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Background Campylobacter spp. infections present an urgent and significant global public health challenge. This study offers a genomic characterization of Campylobacter spp. isolates from human samples and chicken-origin food sources in Italy during 2023. Whole-genome sequencing, cluster analysis, genomic comparison of human and food of animal origin isolates, and characterization of antimicrobial resistance mechanisms were performed on 257 isolates. Results Campylobacter jejuni revealed considerable variability, with predominant types, including ST21, ST50 (ST21 CC), and ST3335 (ST206 CC), in human isolates, whereas ST50 and ST2116 (ST353 CC) prevailed in food sources. In contrast, C coli isolates were associated primarily with the ST828 CC. Cluster analysis based on core-genome multilocus sequence typing (cgMLST) revealed that 40.0% of the isolates formed 23 genetic clusters, suggesting potential outbreak scenarios. The isolates presented a high prevalence of ciprofloxacin resistance linked to mutations in GyrA (80.1%) and extensive tetracycline resistance attributed to tet genes (64.6%). Multidrug resistance was predominantly observed in C. coli (12.1%). Notably, the chromosomal erythromycin resistance gene erm (N) in food C. coli isolates was detected for the first time in Italy and for the first time worldwide in a veterinarian or food isolate. Conclusions This One Health study, which was based on genomic surveillance, improved our understanding of antimicrobial resistance and the epidemiology of Campylobacter spp. This study highlights the emergence of a novel antimicrobial resistance marker in Italy, erm (N), and reveals the prospective occurrence of multiple nationwide outbreak scenarios that demand immediate public health attention.

Public health and regulatory agencies worldwide sequence all Listeria monocytogenes isolates obtained as part of routine surveillance and outbreak investigations. Many of these entities submit the sequences to the National Center for Biotechnology Information Pathogen Detection (NCBI PD) database, which groups the L. monocytogenes isolates into single nucleotide polymorphism (SNP) clusters based on a pairwise SNP difference threshold of 50 SNPs. Our goal was to assess whether isolates with metadata that suggest different sources or locations could show evidence for close genetic relatedness indicating a recent common ancestor and a possible unknown common source. We compared the whole genome sequencing (WGS) data of 249 L. monocytogenes isolates sequenced here, which have detailed metadata, with WGS data of nonclinical isolates on NCBI PD. The 249 L. monocytogenes isolates originated from natural environments (n = 91) as well as from smoked fish (n = 62), dairy (n = 56), and deli meat (n = 40) operations in the United States. Using a combination of subtyping by core genome multilocus sequence typing and high-quality SNP, we observed five SNP clusters in which study isolates and SNP cluster isolates seemed to be closely related and either (i) shared the same geolocation but showed different source types (one SNP cluster); (ii) shared the same source type but showed different geolocations (two SNP clusters); or (iii) shared neither source type nor geolocation (two SNP clusters). For one of the two clusters under (iii), there was, however, no strong bootstrap support for a common ancestor shared between the study isolates and SNP cluster isolates, indicating the value of in-depth evolutionary analyses when WGS data are used for traceback and epidemiological investigations. Overall, our results demonstrate that some L. monocytogenes subtypes may be associated with specific locations or commodities; these associations can help in investigations involving multi-ingredient foods such as sandwiches. However, at least some L. monocytogenes subtypes can be widespread geographically and can be associated with different sources, which may present a challenge to traceback investigations involving these subtypes.

is primarily transmitted via contaminated food and can cause listeriosis, an infection often associated with sepsis and meningitis in at-risk individuals. Accurate outbreak detection relies on whole genome sequencing (WGS) and core genome multilocus sequence typing (cgMLST), which use allele thresholds to identify related strains. This study investigated mutation rates in , focusing on isolates with DNA repair deficiencies. Serial subcultivations were performed, comparing a repair-deficient isolate with a wild-type control. Genetic variability was assessed using WGS and cgMLST. Mutation rates were significantly higher in repair-deficient isolates, exceeding typical cgMLST thresholds currently used in outbreak investigations, leading to a misclassification of related isolates as unrelated. An additional analysis of the Austrian database revealed that such deficiencies are rare among isolates. The standard 7-allele cgMLST threshold effectively identifies related strains in most cases, but may require adjustments for hypermutator strains. Incorporating DNA repair data could improve the accuracy of outbreak investigations, ensuring reliable public health responses.

In industrialized countries, the leading cause of bacterial gastroenteritis is Campylobacter jejuni. However, outbreaks are rarely reported, which may reflect limitations of surveillance, for which molecular typing is not routinely performed. To determine the frequency of genetic clusters among patients and to find links to concurrent isolates from poultry meat, broiler chickens, cattle, pigs, and dogs, we performed whole-genome sequencing on 1,509 C. jejuni isolates from 774 patients and 735 food or animal sources in Denmark during 2015-2017. We found numerous clusters; 366/774 (47.3%) clinical isolates formed 104 clusters of >2 isolates. A total of 41 patient clusters representing 199/366 (54%) patients matched a potential source, primarily domestic chickens/broilers. This study revealed serial outbreaks and numerous matches to concurrent food and animal isolates and highlighted the potential of whole-genome sequencing for improving routine surveillance of C. jejuni by enhancing outbreak detection, source tracing, and potentially prevention of human infections.

Background In 2015, in addition to a United States multistate outbreak linked to contaminated ice cream, another outbreak linked to ice cream was reported in the Pacific Northwest of the United States. It was a hospital-acquired outbreak linked to milkshakes, made from contaminated ice cream mixes and milkshake maker, served to patients. Here we performed multiple analyses on isolates associated with this outbreak: pulsed-field gel electrophoresis (PFGE), whole genome single nucleotide polymorphism (SNP) analysis, species-specific core genome multilocus sequence typing (cgMLST), lineage-specific cgMLST and whole genome-specific MLST (wgsMLST)/outbreak-specific cgMLST. We also analyzed the prophages and virulence genes. Results The outbreak isolates belonged to sequence type 1038, clonal complex 101, genetic lineage II. There were no pre-mature stop codons in inlA . Isolates contained Listeria Pathogenicity Island 1 and multiple internalins. PFGE and multiple whole genome sequencing (WGS) analyses all clustered together food, environmental and clinical isolates when compared to outgroup from the same clonal complex, which supported the finding that L. monocytogenes likely persisted in the soft serve ice cream/milkshake maker from November 2014 to November 2015 and caused 3 illnesses, and that the outbreak strain was transmitted between two ice cream production facilities. The whole genome SNP analysis, one of the two species-specific cgMLST, the lineage II-specific cgMLST and the wgsMLST/outbreak-specific cgMLST showed that L. monocytogenes cells persistent in the milkshake maker for a year formed a unique clade inside the outbreak cluster. This clustering was consistent with the cleaning practice after the outbreak was initially recognized in late 2014 and early 2015. Putative prophages were conserved among prophage-containing isolates. The loss of a putative prophage in two isolates resulted in the loss of the Asc I restriction site in the prophage, which contributed to their Asc I-PFGE banding pattern differences from other isolates. Conclusions The high resolution of WGS analyses allowed the differentiation of epidemiologically unrelated isolates, as well as the elucidation of the microevolution and persistence of isolates within the scope of one outbreak. We applied a wgsMLST scheme which is essentially the outbreak-specific cgMLST. This scheme can be combined with lineage-specific cgMLST and species-specific cgMLST to maximize the resolution of WGS.

The proportion of campylobacteriosis caused by C. coli is increasing rapidly in China. Coincidentally, the dominant species of Campylobacter occurring in poultry products has shifted from C. jejuni to C. coli . Campylobacter is the leading bacterial cause of diarrheal illnesses worldwide. Campylobacter jejuni and C. coli are the most common species accounting for campylobacteriosis. Although the proportion of campylobacteriosis caused by C. coli is increasing rapidly in China, the underlying mechanisms of this emergence remain unclear. In this study, we analyzed the whole-genome sequences and associated environments of 1,195 C. coli isolates with human, poultry, or porcine origins from 1980 to 2021. C. coli isolates of human origin were closely related to those from poultry, suggesting that poultry was the main source of C. coli infection in humans. Analysis of antimicrobial resistance determinants indicated that the prevalence of multidrug-resistant C. coli has increased dramatically since the 2010s, coinciding with the shift in abundance from C. jejuni to C. coli in Chinese poultry. Compared with C. jejuni , drug-resistant C. coli strains were better adapted and showed increased proliferation in the poultry production environment, where multiple antimicrobial agents were frequently used. This study provides an empirical basis for the molecular mechanisms that have enabled C. coli to become the dominant Campylobacter species in poultry; we also emphasize the importance of poultry products as sources of campylobacteriosis caused by C. coli in human patients. IMPORTANCE The proportion of campylobacteriosis caused by C. coli is increasing rapidly in China. Coincidentally, the dominant species of Campylobacter occurring in poultry products has shifted from C. jejuni to C. coli . Here, we analyzed the whole-genome sequences of 1,195 C. coli isolates from different origins. The phylogenetic relationship among C. coli isolates suggests that poultry was the main source of C. coli infection in humans. Further analysis indicated that antimicrobial resistance in C. coli strains has increased dramatically since the 2010s, which could facilitate their adaptation in the poultry production environment, where multiple antimicrobial agents are frequently used. Thus, our findings suggest that the judicious use of antimicrobial agents could mitigate the emergence of multidrug-resistant C. coli strains and enhance clinical outcomes by restoring drug sensitivity in Campylobacter .

ABSTRACT Pseudomonas cichorii is divided into two subclades based on the 16S ribosomal RNA gene sequence and core genome multilocus sequence typing. It was shown that subclade 2 strains utilize d-tartrate as a sole carbon source, whereas subclade 1 strains do not. Draft genome sequencing was performed with P. cichorii strains to identify d-tartrate utilization genes. By genome comparative and homology search studies, an ∼7.1-kb region was identified to be involved in d-tartrate utilization. The region is subclade 2 specific, and contains tarD and dctA genes, which encode a putative enzyme and transporter of d-tartrate, respectively. When the region was introduced into subclade 1 strains, the transformants were able to utilize d-tartrate. Partial fragments of tarD and dctA were amplified from all subclade 2 strains tested in this study by PCR using gene-specific primers, but not from subclade 1 strains. This is the first report on the genetic analysis of biochemical characteristics corresponding to a specific phylogenetic group in P. cichorii. Pseudomonas cichorii subclade 2 utilizes d-tartrate as the sole carbon source, and the DNA region involved in this characteristic was identified by genome comparative and biochemical studies.

Pertussis is a vaccine-preventable disease, and its recent resurgence might be attributable to the emergence of strains that differ genetically from the vaccine strain. We describe a novel pertussis isolate-based surveillance system and a core genome multilocus sequence typing scheme to assess Bordetella pertussis genetic variability and investigate the increased incidence of pertussis in Austria. During 2018-2020, we obtained 123 B. pertussis isolates and typed them with the new scheme (2,983 targets and preliminary cluster threshold of <6 alleles). B. pertussis isolates in Austria differed genetically from the vaccine strain, both in their core genomes and in their vaccine antigen genes; 31.7% of the isolates were pertactin-deficient. We detected 8 clusters, 1 of them with pertactin-deficient isolates and possibly part of a local outbreak. National expansion of the isolate-based surveillance system is needed to implement pertussis-control strategies.

Background Clostridioides difficile infections (CDI) present significant health risks and are among the most important nosocomial infections. Ribotype (RT) 027 poses a particular risk due to its proposed “hypervirulence”. Traditionally, C. difficile isolates are characterized using PCR-ribotyping. More recently, whole genome sequence (WGS) analysis is increasingly used, which may provide a higher discriminatory power. This study aimed to assess the distribution of different C. difficile RTs in hospitals in the Berlin-Brandenburg area, and to analyse the heterogeneity within isolates of different ribotypes using WGS. Methods Between February 2020 and November 2021, stool samples from patients with laboratory-confirmed CDI were collected from 22 hospitals (approximately 13,900 beds) in Berlin and Brandenburg. Toxigenic isolates ( n  = 476) were further characterized by ribotyping, antibiotic susceptibility testing, toxinotyping, and core genome multilocus sequence typing (cgMLST). Results Sixty-five different RTs were detected, with RT014 (16.1%), RT027 (12.8%), and RT001 (7.6%) being the most prevalent. RT027 isolates exhibited resistance to several antibiotics. Further, cgMLST analysis revealed very close genetic relatedness between RT027 isolates despite being epidemiologically unrelated. Similar findings of a monomorphic population were observed for RT078 isolates. In contrast, other RTs showed a heterogenic population structure. Conclusions This study provides first insights into the distribution of C. difficile genotypes, corresponding antimicrobial resistance, and clonal relatedness using cgMLST, highlighting RT027 as the second most common genotype for the studied area. For the monomorphic RT027 and RT078 populations, new definitions of clonal relatedness might be necessary.

Paenibacillus larvae causes the American foulbrood (AFB), a highly contagious and devastating disease of honeybees. Whole-genome sequencing (WGS) has been increasingly used in bacterial pathogen typing, but rarely applied to study the epidemiology of P. larvae . To this end, we used 125 P. larvae genomes representative of a species-wide diversity to construct a stable whole-genome multilocus sequence typing (wgMLST) scheme consisting of 5745 loci. A total of 51 P. larvae isolates originating from AFB outbreaks in Slovenia were used to assess the epidemiological applicability of the developed wgMLST scheme. In addition, wgMLST was compared with the core-genome MLST (cgMLST) and whole-genome single nucleotide polymorphism (wgSNP) analyses. All three approaches successfully identified clusters of outbreak-associated strains, which were clearly separated from the epidemiologically unlinked isolates. High levels of backward comparability of WGS-based analyses with conventional typing methods (ERIC-PCR and MLST) were revealed; however, both conventional methods lacked sufficient discriminatory power to separate the outbreak clusters. The developed wgMLST scheme provides an improved understanding of the intra- and inter-outbreak genetic diversity of P. larvae and represents an important progress in unraveling the genomic epidemiology of this important honeybee pathogen.