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Rectal carriage of sequence type 307 Klebsiella pneumoniae high-risk clone harboring multiple carbapenemase genes in community hospitals Gauteng, South Africa
Salvador-Oke, Kafilat Taiwo; Pitout, Johann D.D.; Peirano, Gisele; Strydom, Kathy-Anne; Kingsburgh, Chanel; Ehlers, Marthie Magdaleen; Kock, Marleen M. (Wiley, 2025-11)
Asymptomatic rectal carriers are recognized as reservoirs of carbapenem-resistant Klebsiella pneumoniae (CRKp), which can spread epidemic high-risk clones [e.g., sequence types (ST)-307] and plasmids [incompatibility group (Inc)-X3] in hospitals, with possible transmission into the community. This study investigated the epidemiology and characteristics of CRKp high-risk clones ST307 among rectal carriage isolates from community hospitals. A carbapenemase positivity rate of 24% was observed for all rectal screening performed during hospital admission (February to August 2021) in Gauteng, South Africa; 252 CRKp isolates were characterized. Antimicrobial susceptibility was performed using the VITEK 2 automated system, and polymerase chain reaction assays were used to detect K. pneumoniae ST307, carbapenemase genes, and associated mobile genetic elements (MGEs e.g., IncX3, IS3000). Of the 252 isolates, 25% (64/252) were ST307 positive and 75% (188/252) were non-ST307. Among the 64 ST307, 45% (29/64) harbored blaOXA-181 on IncX3 plasmids. Occurrence of blaOXA-181 among ST307 (69%; 44/64) when compared to non-ST307 (48%; 91/188) was statistically significant (p-value = 0.002). Fourteen isolates, including two ST307, harbored double carbapenemase genes. Carbapenemase gene combinations include six blaNDM+blaOXA-48-like, four blaNDM +blaOXA-181, three blaKPC+blaOXA-181, and one blaOXA-181+blaVIM. One ST307 isolate harbored three carbapenemase genes (blaNDM+blaOXA-48+blaOXA-181). Level of antimicrobial resistance was significantly (p-value < 0.001) associated with the occurrence of ST307, comprising 73% (47/64) extensively drug resistant. This study highlights the need for rectal screening of XDR clones and plasmids using simple and cost-effective genomic methodologies suitable for low- and middle-income countries for local risk management and control of infectious diseases in hospitals.
Next-generation sequencing in infectious-disease diagnostics : economic, regulatory, and clinical pathways to adoption
Osei Sekyere, John (Wiley, 2025-11)
Next‐generation sequencing (NGS) has emerged as a transformative tool for infectious disease diagnosis, offering broad pathogen detection, antimicrobial resistance profiling, and syndromic panel testing. However, widespread clinical adoption remains hindered by insurance reimbursement challenges, high costs, and regulatory barriers. Unlike polymerase chain reaction (PCR), which enjoys well‐established Current Procedural Terminology (CPT) codes and reimbursement pathways, many NGS‐based tests lack standardized billing mechanisms, discouraging laboratories from integrating NGS into routine diagnostics. This article explores the economic, clinical, and technological considerations of targeted amplicon sequencing (tNGS) versus PCR and whole‐genome sequencing (WGS), demonstrating how optimized multiplexing strategies, emerging NGS platforms, and regulatory advancements can enhance feasibility. It is argued that insurance policies must evolve to recognize NGS's superior clinical utility in detecting polymicrobial infections, emerging pathogens, and antimicrobial resistance determinants, ultimately improving patient outcomes and reducing healthcare costs. Current reagent‐only costs now average US $65 per microbial genome, US $600 per 30× human genome, and US $130–600 per metagenomic sample when multiplexed; these figures continue to fall with higher multiplexing. To accelerate equitable adoption, we recommend near‐term payer coverage pilots for clearly defined clinical indications, dedicated CPT pathways for infectious‐disease sequencing (including metagenomic assays), and pragmatic validation frameworks that acknowledge genotype–phenotype limits while leveraging multiplexing and centralized reference workflows.
Evaluation of six commercial and noncommercial colistin resistance diagnostics
Leshaba, Tumisho Mmatumelo Seipei; Mmatli, Masego; Mbelle, Nontombi Marylucy; Osei Sekyere, John (Wiley, 2025-08)
Resistance to colistin, a last-reserve antibiotic used for treating drug-resistant infections, is rising globally. We compared six commercial and in-house diagnostics-ComASP colistin, CHROMagar COL-APSE, rapid polymyxin NP (Nordmann/Poirel) test, Sensititre, MicroScan, and Vitek 2-against ISO-standard broth microdilution (BMD) using 142 Gram-negative isolates. The isolates (Enterobacterales = 110, Acinetobacter baumannii = 21, Pseudomonas aeruginosa = 11) underwent BMD and conventional multiplex PCR screening for mcr-1-mcr-5. Sensitivity, specificity, categorical agreement (CA), major error (ME), and very major error (VME) were calculated for each test. Vitek 2 and sensititre yielded the highest CA (≥ 98%) and the lowest VME (≤ 0.0%) across taxa. ComASP showed excellent performance for A. baumannii (100% sensitivity/specificity) but slightly lower CA for P. aeruginosa (73%). CHROMagar COL-APSE demonstrated acceptable sensitivity (92%) but low specificity (69%) in Enterobacterales. MicroScan had reduced specificity in Enterobacterales (87.80%). The CHROMAgar COL-APSE efficiently identified the species with their unique colours but was the least specific (68.63%), with the highest ME in Enterobacterales. The rapid NP test provided rapid results within 4 h but showed a relatively high VME (7.84%), despite maintaining an acceptable sensitivity (92.16%) and specificity (96.08%). For laboratories with automated platforms, Vitek 2 remains optimal for colistin MIC testing; Sensititre and ComASP are suitable low-cost BMD alternatives. The Rapid NP test provides a same-day screen, but confirmatory MIC testing is advised. CHROMagar COL-APSE should be used with a ≤ 1 CFU mL⁻¹ inoculum to minimise false resistance calls. Knowing the comparative performance of these different tests will assist in choosing the best test for every species, improving on efficient diagnosis and healthcare outcomes.
Virus-neutralizing monoclonal antibodies against bovine viral diarrhea virus and classical swine fever virus target conformational and linear epitopes on E2 glycoprotein subdomains
Roman-Sosa, Gleyder; Meyer, Denise; Dellarole, Mariano; Wengen, Doris a; Lerch, Susanne; Postel, Alexander; Becher, Paul (American Society for Microbiology, 2025-04)
The envelope glycoprotein E2 of pestiviruses plays a crucial role in viral entry and elicits a virus-neutralizing humoral immune response. Consequently, the epitopes recognized by monoclonal antibodies (mAbs) on E2 are a significant focus in pestivirus research and diagnostics. In this study, we characterized a panel of murine mAbs against the E2 protein of classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV), two major pathogens for swine and cattle, respectively. The majority of mAbs neutralized the virus in vitro and recognized conformational epitopes, which were also detected by sera from infected animals. Notably, binding to these epitopes was retained after low-pH treatment, although conformational epitopes were disrupted upon disulfide bond reduction. The epitopes of the anti-CSFV mAbs were located in various domains of E2, including the interdomain linker sequences. Conversely, all but one of the anti-BVDV mAb epitopes were located in domain A. Moreover, the reactivity of one mAb suggests a conformational interdependence among the linker sequences of pestivirus E2. The panel of mAbs characterized in this study holds potential to support basic research on the mechanism of early pestivirus invasion and to assist in the design of E2-based diagnostic tools and vaccines. IMPORTANCE : Classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV), which belong to the family Flaviviridae, cause economically significant diseases in pigs and cattle. The pestivirus glycoprotein E2 is located on the viral surface and is targeted by antibodies that neutralize virus infection. Due to its variability, E2 is a useful antigen for the development of diagnostic tests to differentiate between infections caused by different pestiviruses. In the present study, two panels of monoclonal antibodies (mAbs) specifically reactive with either CSFV or BVDV E2 were characterized. Interestingly, the majority of mAbs neutralized the respective virus in vitro. Epitope mapping revealed that the mAbs recognized low-pH-resistant epitopes of conformational nature located in different domains of CSFV E2 (anti-CSFV mAbs) or in domain A of BVDV E2 (anti-BVDV mAbs). The recombinant proteins along with the characterized mAbs have the potential to develop improved pestivirus-specific diagnostic tests and vaccines.
The biocontrol potential of endophyte Bacillus velezensis to reduce post-harvest tomato infection caused by Rhizopus microsporus
Kock, Alicia; Napo, Mmanoko; Viviers, Dionné; Akinmoladun, Oluwakemi V.; Alayande, Kazeem A.; Yusuf, Abdullahi Ahmed; Uehling, Jessie; Pawlowska, Teresa E.; Adeleke, Rasheed A. (American Society for Microbiology, 2025-12)
Rhizopus microsporus is a necrotrophic post-harvest pathogen that causes significant economic losses in the agricultural sector. To explore alternatives to conventional management strategies for the mitigation of post-harvest infections, we investigated the potential of two previously identified endophytic Bacillus velezensis strains as biological control agents. Through in vitro and in vivo experiments, we examined the mechanisms of biocontrol displayed by two B. velezensis strains (KV10 and KV15) against three R. microsporus strains (W2-50, W2-51, and W2-58). In vitro assays assessed co-cultivability and the inhibitory effects of B. velezensis against R. microsporus. The results demonstrated strain-specific antifungal activity with a reduction in fungal growth across treatments. Further analysis revealed that volatile organic compounds produced by B. velezensis contributed to its antifungal properties. To evaluate the biocontrol efficacy in vivo, tomato fruits were inoculated with R. microsporus and subsequently treated with B. velezensis. The results support the strain-specific reduction in tomato spoilage, yielding various spoilage rates observed across treatments. Our findings highlight the potential of B. velezensis as a promising biocontrol agent for the management of R. microsporus post-harvest infections in tomatoes. Further research is warranted to optimize the application of B. velezensis as a sustainable and environmentally friendly approach for controlling post-harvest diseases in tomatoes.