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ABSTRACT Plasmid-mediated AmpC β-lactamases are a cause of acquired cephalosporin resistance in Gram-negative bacteria. However, consensus regarding the optimal detection method is yet to be achieved and varies depending on local epidemiology and laboratory capacity. We determined the acquired genotypic resistance mechanisms of 250 Escherichia coli isolates with a positive AmpC screen, defined as cefoxitin MIC ≥ 8 mg/L and a positive AmpC double- disc diffusion test, using in-house designed high-resolution melt PCR, detecting plasmid-acquired genes from the CIT and DHA families. A proportion of these isolates (n = 170, 68%) underwent further genotypic characterization using whole- genome sequencing (WGS). Of 250 isolates with a positive screening test, 72 (28.8%) were determined to carry an acquired AmpC gene. There was 100% concordance between PCR and WGS in the identification of acquired AmpC genes. The phenotypic criteria were then assessed to determine their utility in predicting acquired AmpC gene carriage. Criteria 1 (cefoxitin MIC > 8 mg/L plus ceftriaxone MIC > 1 mg/L and/or ceftazidime MIC > 1 mg/L) yielded a sensitivity of 93.1% and a specificity of 47.8%. Criteria 2 (cefoxitin MIC > 16 mg/L plus ceftriaxone MIC > 4 mg/L) had a sensitivity of 33.3% and a specificity of 98.9%. DHA genes, whose expression may be induced following antibiotic exposure, were present in 19% of isolates testing susceptible to ceftriaxone (MIC ≤ 1 mg/L) and were significantly more likely than CIT genes to be detected in susceptible isolates (P < 0.0001). These findings highlight the importance of using genotypic methods to detect acquired AmpC resistance in E. coli isolates that meet phenotypic screening criteria.IMPORTANCEDetection of transmissible AmpC resistance remains a challenging problem for diagnostic laboratories, especially in Escherichia coli where the expression of its intrinsic AmpC gene can result in phenotypic resistance patterns indistinguishable from plasmid-mediated resistance. In conjunction with whole- genome sequencing (WGS), we describe the development and performance of a novel melt-curve PCR to identify the two most prevalent plasmid-mediated AmpC gene families: CIT and DHA. We then describe phenotypic testing algorithms that incorporate this PCR and can differentiate these from non-acquired resistance in E. coli. It is important to distinguish these, not only to spare patients from unnecessarily being treated with infection control precautions, but also to identify plasmid-mediated genes, especially of the DHA family, that have been associated with inducible drug resistance to third- generation cephalosporins.

Summary: Background: Oropharyngeal carriage of Neisseria meningitidis is frequent during adolescence, representing a major source of invasive meningococcal disease. This study examined the impact of a serogroup B vaccination (Bexsero, GSK 4CMenB) programme on adolescent N. meningitidis carriage using genomic data. Methods: A total 34,489 oropharyngeal samples were collected as part of a state-wide cluster randomised-controlled trial in South Australia during 2017 and 2018 (NCT03089086). Samples were screened for the presence of N. meningitidis DNA by porA PCR prior to culture. Whole genome sequencing was performed on all 1772 N. meningitidis culture isolates and their genomes were analysed. Findings: Unencapsulated meningococci were predominant at baseline (36.3% of isolates), followed by MenB (31.0%), and MenY (20.5%). Most MenB were ST-6058 from hyperinvasive cc41/44, or ST-32 and ST-2870 from cc32. For MenY, ST-23 and ST-1655 from cc23 were prevalent. Meningococcal carriage was mostly unchanged due to the vaccination programme; however, a significant reduction in ST-53 capsule-null meningococci prevalence was observed in 2018 compared to 2017 (OR = 0.52; 95% CI: 0.30–0.87, p = 0.0106). This effect was larger in the vaccinated compared to the control group (OR = 0.37; 95% CI: 0.12–0.98, p = 0.0368). Interpretation: While deployment of the 4CMenB vaccination did not alter the carriage of hyperinvasive MenB in the vaccinated population, it altered the carriage of other N. meningitidis sequence types following the vaccination program. Our findings suggest 4CMenB vaccination is unlikely to reduce transmission of hyperinvasive N. meningitidis strains and therefore ongoing targeted vaccination is likely a more effective public health intervention. Funding: This work was funded by GlaxoSmithKline Biologicals SA.

The gold standard for diagnostics of SARS-CoV-2 (COVID-19) virus is based on real-time polymerase chain reaction (RT-PCR) using centralized PCR facilities and commercial viral RNA extraction kits. One of the key components of these kits are magnetic beads composed of silica coated magnetic iron oxide (Fe2O3 or Fe3O4) nanoparticles, needed for the selective extraction of RNA. At the beginning of the pandemic in 2019, due to a high demand across the world there were severe shortages of many reagents and consumables, including these magnetic beads required for testing for SARS-CoV-2. Laboratories needed to source these products elsewhere, preferably at a comparable or lower cost. Here, we describe the development of a simple, low-cost and scalable preparation of magnetic nanoparticles (MNPs) from biowaste and demonstrate their successful application in viral RNA extraction and the detection of COVID-19. These MNPs have a unique nanoplatelet shape with a high surface area, which are beneficial features, expected to provide improved RNA adsorption, better dispersion and processing ability compared with commercial spherical magnetic beads. Their performance in COVID-19 RNA extraction was evaluated in comparison with commercial magnetic beads and the results presented here showed comparable results for high throughput PCR analysis. The presented magnetic nanoplatelets generated from biomass waste are safe, low-cost, simple to produce in large scale and could provide a significantly reduced cost of nucleic acid extraction for SARS-CoV-2 and other DNA and RNA viruses.