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Abstract 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.

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.

Reported cases of listeriosis from food of non-animal origin (FNAO) are increasing. In order to assess the risk of exposure to Listeria monocytogenes from FNAO, the genetic characterization of the pathogen in FNAO products and in primary production and processing plants needs to be investigated. For this, 123 samples of fresh and frozen soft fruit and 407 samples of 39 plants in Bavaria, Germany that produce and process FNAO were investigated for Listeria contamination. As a result, 64 Listeria spp. isolates were detected using ISO 11290-1:2017. Environmental swabs and water and food samples were investigated. L. seeligeri (36/64, 56.25%) was the most frequently identified species, followed by L. monocytogenes (8/64, 12.50%), L. innocua (8/64, 12.50%), L. ivanovii (6/64, 9.38%), L. newyorkensis (5/64, 7.81%), and L. grayi (1/64, 1.56%). Those isolates were subsequently sequenced by whole-genome sequencing and subjected to pangenome analysis to retrieve data on the genotype, serotype, antimicrobial resistance (AMR), and virulence markers. Eight out of sixty-four Listeria spp. isolates were identified as L. monocytogenes. The serogroup analysis detected that 62.5% of the L. monocytogenes isolates belonged to serogroup IIa (1/2a and 3a) and 37.5% to serogroup IVb (4b, 4d, and 4e). Furthermore, the MLST (multilocus sequence typing) analysis of the eight detected L. monocytogenes isolates identified seven different sequence types (STs) and clonal complexes (CCs), i.e., ST1/CC1, ST2/CC2, ST6/CC6, ST7/CC7, ST21/CC21, ST504/CC475, and ST1413/CC739. The core genome MLST analysis also showed high allelic differences and suggests plant-specific isolates. Regarding the AMR, we detected phenotypic resistance against benzylpenicillin, fosfomycin, and moxifloxacin in all eight L. monocytogenes isolates. Moreover, virulence factors, such as prfA, hly, plcA, plcB, hpt, actA, inlA, inlB, and mpl, were identified in pathogenic and nonpathogenic Listeria species. The significance of L. monocytogenes in FNAO is growing and should receive increasing levels of attention.

Interpretation of whole-genome sequencing (WGS) data for foodborne outbreak investigations is complex, as the genetic diversity within processing plants and transmission events need to be considered. In this study, we analyzed 92 food-associated Listeria monocytogenes isolates by WGS-based methods. We aimed to examine the genetic diversity within meat and fish production chains and to assess the applicability of suggested thresholds for clustering of potentially related isolates. Therefore, meat-associated isolates originating from the same samples or processing plants as well as fish-associated isolates were analyzed as distinct sets. In silico serogrouping, multilocus sequence typing (MLST), core genome MLST (cgMLST), and pangenome analysis were combined with screenings for prophages and genetic traits. Isolates of the same subtypes (cgMLST types (CTs) or MLST sequence types (STs)) were additionally compared by SNP calling. This revealed the occurrence of more than one CT within all three investigated plants and within two samples. Analysis of the fish set resulted in predominant assignment of isolates from pangasius catfish and salmon to ST2 and ST121, respectively, potentially indicating persistence within the respective production chains. The approach not only allowed the detection of distinct subtypes but also the determination of differences between closely related isolates, which need to be considered when interpreting WGS data for surveillance.

Tissue-specific transcripts are likely to be of importance for the corresponding organ. While attempting to define the specific transcriptome of the human lung, we identified the transcript of a yet uncharacterized protein, SFTA2. In silico analyses, biochemical methods, fluorescence imaging and animal challenge experiments were employed to characterize SFTA2. Human SFTA2 is located on Chr. 6p21.33, a disease-susceptibility locus for diffuse panbronchiolitis. RT-PCR verified the abundance of SFTA2-specific transcripts in human and mouse lung. SFTA2 is synthesized as a hydrophilic precursor releasing a 59 amino acid mature peptide after cleavage of an N-terminal secretory signal. SFTA2 has no recognizable homology to other proteins while orthologues are present in all mammals. SFTA2 is a glycosylated protein and specifically expressed in nonciliated bronchiolar epithelium and type II pneumocytes. In accordance with other hydrophilic surfactant proteins, SFTA2 did not colocalize with lamellar bodies but colocalized with golgin97 and clathrin-labelled vesicles, suggesting a classical secretory pathway for its expression and secretion. In the mouse lung, Sfta2 was significantly downregulated after induction of an inflammatory reaction by intratracheal lipopolysaccharides paralleling surfactant proteins B and C but not D. Hyperoxia, however, did not alter SFTA2 mRNA levels. We have characterized SFTA2 and present it as a novel unique secretory peptide highly expressed in the lung.