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Two physical treatments (heat via water bath and cold air) with various temperatures (20/70/75/80°C and - 80/-90°C) and exposure times (20, 30, 40 s) were carried out to identify a decontaminating effect on zoonotic pathogens on broiler carcasses. Subsequently, carcasses were analyzed for thermotolerant Campylobacter (C.), Salmonella, Escherichia (E.). coli and total colony count (TCC). Moreover, for the hot water treatment, qPCR with viable/dead differentiation (v-qPCR) was applied to detect viable but non-culturable cells (VBNC) of Campylobacter, referred to as intact but putatively infectious units (IPIU). Hot water immersion was tested on carcasses inoculated with C. jejuni and Salmonella, while cold air treatment was evaluated for naturally contaminated carcasses of broiler flocks colonized with Campylobacter. For hot water treatment, the statistically significant reducing effect was about 1 log10 CFU/ml for both Salmonella and Campylobacter for 70-80°C and 20/30 s treatments. The effect of heat treatment for Campylobacter was smaller when samples were analyzed with v-qPCR with reductions of 0.5-0.8 log10 IPIU/ml in mean. Cold air treatments at -90°C were effective in reducing the mean contamination level of Campylobacter by 0.4-0.5 log10 CFU/ml at all exposure times (p < 0.05). Hot water treatments showed a decreasing trend on TCC by 0.6-0.9 log10 CFU/ml (p < 0.05). TCC counts were not significantly affected by cold air treatment. For E. coli no statistically significant reductions were observed by hot water treatment. The cold air treatment at -90°C for 20 and 40 s led to a reduction of E. coli by 0.4 and 0.8 log10 CFU/ml (p < 0.05), respectively. Treatment of carcasses with higher bacterial levels tended to show higher reduction. The research demonstrated that the efficacy of physical treatments for decontamination of broiler carcasses was more pronounced for hot water immersion than for cold air exposure. In conclusion, the results shed light on the potential application of these physical treatments in practice to reduce the quantitative load of contaminating pathogens to enhance food safety in the broiler meat production. [ABSTRACT FROM AUTHOR]

Campylobacter spp. cannot grow in raw milk, but it is able to transform into a viable but non‐culturable (VBNC) state enabling the survival in such harsh conditions. In this study, Campylobacter jejuni survival in raw milk was investigated taken into consideration colony‐forming units (CFUs) and VBNC cells. CFU from two different strains of C. jejuni (DSM 4688 and BfR‐CA‐18043) were enumerated at three temperatures (5°C, 8°C, and 12°C). In parallel, a viability real‐time PCR was conducted to quantify intact and putatively infectious units (IPIUs) (comprising CFU and VBNC bacteria). The data generated were used to model the viability of C. jejuni during raw milk storage. Here, a one‐step fitting approach was performed using parameter estimates from an intermediate two‐step fit as starting values to generate tertiary models. Different primary model equations (Trilinear and Weibull) were required to fit the CFU and the IPIU data. Strain‐specific linear secondary models were generated to analyze the effect of storage temperature on the maximum specific inactivation rate of the CFU data. The time of the first decimal reduction parameter of the IPIU models could be modeled by a strain‐independent linear secondary model. The developed tertiary models for CFU and IPIU differ significantly in their predictions, for example, for the time required for a one log10 reduction. Taken into consideration that VBNC could revert to a culturable state during the raw milk storage, our results underline the importance of considering IPIU and not only CFU to avoid underestimation of the survival of C. jejuni in raw milk. [ABSTRACT FROM AUTHOR]

Thermotolerant Campylobacter spp. are the most frequent cause of foodborne bacterial diarrhea and high-priority antibiotic-resistant pathogens, according to the World Health Organization (WHO). Monitoring revealed current low prevalence of gentamicin resistance in European Campylobacter spp. isolates but substantial presence of gentamicin modifying genes circulating globally. Using a combined approach of natural transformation and whole-genome sequencing, we revealed a novel gentamicin resistance mechanism, namely the point mutation A1387G in the 16S rRNA gene, originally identified in a C. coli isolate from turkey caecal content. The transformation rate of the resistance using genomic DNA of the resistant donor to sensitive recipient C. jejuni and C. coli was ~2.5 log 10 lower compared to the control rpsL -A128G point mutation conferring streptomycin resistance. Antimicrobial susceptibility tests showed cross-resistance to apramycin, kanamycin, and tobramycin, with transformants exhibiting more than 4- to 8-fold increased MICs to apramycin and tobramycin and over 64-fold higher MICs to kanamycin compared to wild-type isolates. Although transformants showed 177-1,235 variations relative to the recipient, only the A1387G point mutation in the 16S rRNA was in common. This mutation was causal for resistance, as transformation of a 16S rRNA&#95;A1387G PCR fragment into susceptible isolates also led to resistant transformants. Sanger sequencing of the 16S rRNA genes and Oxford nanopore whole-genome sequencing of transformants identified clones harboring either all three copies with A1387G or a mixed population of wild-type and mutated 16S rRNA gene alleles. Within 15 passages on non-selective medium, transformants with mixed populations of the 16S rRNA gene copies partially reverted to wild type, both geno- and phenotypically. In contrast, transformants harboring the A1387G point mutation in all three 16S rRNA gene copies kept full resistance within at least 45 passages. We speculate that partial acquisition and rapid loss of the point mutation limited its spread among C . spp. isolates. In-depth knowledge on resistance mechanisms contributes to optimal diagnosis and preventative measures., Competing Interests: The authors declare no conflict of interest.

Campylobacter jejuni and Arcobacter butzleri are microaerobic food-borne human gastrointestinal pathogens that mainly cause diarrheal disease. These related species of the Campylobacteria class face variable atmospheric environments during infection and transmission, ranging from nearly anaerobic to aerobic conditions. Consequently, their lifestyles require that both pathogens need to adjust their metabolism and respiration to the changing oxygen concentrations of the colonization sites. Our transcriptomic and proteomic studies revealed that C. jejuni and A. butzleri, lacking a Campylobacteria -specific regulatory protein, C. jejuni Cj1608, or a homolog, A. butzleri Abu0127, are unable to reprogram tricarboxylic acid cycle or respiration pathways, respectively, to produce ATP efficiently and, in consequence, adjust growth to changing oxygen supply. We propose that these Campylobacteria energy and metabolism regulators (CemRs) are long-sought transcription factors controlling the metabolic shift related to oxygen availability, essential for these bacteria's survival and adaptation to the niches they inhabit. Besides their significant universal role in Campylobacteria , CemRs, as pleiotropic regulators, control the transcription of many genes, often specific to the species, under microaerophilic conditions and in response to oxidative stress., Importance: C. jejuni and A. butzleri are closely related pathogens that infect the human gastrointestinal tract. In order to infect humans successfully, they need to change their metabolism as nutrient and respiratory conditions change. A regulator called CemR has been identified, which helps them adapt their metabolism to changing conditions, particularly oxygen availability in the gastrointestinal tract so that they can produce enough energy for survival and spread. Without CemR, these bacteria, as well as a related species, Helicobacter pylori , produce less energy, grow more slowly, or, in the case of C. jejuni , do not grow at all. Furthermore, CemR is a global regulator that controls the synthesis of many genes in each species, potentially allowing them to adapt to their ecological niches as well as establish infection. Therefore, the identification of CemR opens new possibilities for studying the pathogenicity of C. jejuni and A. butzleri ., Competing Interests: The authors declare no conflict of interest.

Bacterial plasmids play a major role in the spread of antibiotic resistance genes. However, their characterization via DNA sequencing suffers from the low abundance of plasmid DNA in those samples. Although sample preparation methods can enrich the proportion of plasmid DNA before sequencing, these methods are expensive and laborious, and they might introduce a bias by enriching only for specific plasmid DNA sequences. Nanopore adaptive sampling could overcome these issues by rejecting uninteresting DNA molecules during the sequencing process. In this study, we assess the application of adaptive sampling for the enrichment of low-abundant plasmids in known bacterial isolates using two different adaptive sampling tools. We show that a significant enrichment can be achieved even on expired flow cells. By applying adaptive sampling, we also improve the quality of de novo plasmid assemblies and reduce the sequencing time. However, our experiments also highlight issues with adaptive sampling if target and non-target sequences span similar regions., Importance: Antimicrobial resistance causes millions of deaths every year. Mobile genetic elements like bacterial plasmids are key drivers for the dissemination of antimicrobial resistance genes. This makes the characterization of plasmids via DNA sequencing an important tool for clinical microbiologists. Since plasmids are often underrepresented in bacterial samples, plasmid sequencing can be challenging and laborious. To accelerate the sequencing process, we evaluate nanopore adaptive sampling as an in silico method for the enrichment of low-abundant plasmids. Our results show the potential of this cost-efficient method for future plasmid research but also indicate issues that arise from using reference sequences., Competing Interests: J.U.U. and B.Y.R. have filed a patent application on selective nanopore sequencing approaches.