infection is a major global health concern, and its increasing antibiotic resistance poses significant challenges to eradication therapy. Traditional methods for detecting resistance are time-consuming and labour-intensive. The limitations of traditional methods highlight a critical need for a rapid, accurate and comprehensive approach to detect resistance that can inform personalized treatment strategies and improve eradication outcomes. This study aimed to explore the potential of Raman spectroscopy as a rapid and accurate method for detecting resistance to clarithromycin and levofloxacin. We employed Raman spectroscopy to analyse the metabolic fingerprints of strains treated with different concentrations of antibiotics. Principal component analysis and deuterium oxide labelling techniques were used to differentiate between resistant and susceptible strains. Our results demonstrated that Raman spectroscopy can accurately predict antibiotic resistance within 4-6 h, significantly reducing detection time compared with traditional methods. This study provides a promising approach for rapid and accurate detection of antibiotic resistance, enabling personalized treatment strategies and improving eradication outcomes.

Carbapenem-resistant (CRAB) represents a critical healthcare threat with limited treatment options, particularly challenging in Southeast Asia where resistance rates exceed 65%. Current synergy testing methods are labour-intensive and poorly standardized, limiting their clinical application. We hypothesize that synergistic interactions between antibiotics follow predictable mathematical patterns derivable from separate MIC determinations for each antibiotic. To evaluate the efficacy of colistin-meropenem-ampicillin/sulbactam combinations at standard dosages and develop a predictive mathematical model for synergistic interactions against CRAB. A cross-sectional study was conducted using 61 CRAB clinical isolates from a Vietnamese tertiary hospital. MICs were determined using broth microdilution, and synergy was assessed via checkerboard assays. Mathematical models were developed to predict fractional inhibitory concentration (FIC) values from separate MIC determinations for each antibiotic. All isolates demonstrated high-level meropenem resistance (MICs 32-≥128 µg ml) and ampicillin/sulbactam resistance (98.4% with MICs≥64/32 µg ml) but remained intermediate to colistin (MICs 0.0625-0.25 µg ml). The triple combination achieved 100% synergy at standard ampicillin/sulbactam doses (8/4 µg ml). Our log-transformed power model accurately predicted synergistic interactions (²=0.928) using the equation log(FIC) = -2.52+(-1.02) × 1/√Mero+0.43×1/√Col+4.32×1/√As. The triple combination achieves universal synergy at standard dosages, potentially reducing toxicity risks. Our mathematical model enables rapid prediction of effective combinations from routine susceptibility tests, offering a transformative approach to optimizing therapy against multidrug-resistant pathogens.

Peri-implantitis is a prevalent and challenging complication in implant dentistry, primarily induced by biofilm-associated pathogens. Among these, and have emerged as key contributors, with evidence suggesting their potential synergistic role in exacerbating peri-implant inflammation and tissue destruction. While is a well-characterized periopathogen, the specific role of , alone or in combination with , in peri-implantitis remains underexplored. This study addresses the gap in quantifying their presence in diseased versus healthy peri-implant sites. To assess the prevalence and microbial load of and in peri-implantitis and healthy peri-implant sites using quantitative real-time PCR (qPCR) and to investigate their correlation with clinical parameters. This cross-sectional study included 110 participants: 52 diagnosed with peri-implantitis and 58 with healthy peri-implant tissues. Clinical examination recorded probing depth (PD), clinical attachment level (CAL) and bleeding on probing (BOP). Submucosal biofilm samples were collected and analysed using species-specific qPCR. Statistical analysis employed the Mann-Whitney test for intergroup comparisons and Spearman's rank correlation for associations between microbial levels and clinical indices. Both and were significantly elevated in peri-implantitis sites compared to healthy controls. Mean levels were 4.80×10⁶ ± 4.78×10⁶ copies µl in peri-implantitis and 2.09×10³ ± 1.26×10³ copies µl in healthy sites (<0.001). levels averaged 4.58×10⁵ ± 3.40×10⁵ copies µl in peri-implantitis and 2.45×10³ ± 1.64×10³ copies µl in healthy sites (<0.001). showed strong positive correlations with PD, CAL and BOP, while correlated moderately with PD and CAL but not significantly with BOP. The significant elevation of and in peri-implantitis supports their potential synergistic involvement in disease pathogenesis. These findings underscore the need for antimicrobial strategies that target both organisms and disrupt their cooperative biofilm behaviour. Further research should clarify their pathogenic interplay and inform the development of precise therapeutic interventions.

Pleural empyema is common but microbiological diagnosis is challenging, often leading to prolonged empiric broad-spectrum antimicrobial use. 16S rRNA PCR on operative pleural tissue will increase diagnostic yield and improve antibiotic stewardship in patients with surgically managed pleural empyema. The objective was to characterize the clinical and microbiological characteristics of surgically managed pleural empyema, assess the diagnostic yield of 16S rRNA PCR and determine its impact on antimicrobial use. This retrospective study identified patients with pleural empyema at a tertiary thoracic referral unit in North London, UK, between March 2019 and March 2023. Demographic, clinical and microbiological characteristics were collected. One hundred eighty-three patients underwent surgery for pleural empyema at University College Hospital at Westmoreland Street over a 4-year period. Most empyemas were community-acquired, and the main surgical modality was minimally invasive video-assisted thoracoscopic surgery (151/183, 82.5%). Thirty-day all-cause post-operative mortality was 0.6%. Microbial aetiology was ascertained in 47%, with group, and anaerobes most frequently identified. Operative pleural tissue 16S rRNA PCR had a high diagnostic yield compared to conventional culture alone (68.2% vs. 26.4%). Post-operatively, antibiotics were prescribed for a median of 14 days (interquartile range 10-18), the majority broad spectrum (169/181, 93.4%). 16S PCR impacted antimicrobial prescribing in one-tenth of cases when used (4/32, 12.5%); however, turnaround times limited its impact. Molecular diagnostics improve bacterial detection and antibiotic stewardship in pleural empyema. Streamlining molecular diagnostics pathways or developing pleural-specific multiplex PCR panels could further enhance management.

Following two large foodborne outbreaks of the gastrointestinal pathogen, enteroaggregative (EAEC), in Germany in 2011 and the UK in 2014, the UK Health Security Agency (UKHSA) implemented enhanced surveillance strategies for EAEC. The surveillance of diarrhoeagenic in England focuses on Shiga toxin-producing (STEC), and the true clinical and community burden of EAEC is unknown. This gap extends globally, as many countries lack the infrastructure, diagnostic tools and healthcare facilities to resource surveillance programmes for EAEC. The aim of the study was to review the microbiological typing data and demographic data linked to isolates and cases diagnosed from 2016 to 2023 and to assess the risk to public health. Faecal samples that tested positive by PCR for diarrhoeagenic at local microbiology diagnostic laboratories were referred to the UKHSA for confirmation and culture. Isolates identified as EAEC were sequenced on the Illumina HiSeq and NextSeq platforms. Sequence type, serotype and antimicrobial resistance (AMR) profile were derived from the genome sequence. Age, sex and travel histories were linked to the typing data. There was a total of 1,402 notifications of EAEC, exhibiting a fivefold increase in diagnoses from 93 in 2016 to 524 in 2023. The most common sequence types (STs) were ST34 (=202/1,402, 14.4%), ST10 (=185/1,402, 13.2%), ST200 (=183/1,402, 13.1%) and ST678 (=101/1,402, 7.2%), and the most common serotypes were O92:H33 (=130/1,402, 9.3%), O175:H31 (=78/1,402, 5.6%) and O99:H10 (=78/1,402, 5.6%). Most cases were female (=748/1,372, 54.5%) and/or were aged <10 (=387/1,372, 28.2%), within which 299 out of 387 (77.3%) were <5 years old. Of the 756 out of 1,386 (54%) cases that had a travel history, 597 out of 756 (79%) reported foreign travel within 7 days of onset of symptoms. AMR was detected in 1,030 out of 1,402 (73.5%) isolates with resistance to fluoroquinolone (=810/1,402, 57.8%) and beta-lactam (=807/1,402, 57.6%) antibiotics being the most common. Given the burden of disease caused by EAEC in the community, the high proportion of infections in children and travellers, the risk of the emergence of hybrid STEC/EAEC pathotypes and the high proportion of AMR, we recommend that EAEC should be part of the diagnostic algorithm in the UK.

In antibiotic chemotherapy, an antimicrobial agent is selected based on MIC, which is determined by a test method using the principle of the broth microdilution. However, it does not provide a viable count in the mutant selection window. In this study, we used turbidimetry and visual judgement to examine the validity of the selection of various antibiotics to treat based on MIC determination after 24 h of incubation. The antibiotics used in this study were piperacillin (PIPC), imipenem (IPM), meropenem, ciprofloxacin and amikacin (AMK). The strains used were 30 . strains clinically isolated that were susceptible to all the antibiotics used, and the standard PAO1 strain. The viable count was measured after exposure for 3 and 24 h to therapeutic concentrations of various antibiotics, at which time turbidity was examined visually or by transmittance. In addition, the MPCs of IPM and PIPC were measured. In this study, 10²-10⁸ c.f.u. ml of survived exposure to PIPC, IPM and AMK at concentrations 2.5-80 times the MIC despite high drug concentrations. No turbidity was observed in the culture medium. Furthermore, both IPM and PIPC showed high mutant prevention concentration (MPC), with 64.5% of strains in IPM and 10% of strains in PIPC showing intermediate or resistance after 24 h. Choosing an appropriate antibiotic based on exceeding the MIC may be insufficient. While the PK/PD theory focuses on MIC, measuring MPC alongside MIC is urgent in clinical practice for optimal antibiotic selection.

, and are closely related species (collectively referred to as 'PQV') but exhibit distinct clinical presentations and epidemiological profiles. These distinctions are important for proper diagnosis and treatment of infections and other -related diseases. The ability to differentiate between these species is becoming more important as we better understand their unique roles in disease. Despite the importance of accurate taxonomic classifications, both traditional and modern laboratory techniques fail to accurately delineate between the PQV species, which can lead to treatment failures. Conversely, taxonomy via whole-genome sequencing is highly accurate but is both costly and resource-intensive. Thus, there is a lack of a widely adopted method that balances both cost-effectiveness and accuracy for differentiating PQV species. To assess the taxonomic accuracy of existing genome databases and classification tools for PQV species and to develop a rapid, cost-effective method for accurate species differentiation. To address these challenges, we extracted 78 representative PQV genomes from the Integrated Microbial Genomes and Microbiomes database. We used multiple comparative genomic comparison techniques, phylogenetic tree constructions and pangenome profiling to accurately phylogenetically and subsequently taxonomically classify the genomes. After establishing accurate taxonomic classifications, we identified species-specific marker genes (SSMGs) represented by KEGG Orthologies (KOs). The Genome Taxonomy Database (GTDB) classifications were consistent with our comparative genomic benchmarks, while the National Center for Biotechnology Informationdatabase originally contained six misclassified genomes. The GTDB-Tk tool also showed reliability for systematic classification of the genomes. Our KO-based screening identified 22 candidate SSMGs. Four markers (K05306, K07507, K13795 and K09955) exhibited significant specificity. ATP-binding proteins showed slightly higher maximum percentage identity values due to conserved domains but were still valuable within multi-locus SSMG panels. Our findings establish the GTDB as the gold standard taxonomic reference for PQV classification when complete genomes are available. Additionally, we developed a practical panel of genetic markers that enables rapid, cost-effective and accurate species differentiation. These SSMGs represent practical tools that can be implemented in diagnostic laboratories for both clinical specimens and environmental surveillance, addressing a critical gap in clinical microbiology.

The and genes encode the CagA and VacA proteins, which are the two main toxins of . Regardless of whether the illness is benign or malignant, the majority of Asian strains are () and s1 ( signal region 1 allele); hence, these genotypes cannot account for the severity of gastroduodenal disease. The gene encodes the important adhesin BabA of , which is crucial for persistent colonization and facilitates the translocation of CagA into host gastric epithelial cells. The synergic interaction of toxins, including CagA, VacA and BabA, could significantly contribute to the pathogenesis of . The investigation of , and genes in clinical isolates in Asian nations, particularly Vietnam, is insufficient. To investigate the , and genotypes to further understand their synergistic interaction in the development of gastroduodenal disease in Vietnamese populations. A cross-sectional study was conducted on 169 . strains isolated from patients with gastroduodenal disease. The PCR assays were performed to determine the , and genotypes on DNA extracted from cultured isolates. The research showed that the percentage of the (+), (+), s1m1 and s1m2 was 87.6%, 73.4%, 52.1% and 44.4%, respectively. The frequencies of (+)/(+)/s1m1 and (+)/(+)/s1m2 combinations were 44.4% and 28.4 %, respectively. The (+)/(+)/s1m2 combination was associated with peptic ulcer disease [adjusted odds ratio (aOR)=5.53, 95 % confidence interval (CI) 1.09-28.16, =0.039] in male patients and chronic gastritis with precancerous lesions (aOR=5.31, 95 % CI 1.23-22.89, =0.025) in female patients. The (+)/(+)/s1m1 and (+)/(+)/s1m2 combinations were found to be quite prevalent among Vietnamese strains. The synergistic effect of (+), (+) and s1m2 in increasing the odds of both peptic ulcer disease and gastric precancerous lesions has been observed.

Antimicrobial resistance (AMR) is a global threat to health, food security and economies across the world. To strengthen UK surveillance efforts, the Microbiology Society and the Pathogen Surveillance in Agriculture, Food and Environment Programme (PATH-SAFE) convened a One Health AMR Surveillance Roundtable in December 2024, bringing together 16 government agencies from the human, animal, plant, environmental and food sectors across the UK central government and devolved nations. The workshop identified key questions for a UK-focused national One Health AMR surveillance system and explored opportunities and challenges for implementation. Key challenges include fragmented data with limited sharing and underutilized microbiological samples. Participants called for greater cross-sector collaboration, a shared AMR vocabulary, sustained investment and the development of a national AMR data catalogue. Coordinated One Health surveillance, backed by political commitment, is essential to tackle AMR effectively.

The abnormal proliferation of , a human skin commensal bacterium, aggravates acne. Prompt and aggressive treatment of acne in its initial stages is essential because of the increased psychological distress experienced by patients. Over-the-counter medications containing disinfectants are sometimes used to treat acne because they are widely available, exhibit broad-spectrum activity and have a likelihood of causing resistance. Limited information is available regarding the bactericidal effects of disinfectants against . In the present study, we comprehensively evaluated the efficacy of various disinfectants. ATCC6919 and eight clinical isolates with different genotypes obtained from patients with acne were used. Benzethonium, chlorhexidine, resorcinol, homosulfamine, isopropyl methylphenol and olanexidine were used as disinfectants. The bactericidal activity of these disinfectants against was assessed using a time-kill kinetic assay. Three independent experiments were conducted to ensure reproducibility of the results. Benzethonium, chlorhexidine, isopropyl methylphenol and olanexidine exhibited high efficacy. However, under acne conditions, with the addition of artificial sebum, the bactericidal efficacy of the disinfectants was significantly reduced. However, olanexidine retained bactericidal activity at concentrations as low as 0.2%, comparable with that at 1.5%. This study showed that the disinfectants benzethonium, isopropyl methylphenol, chlorhexidine and olanexidine have high bactericidal activity against . In particular, olanexidine demonstrated a strong bactericidal effect even under acne conditions. Further validation is required to determine whether olanexidine could be a new treatment option for managing acne.

is a pathogenic yeast in humans, recognized for its genomic plasticity and increasing prevalence of antifungal resistance, including multidrug-resistant phenotypes, especially in the US and European countries. This study hypothesizes that the resistance mechanisms in clinically resistant strains of differ from laboratory-generated resistant strains. This study aims to understand the resistance mechanism in Indian clinical isolates of A total of 240 clinical isolates of were tested for antifungal susceptibility and one resistant strain was artificially synthesized in the laboratory. Both clinical and lab-generated resistant strains were analysed for antifungal resistance using methods such as phenotypic assays, real-time quantitative PCR, Fluorescence-activated cell sorting (FACS) analysis and targeted gene sequencing. Mechanisms involving drug efflux pumps, mismatch repair pathways, ergosterol biosynthesis pathway and biofilm formation were systematically studied. Among clinical isolates, one susceptible-dose dependent strain and three fluconazole-resistant strains were identified. Both clinical and lab-generated resistant strains demonstrated antifungal resistance phenotypically, with increased expression of . Targeted gene sequencing revealed novel mutations in , while mutations in served as genotypic markers for resistance. Overexpression of was seen in a lab-generated resistant strain where a specific mutation was identified. Biofilm activity contributed to resistance in one of the clinical strains. This study reports for the first time the fluconazole resistance mechanism in from India. The findings underscore the diversity of resistance mechanisms among clinical and lab-generated isolates, emphasizing the need for novel antifungal therapies to address these emerging resistance profiles effectively.

Bloodstream infections (BSIs) caused by multidrug-resistant Gram-negative bacteria (MDR-GNB) are a major cause of mortality, with limited treatment options. Despite the global rise of MDR-GNB, prospective data on their clinical impact in North Africa remain scarce, restricting evidence-based guidance for empiric therapy. To assess the prevalence, resistance patterns and clinical outcomes of MDR-GNB BSIs in Libya, and to identify predictors of 30-day mortality. A prospective cohort study was conducted in two Libyan hospitals between November 2022 and November 2024. Adult patients with positive blood cultures were enrolled. Isolates were identified using standard microbiological methods, and antimicrobial susceptibility testing was interpreted according to Clinical and Laboratory Standards Institute (CLSI) 2023 guidelines. Multivariable logistic regression was applied to identify independent predictors of 30-day mortality. Among 673 BSI episodes, 37.4% were multidrug resistance (MDR). (32.0%) and (25.9%) predominated, while carbapenem resistance was highest in (42.4%). Overall, 30-day mortality was 23.8%, and was significantly higher in MDR infections (32.1% vs 18.8%; <0.001). Independent predictors of mortality were MDR infection [adjusted odds ratio (aOR), 1.9], Intensive care Unit (ICU) admission (aOR, 2.6), Charlson Comorbidity Index ≥3 (aOR, 1.7) and inappropriate empiric therapy (aOR, 2.3). MDR-GNB BSIs are highly prevalent in Libya and substantially worsen outcomes. These findings highlight the urgent need for improved empiric therapy, antimicrobial stewardship and infection control programmes.

Gastrointestinal infections remain a leading cause of morbidity and mortality within the UK. The Luminex NxTAG Gastrointestinal Pathogen Panel (NxTAG GPP) multiplex reverse transcriptase PCR assay performs equivalently to standard-of-care diagnostic approaches. To compare the analytical performance of the NxTAG GPP assay versus routine diagnostic testing methods in a district general hospital setting. Gastrointestinal pathogens in 159 faecal specimens from hospital inpatients and outpatient clinics were comparatively analysed using the NxTAG GPP assay versus traditional culture, enzyme immunoassay and molecular methods. Positive results were detected in 45 out of 159 specimens (28.3%) by NxTAG GPP, which was a higher positivity rate when compared with traditional diagnostic methods which detected 31 out of 159 (19.5%) positive infections (=0.087 by Fisher's exact test). Infections were caused by a single organism in 40 out of 45 (88.9%) cases, but 5 out of 45 (11.1%) infections detected were due to coinfections. No coinfections were detected by traditional methods. Group was the most common enteropathogen detected with 15 out of 52 (28.9%) infections. Viruses caused 26.9% of infections, including 15.4% being norovirus. Overall sensitivity, specificity and accuracy for the NxTAG GPP assay were 97.6%, 99.7% and 99.5%, respectively, for enteropathogenic bacteria and viruses detected during this study. No parasites were detected during this study and were not included in comparisons. The NxTAG GPP assay demonstrated high sensitivity and specificity for identifying gastrointestinal pathogens, with comparable accuracy as more resource-intensive and time-consuming standard diagnostic approaches. The NxTAG GPP has the potential to enhance patient diagnosis, reduce turnaround time and improve clinical outcomes compared to routine diagnostic methods.

Six to seven million individuals are infected with , the causative agent of Chagas disease. With 12,000 deaths annually, chronic Chagas disease remains a significant global health challenge due to persistent vector transmission, increasing non-vector transmission and limited therapeutic options. Chronic Chagas cardiomyopathy is a leading cause of morbidity and mortality, yet the underlying mechanisms remain poorly understood. Since its initial description more than 100 years ago, research efforts into the cardiomyopathy found in chronic Chagas disease have primarily focused on the contributions of immune cells, cardiomyocytes and cardiac fibroblasts, leaving a significant gap in understanding the role of microvascular endothelial dysfunction in disease progression. The aim of this study was to identify any morphological or functional changes to cardiac microvascular endothelial cells induced by infection with the potential to contribute to the pathologies found in chronic Chagas disease. We cultured primary cardiac microvascular endothelial cell monolayers and infected them with trypomastigotes or exposed them to conditioned media collected from control or infected endothelial cells. Cells were analysed for changes in morphology and proliferation, by wound healing assays for measurements of migratory capacity and by tube-forming assay to characterize their ability to form capillary-like structures. We show that infection leads to the development of hypertrophic multinuclear cells, inhibits endothelial proliferation, increases endothelial migration and results in changes in several aspects of angiogenesis. We present data to demonstrate morphological and functional changes in cardiac endothelial cells that occur as a result of infection and propose that these changes may contribute to endothelial dysfunction and the development of chronic Chagas cardiomyopathy.

The TriVerity™ test (Inflammatix Inc.) uses the patient's mRNA expression patterns from whole blood to provide a site-agnostic likelihood of bacterial infection and/or viral infection. The test also reports an illness severity score, an all-cause predictor of the need for critical care (mechanical ventilation, vasopressor therapy or renal replacement therapy) within 7 days of testing. Differentiating the source and assessing disease severity in patients presenting to the emergency department (ED) with signs and symptoms of sepsis remains an unmet medical need. The results of the TriVerity test were compared to the EPIC sepsis score, systemic inflammatory response syndrome criteria, discharge diagnoses, location of patient discharge and an assessment of potential cost savings in unnecessary resource utilization. Blood was retained from 30 patients presenting to the ED with signs and symptoms suggestive of sepsis. Remnant EDTA-preserved blood from routine medical testing was transferred into PAXgene tubes, stored frozen and retrospectively tested with TriVerity. TriVerity was correctly correlated with clinical diagnosis and outcomes in 29 of 30 patients with one false negative (low severity score for sepsis diagnosis, admitted for bladder cancer and on pre-existing antibiotic therapy). For two patients with a positive TriVerity bacterial and severity score, antibiotic treatment may have been initiated 6-9 h earlier if tested prospectively. Twelve patients were discharged home with a mean ED length of stay of 7.25 h, of which 7 (58 %) had a positive EPIC sepsis score (>5) and all had a low TriVerity severity score. If this test were performed prospectively at ED presentation, a net savings of $351 per patient is estimated in direct laboratory, radiology and ED costs alone, without consideration of potentially shorter hospital stays or changed disposition. The TriVerity test may enable early consideration of ruling in or out of bacterial and/or viral infection, provide severity assessment of adult patients presenting to the ED suspected of, and help reduce unnecessary utilization of laboratory and ED resources.

Intestinal dysbiosis is associated with systemic health, and approaches targeting the microbiome can influence the host. Oral and intestinal microbiota are interrelated; therefore, we aimed to determine whether non-surgical periodontal treatment (NSPT) affects systemic health through its impact on the intestinal microbiota. Although the association between oral and gut microbiota has been suggested, there is limited evidence regarding how periodontal therapy may influence intestinal microbial composition. We hypothesized that NSPT in patients with periodontitis would lead to favourable changes in the gut microbiome, which may parallel improvements in clinical periodontal parameters. This study aimed to investigate the effect of NSPT on both oral and intestinal microbiota and to evaluate whether changes in gut microbial composition correlate with periodontal clinical outcomes. Five systemically healthy individuals with grade C periodontitis and five systemically and periodontally healthy individuals were included. Saliva and stool samples were collected at baseline and 1 month after NSPT. DNA extractions were performed and subjected to 16S ribosomal RNA gene sequencing on the Illumina Novaseq at the V3-V4 hypervariable regions. Grade C periodontitis patients displayed distinct oral and gut microbiomes compared to healthy individuals. NSPT resulted in a reduction in the diversity of both saliva and stool samples in healthy individuals (>0.05). Salivary levels (<0.05) and the gut / ratio decreased after NSPT. Moreover, changes in gut microbiota significantly correlated with improvements in periodontal probing depth and clinical attachment level in periodontitis patients. The improvement in clinical periodontal parameters after NSPT correlates with a positive shift in the gut microbiome towards health. Although the number of participants was limited, these findings support a strong relationship between periodontal and gut status. Further studies with larger cohorts and long-term follow-up are required to confirm these results.

Schmallenberg virus (SBV) is an arthropod-borne virus and belongs to the Simbu serogroup within the family , genus . Infection of naïve ruminants at critical stages of gestation can result in severe congenital malformations or abortion. Tools to measure virus infection parameters in cell culture such as replication efficiency, as well as neutralization assays, are mainly available in the form of assays based on the evaluation of cytopathic effects. The methods are labour-intensive and low-throughput, as they require long incubation periods of several days. Tools such as tagged SBV that allow for fast and automated readout are missing. We aimed to develop a tagged SBV that can be used for assays with fast and automated read-out. We report the construction of a recombinant SBV stably expressing the nanoluciferase (nluc) enzyme (rSBV_nluc). Using reverse genetics, the nluc gene was integrated into the genome of an SBV variant naturally harbouring a large deletion within the Gc-head domain. The nluc gene was inserted into this locus. The nluc-tagged virus showed no signs of attenuation and identical replication properties when compared to the parental virus in baby hamster kidney (BHK-21) cells. Our results demonstrate a new approach for rapid access to SBV replication. By performing nluc assays, we were able to track viral replication and also virus uptake in detail. We further evaluated neutralization properties of an SBV variant, which is lacking a major part of its antigenic domain (Gc-head) and developed a nanoluciferase activity-based serum neutralization assay. Overall, the nluc-tagged SBV is a suitable tool that further facilitates the study of viral infection dynamics and allows for high-throughput assays.

Antimicrobial susceptibility testing (AST) of isolates is recommended in the UK to ensure antimicrobial stewardship and detection of multi-drug and extensively resistant cases. In diagnostic and reference laboratories, this testing is primarily carried out via a gradient strip. However, agar dilution methodology may also be used for high-throughput testing. is not a validated species on all ETEST (bioMérieux, France) gradient strip formulations available, and, therefore, additional comparative validation data are required to support use in clinical laboratory settings. To determine the reproducibility of ETEST for AST of , and to demonstrate the comparability of susceptibility results obtained using agar dilution and gradient strip methods. Modal ETEST MICs for six well-characterized World Health Organization control strains, against eight antimicrobials, were calculated. ETEST modal MICs were compared to published and local, historical agar dilution MICs. MICs were interpreted using European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. Concordance of ETEST modal MICs, within essential and categorical agreement, for each strain was calculated. Overall, 95.80% of modal ETEST MICs were within essential agreement with published MICs. Where variance from exact concordance was noted, a systematic shift was observed to lower MICs in this study. On three occasions, variance from the published MIC resulted in a categorical classification change. On two occasions, the modal ETEST MIC in this study was two doubling dilutions lower than the published MIC. Neither resulted in a categorical classification change. When modal ETEST and agar dilution MICs were compared, overall essential agreement was 83.30%. However, this increased to 94.4% when concordance was analysed for clinically important antimicrobials: azithromycin, cefixime and ceftriaxone. Again, a systematic shift to lower MICs for ETEST was observed in this study. Categorical agreement was 83.3% for all antimicrobials and 100% for clinically important antimicrobials. Excellent concordance was demonstrated for MICs generated using ETEST with published MICs. Good concordance was observed for MICs generated using two different susceptibility testing methodologies. Where variance was noted, MICs generally read lower on ETEST in this study. ETEST remains fit for purpose for the AST of , a clinically important pathogen.

complex disease is difficult to treat, with high failure and recurrence rates despite multidrug, macrolide-based treatments. The bacterial mechanisms involved in this drug tolerance and persistence are incompletely understood. Recent evidence has suggested persistence through metabolic adaptations indicative of the viable but nonculturable state, including a decreased respiratory rate and a switch to lipid accumulation and metabolism. To assess the contribution of switching to viable but nonculturable state to macrolide tolerance, we performed time-kill kinetics assays for clarithromycin against . In these experiments, we performed Auramine-O (for acid-fastness, representing active mycobacteria) and Nile red (for lipid accumulation) staining and stimulation using resuscitation-promoting factors of . Loss of auramine staining, increased Nile red staining and increased population sizes after stimulation with resuscitation-promoting factors support the hypothesis that clarithromycin induces a viable but nonculturable state in . Induction of a viable but nonculturable state is one of the mechanisms of macrolide tolerance in . It might be one of the drivers of the high failure and recurrence rates of macrolide-based treatments. Antimicrobials active against viable but nonculturable may improve treatment outcomes.

(Kp) is a major cause of nosocomial infections, with its evolving pathotypes including multidrug-resistant, hypervirulent (hvKp) and convergent strains posing significant diagnostic and treatment challenges due to combined antimicrobial resistance and virulence. While there is a pressing requirement for thorough detection of Kp pathotypes, current assays in resource-limited environments are unable to effectively focus on essential carbapenemase and hypervirulence genes with the necessary reliability and precision. To develop and validate a multiplex PCR (m-PCR) assay capable of simultaneously detecting Kp isolates including those carrying partial or full virulence markers, alongside antimicrobial resistance. In this study, an m-PCR assay was designed and optimized for the simultaneous detection of key biomarkers associated with hypervirulent (, , , and ), carbapenem-resistant ( , and ) and convergent Kp pathotypes in clinical isolates. The assay was evaluated on clinical isolates and validated against whole-genome sequencing (WGS) data for accuracy, specificity and sensitivity. The developed m-PCR assay exhibited 100% specificity when compared to WGS data, successfully detecting all target genes without cross-amplification in ATCC control strains. The assay demonstrated high sensitivity, efficiently amplifying bacterial genomes from minimal DNA input as low as 1 ng µl. Additionally, validation through sequencing confirmed the accuracy of detected amplicons. This m-PCR assay offers a rapid, sensitive and specific diagnostic tool for differentiating Kp pathotypes in clinical settings, aiding in timely intervention and improved infection control measures.