Salmonella enterica serotype Enteritidis (SE) is a common foodborne pathogen that can cause human salmonellosis. Identifying closely related cases is essential to control the pathogen through, e.g. outbreak investigation, but it is often challenging due to the low genetic diversity of SE, particularly with traditional typing methods. This study aimed to investigate the population structure of SE genomes collected during routine surveillance in the Netherlands using whole-genome sequencing (WGS), their clustering, temporal distribution and the association between epidemiological and phenotypic antimicrobial resistance (AMR) factors and the persistence of SE clusters. We also investigated the distribution of genotypic AMR markers among these isolates. The study collection comprised 1,669 unique SE isolates from human infections collected from Dutch surveillance between 2019 and 2023, and their relatedness was derived using core-genome multi-locus sequence typing and Hamming distances. Based on the results, the 216 clusters comprised 1,085 sequences, in addition to 584 sequences depicted as singletons. These clusters predominantly fell within three major lineages, of which two were the previously described Global and Atlantic lineages. Of these clusters, approximately a third persisted for more than 1 year during the 5-year study period. However, no statistically significant associations were found between epidemiological factors, such as age, gender and travel history, or phenotypic AMR and the persistence of SE clusters. The most common AMR genetic markers observed were related to antimicrobial classes of (fluor)quinolones, β-lactamases and aminoglycosides. This study provides a better understanding of the genomic epidemiology of SE in the Netherlands based on WGS. Further analysis that includes samples from the food-chain supply, along with higher resolution methods during a post-Coronavirus Disease of 2019 (COVID-19) period, may provide more insights into the possible causes of the persistence of SE clusters.

Antimicrobial peptides (AMPs) are often produced by eukaryotes to control bacterial populations in both pathogenic and mutualistic symbioses. Several pathogens and nitrogen-fixing legume symbionts depend on transporters called SbmA (or BacA) or BclA (BacA-like) to survive exposure to AMPs. However, how broadly these transporters are distributed amongst bacteria, and their evolutionary history, is poorly understood. We used computational approaches, including phylogenetic and sequence similarity analyses, to examine the distribution of SbmA/BacA and BclA proteins across 1,255 species spanning the domain Bacteria, leading to the identification of 71 and 177 SbmA/BacA and BclA proteins, respectively. In vitro sensitivity assays using legume AMPs and several BclA proteins confirmed that AMP transport is a common feature of BclA homologues. Our analyses indicated that SbmA/BacA homologues are encoded only by species in the phylum Pseudomonadota and are primarily found in just two orders: Hyphomicrobiales and Enterobacterales. BclA homologues are somewhat more broadly distributed and were found in clusters across four phyla. These included several orders of the phyla Pseudomonadota and Cyanobacteriota, the order Mycobacteriales (phylum Actinomycetota) and the class Negativicutes (phylum Bacillota). Many of the clades enriched for species encoding SbmA/BacA or BclA homologues are rich in species that interact with eukaryotic hosts in mutualistic or pathogenic interactions. These observations suggest that SbmA/BacA and BclA proteins have been repeatedly co-opted to facilitate associations with eukaryotic hosts by allowing bacteria to cope with host-encoded AMPs.

RP4, RP1, RK2 and R68 were isolated from the multidrug-resistant bacterial wound isolates in 1969 in the Birmingham Accident Hospital, Birmingham, England, and collectively called Birmingham-group IncP-1α plasmids. These plasmids have been widely used as models to study different aspects of plasmid biology, develop genetic delivery systems and design plasmid vectors. Early studies showed that these plasmids conferred the same antibiotic resistance profile, had a similar size and were undistinguishable from each other using DNA heteroduplex electron microscopy and restriction endonuclease analyses. These observations have led to the widely held assumption that they are identical, although there has been no conclusive supporting evidence. In this work, we sequenced the plasmids RP1 and RP4 from our laboratory strain collection and compared these new sequences with the plasmids RP4 and RK2 assembled from a publicly available sequencing database, showing that the RP1, RP4 and RK2 plasmids are 60 095 bp in length and identical at the nucleotide resolution. Noteworthily, the plasmid sequence is highly conserved despite having been distributed to different labs over 50 years and propagated in different bacterial hosts, strengthening the previous observation that the bacterial host adapts to the RP4/RP1/RK2 plasmid rather than the opposite. In the updated RP4/RP1/RK2 sequence, we found a fusion gene, called pecM-orf2, that was formed putatively by a genetic deletion event. By searching for pecM-orf2 in the National Center for Biotechnology Information database, we detected remnants of the RP4/RP1/RK2 plasmid that carry features of laboratory-engineered vectors in bacterial environmental isolates, either in their chromosome or as a plasmid. This suggests a leak of these plasmids from the laboratory into the environment, which may subsequently impact bacterial evolution and raises concerns about the biocontainment of engineered plasmids when being handled in laboratory settings.

The sequence of the 16S region is taxonomically informative and widely used for genotyping microbes. While it is easy and inexpensive to genotype several isolates by Sanger sequencing the 16S region, this method becomes quite costly if scaled to many isolates. High-throughput sequencing provides one potential avenue for obtaining 16S sequences at scale but presents additional challenges. First, DNA purification workflows for high-throughput sample preparation are labour-intensive and expensive. Second, cost-effective multiplexing and library preparation schemes are difficult to implement for many libraries on a single sequencing run. Therefore, we implemented a scalable protocol for isolate genotyping involving colony polymerase chain reaction (PCR) with simple cell lysis as well as a four-barcode indexing scheme that enables scalable multiplexing and streamlined library preparation by amplifying with four primers simultaneously in a single reaction. We tested this protocol on 93 colonies cultured from environmental samples, and we were able to ascertain the identity of ~90% of microbial isolates.

Staphylococcus aureus (SA) is an opportunistic pathogen and human commensal that is frequently present in the upper respiratory tract, gastrointestinal tract and skin. While SA can cause diseases ranging from minor skin infections to life-threatening bacteraemia, it can also be carried asymptomatically. Indigenous individuals in the Southwest USA experience high rates of invasive SA disease. As carriage is the most significant risk factor for disease, understanding the dynamics of SA carriage, and in particular co-carriage of multiple strains, is important to develop strategies to prevent transmission in vulnerable communities. Here, we investigated SA co-carriage and intrahost evolution by sampling several colonies from multiple anatomical sites and whole-genome sequencing (WGS) on 310 SA isolates collected from 60 Indigenous adults participating in a cross-sectional carriage study. We assessed the richness and diversity of SA isolates via differences in multilocus sequence type, core-genome SNPs and genome content. Using WGS data, we identified 95 distinct SA intra-subject lineages (ISLs) among 60 participants; co-carriage was detected in 42% (25/60). Notably, two participants each carried four distinct SA ISLs. Variation in antibiotic resistance determinants among carried strains was identified among 42% (25/60) of participants. Lastly, we found unequal distribution of clonal complex by body site, suggesting that certain lineages may be adapted to specific anatomical sites. Together, these findings suggest that co-carriage may occur more frequently than previously appreciated and further our understanding of SA intrahost diversity during carriage, which has implications for surveillance activities and epidemiological investigations.

A healthy early-life gut microbiota plays an important role in maintaining immediate and long-term health. Perturbations, particularly in low- to middle-income communities, are associated with increased infection risk. Thus, a promising avenue for restoring a healthy infant microbiota is to select key beneficial bacterial candidates from underexplored microbiomes for developing new probiotic-based therapies. This study aimed to recover bifidobacteria and lactic acid bacteria from the faeces of healthy Cameroonian infants and unravel the genetic basis of their beneficial properties. Faecal samples were collected from 26 infants aged 0-5 months recruited in Dschang (Cameroon). Recovered bacterial isolates were subjected to whole-genome sequencing and in silico analysis to assess their potential for carbohydrate utilization, their antimicrobial capacities, host-adaptation capabilities and their safety. From the range of infant-associated Bifidobacterium and Enterococcus strains identified, Bifidobacterium species were found to harbour putative gene clusters implicated in human milk oligosaccharide metabolism. Genes linked to the production of antimicrobial peptides such as class IV lanthipeptides were found in Bifidobacterium pseudocatenulatum, while those implicated in biosynthesis of cytolysins, enterolysins, enterocins and propeptins, among others, were identified in enterococci. Bifidobacterial isolates did not contain genes associated with virulence; however, we detected the presence of putative tetracycline resistance genes in several strains belonging to Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. longum. Among the enterococci, Enterococcus mundtii PM10 did not carry any genes associated with antimicrobial resistance or virulence. The latter, together with all the Bifidobacterium strains, also encoded several putative adaptive and stress-response-related genes, suggesting robust gastroinstestinal tract colonization potential. This work provides the first genomic characterization of Bifidobacterium and Enterococcus isolates from Cameroonian infants. Several strains showed the genomic potential to confer beneficial properties. Further phenotypic and clinical investigations are needed to confirm their suitability as customized probiotics.

Microbiology Society journals contain high-quality research papers and topical review articles. We are a not-for-profit publisher and we support and invest in the microbiology community, to the benefit of everyone. This supports our principal goal to develop, expand and strengthen the networks available to our members so that they can generate new knowledge about microbes and ensure that it is shared with other communities.

A diverse array of micro-organisms can be found on food, including those that are pathogenic or resistant to antimicrobial drugs. Metagenomics involves extracting and sequencing the DNA of all micro-organisms on a sample, and here, we used a combination of culture and culture-independent approaches to investigate the microbial ecology of food to assess the potential application of metagenomics for the microbial surveillance of food. We cultured common foodborne pathogens and other organisms including Escherichia coli, Klebsiella/Raoultella spp., Salmonella spp. and Vibrio spp. from five different food commodities and compared their genomes to the microbial communities obtained by metagenomic sequencing following host (food) DNA depletion. The microbial populations of retail food were found to be predominated by psychrotrophic bacteria, driven by the cool temperatures in which the food products are stored. Pathogens accounted for a small percentage of the food metagenome compared to the psychrotrophic bacteria, and cultured pathogens were inconsistently identified in the metagenome data. The microbial composition of food varied amongst different commodities, and metagenomics was able to classify the taxonomic origin of 59% of antimicrobial resistance genes (ARGs) found on food to the genus level, but it was unclear what percentage of ARGs were associated with mobile genetic elements and thus transferable to other bacteria. Metagenomics may be used to survey the ARG burden, composition and carriage on foods to which consumers are exposed. However, food metagenomics, even after depleting host DNA, inconsistently identifies pathogens without enrichment or further bait capture.

Microbiology Society journals contain high-quality research papers and topical review articles. We are a not-for-profit publisher and we support and invest in the microbiology community, to the benefit of everyone. This supports our principal goal to develop, expand and strengthen the networks available to our members so that they can generate new knowledge about microbes and ensure that it is shared with other communities.

Ascochyta blight caused by the ascomycete Ascochyta rabiei poses a major biotic threat to chickpea (Cicer arietinum) industries worldwide and incurs substantial costs to the Australian multimillion-dollar chickpea industry in both disease control and yield loss. The fungus was introduced to Australia in the 1970s from an unknown source population and, within a few decades, successfully established in all Australian agroecological chickpea-growing regions. Although genetically highly clonal, a broad range of phenotypic variation in terms of aggressiveness exists among the Australian A. rabiei isolates. More recently, highly aggressive isolates capable of causing severe disease symptoms on moderate to highly resistant chickpea cultivars have increased in frequency. To identify genetic loci potentially associated with A. rabiei aggressiveness on Australian chickpea cultivars, we performed deep genome sequencing of 230 isolates collected from a range of agroecological chickpea-growing regions between 2013 and 2020. Population genetic analyses using genome-wide SNP data identified three main clusters of genetically closely related isolates in Australia. Phylogenetic analyses showed that highly aggressive phenotypes developed multiple times independently throughout the phylogeny. The results point to a minor contribution of multiple genetic regions and most likely epigenomic variations to aggressiveness of A. rabiei isolates on Australian chickpea cultivars.