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Background: Campylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide., Methods: We systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids., Results: Overall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3')-IIIa, aph(2'')-If, catA, lnu(C), bla OXA , sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter., Conclusions: The results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety., (© 2024. The Author(s).)

Campylobacter jejuni and Campylobacter coli are the predominant thermophilic species responsible for foodborne gastroenteritis worldwide. Elevated resistance to certain antibiotics was observed due to antimicrobial therapy in farm animals and humans, while reduced antimicrobial usage partially reduced antibiotic resistance. Monitoring the antimicrobial resistance demonstrated a substantial fraction of multi-resistant isolates, indicating the necessity of reliable tools for their detection. In this study, resistance determinants in 129 German and 21 Vietnamese isolates were selected to establish a novel multiplex real-time PCR (qPCR), facilitating the simultaneous detection of four resistance determinants. These comprised tet (O) gene variants associated with tetracycline resistance, point mutations GyrA_T86I and GyrA_T86V associated with ciprofloxacin resistance, and the erm (B) gene together with the point mutation A2075G in the 23S rRNA gene, associated with erythromycin resistance. Moreover, the performance of the qPCR assay was evaluated by comparing the results of qPCR to phenotypic antimicrobial resistance profiles, obtained with standardized EUCAMP3 microdilution panel, which showed 100% similarity (inclusivity and exclusivity). Variation in measurement methods, including qPCR machines and master mixes showed robustness, essential for laboratories. The assay can be used for the rapid detection of resistance determinants, and is beneficial for monitoring the spread of antibiotic resistance in C. jejuni and C. coli.

Introduction: The lack of definitive means to prevent or treat cognitive impairment or dementia is driving intense efforts to identify causal mechanisms. Recent evidence suggests clinically meaningful declines in cognition might present as early as middle age. Studying cognitive changes in middle adulthood could elucidate modifiable factors affecting later cognitive and health outcomes, yet few cognitive ageing studies include this age group. The purpose of the MidCog study is to begin investigations of less-studied and potentially modifiable midlife determinants of later life cognitive outcomes., Methods and Analysis: MidCog is a prospective cohort study of adults ages 35-64, with two in-person interviews 2.5 years apart. Data will be collected from interviews, electronic health records and pharmacy fill data. Measurements will include health literacy, self-management skills, cognitive function, lifestyle and health behaviours, healthcare use, health status and chronic disease outcomes. Associations of health literacy and self-management skills with health behaviours and cognitive/health outcomes will be examined in a series of regression models, and moderating effects of modifiable psychosocial factors.Finally, MidCog data will be linked to an ongoing, parallel cohort study of older adults recruited at ages 55-74 in 2008 ('LitCog'; ages 70-90 in 2023), to explore associations between age, health literacy, self-management skills, chronic diseases, health status and cognitive function among adults ages 35-90., Ethics and Dissemination: The Institutional Review Board at Northwestern University has approved the MidCog study protocol (STU00214736). Results will be published in peer-reviewed journals and summaries will be provided to the funders of the study as well as patients., Competing Interests: Competing interests: SCB reports grants from the NIH, Merck, Pfizer, Gordon and Betty Moore Foundation, Retirement Research Foundation for Aging, Lundbeck, Gilead, and Eli Lilly via her institution and personal fees from Sanofi, Pfizer, University of Westminster, Lundbeck, Gilead, and Luto UK outside the submitted work. PCZ reports grants from the NIH and Vanda via her institution and personal consulting fees from Jazz, Eisai, Harmony and Sleep Number that are outside the submitted work. MW reports grants from the NIH, Gordon and Betty Moore Foundation, and Eli Lilly, and personal fees from Pfizer, Sanofi, Luto UK, University of Westminster, and Lundbeck outside the submitted work. RO’C is supported by a training grant from the National Institute on Aging (K01AG070107). All the other authors report no conflict of interest., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the "gold standard" in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log 10 Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log 10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination. Campylobacter served as a model organism, challenging CFU as "gold standard" and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Quantification of Campylobacter is challenging and one major reason is the fact that bacteria lose cultivability due to cold or oxygen stress during storage at retail. Alternative live/dead discriminatory qPCR currently lacks standardization and might overestimate live cells in the presence of dead cells. In this study an internal sample process control (ISPC) was developed. The ISPC consists of a specified number of peroxide-killed C. sputorum cells to be added to each sample in order to monitor (i) the level of reduction of the signal from dead cells and (ii) DNA losses during sample processing. A species-specific fragment of the 16S rRNA gene of C. sputorum was selected as real-time PCR target, based on its similar size and gene copy number compared to the C. jejuni/coli/lari target and confirmed in an exclusivity study. Extension of the amplification oligonucleotides for the target of thermotolerant Campylobacter improved real-time PCR efficiency, rendering the method suitable for quantification according to international standards. Concordant PCR signal variation of both C. jejuni and C. sputorum targets in co-inoculated chicken rinses verified the suitability of the ISPC. This provides a crucial step towards implementation of cultivation-independent quantification for improved food safety of fastidious bacteria., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The w Mel strain of Wolbachia can reduce the permissiveness of Aedes aegypti mosquitoes to disseminated arboviral infections. Here, we report that w Mel-infected Ae. aegypti (Ho Chi Minh City background), when directly blood-fed on 141 viremic dengue patients, have lower dengue virus (DENV) transmission potential and have a longer extrinsic incubation period than their wild-type counterparts. The w Mel-infected mosquitoes that are field-reared have even greater relative resistance to DENV infection when fed on patient-derived viremic blood meals. This is explained by an increased susceptibility of field-reared wild-type mosquitoes to infection than laboratory-reared counterparts. Collectively, these field- and clinically relevant findings support the continued careful field-testing of w Mel introgression for the biocontrol of Ae. aegypti -born arboviruses., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)