounding can increase apparent essential agreement, essential agreement based on the original continuous readings was also assessed and was 102/111 (91.9%). Using a binary interpretation based on the revised susceptible breakpoint (≤0.125 mg/L, displayed by CLSI as ≤0.12 mg/L, vs >0.125 mg/L), gradient strip and agar dilution classifications agreed for all 111 isolates (100.0%). Complete CLSI 2026 S/I/R categorical agreement was 105/111 (94.6%). The six discrepancies were all minor errors in which gradient strip testing classified an isolate as intermediate, whereas agar dilution classified it as resistant; no major or very major errors occurred. Gradient strip testing identified 12 of 18 isolates classified as resistant by agar dilution, corresponding to 66.7% sensitivity for the resistant category. Both methods classified 28 of 111 isolates (25.2%) as having MICs above the revised susceptible breakpoint; this proportion represents a classification frequency in a method-comparison data set, not a regional prevalence estimate.

Urethritis is most commonly caused by sexually transmitted pathogens; however, refractory presentations may indicate an atypical etiology that does not respond to empiric therapy. Acinetobacter species are Gram-negative bacilli classically associated with nosocomial infections and are an uncommon cause of community-acquired lower urinary tract infections in immunocompetent women. We present a case of severe community-acquired urethritis caused by Acinetobacter baumannii complex in a previously healthy 44-year-old woman, with concurrent isolation of Acinetobacter lwoffii of uncertain pathogenic significance. The patient presented with dysuria and pelvic pain that persisted despite a single 500-mg intramuscular dose of ceftriaxone. Nucleic acid amplification testing was negative for Chlamydia trachomatis and Neisseria gonorrhoeae. Urinalysis demonstrated microscopic hematuria without pyuria, and urine cultures showed no bacterial growth. Urethral swab culture demonstrated moderate growth of both organisms, with identification and antimicrobial susceptibility testing performed using the VITEK 2 automated system. Both isolates were susceptible to trimethoprim-sulfamethoxazole (TMP-SMX), and a 10-day course resulted in complete resolution of symptoms. This case expands the recognized clinical spectrum of Acinetobacter infections by demonstrating their potential to cause community-acquired urethritis in immunocompetent women. It highlights the diagnostic value of urethral swab culture in patients with refractory lower urinary tract symptoms and negative urine cultures and proposes a stepwise clinical framework for evaluating urethritis that fails to respond to empiric therapy.

False-positive Neisseria gonorrhoeae (NG) screening results require supplemental nucleic acid amplification testing for confirmation; however, Alinity m multi-Collect media (Alinity media) forms a precipitate when combined with guanidinium-based lysis buffers, interfering with nucleic acid extraction and causing Xpert CT/NG assay failures. This study evaluated a simple pre-extraction method to enable supplemental NG testing from Alinity media. A guanidine hydrochloride pre-extraction step was developed to remove precipitate prior to testing. Sixteen urine specimens and a swab dilution series were inoculated into both Alinity media and cobas PCR media as a control. Samples were tested using Xpert CT/NG (NG2 and NG4 targets) and extracted on the MagNA Pure 96 platform for supplemental testing with ResistancePlus GC (RP-GC), targeting opa and porA. Qualitative results from Alinity media and cobas PCR media were in complete agreement, with paired quantification cycle (Cq) values showing high concordance across all targets and internal controls. RP-GC opa and porA targets demonstrated mean Cq differences of -0.45 and - 0.47, respectively, indicating a small but statistically significant earlier Cq for Alinity media (p = 0.004 and p = 0.018). Despite reaching statistical significance, these differences were less than 0.5 cycles and were not considered analytically meaningful. Xpert NG2 and NG4 targets showed negligible, non-significant Cq shifts. These findings demonstrate that the guanidine hydrochloride pre-extraction method effectively removes precipitate from Alinity media and enables reliable supplemental NG testing, with performance equivalent to cobas PCR media across both extraction-based and sample-to-answer platforms.