- Volume 11, Issue 2, 2025

Rates of bacterial sexually transmitted infections (STIs) are rising, and accessing their genomes provides information on strain evolution, circulating strains and encoded antimicrobial resistance (AMR). Notable pathogens include Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Treponema pallidum (TP), globally the most common bacterial STIs. Mycoplasmoides (formerly Mycoplasma) genitalium (MG) is also a bacterial STI that is of concern due to AMR development. These bacteria are also fastidious or hard to culture, and standard sampling methods lyse bacteria, completely preventing pathogen culture. Clinical samples contain large amounts of human and other microbiota DNA. These factors hinder the sequencing of bacterial STI genomes. We aimed to overcome these challenges in obtaining whole-genome sequences and evaluated four approaches using clinical samples from Argentina (39), and Switzerland (14), and cultured samples from Finland (2) and Argentina (1). First, direct genome sequencing from swab samples was attempted through Illumina deep metagenomic sequencing, showing extremely low levels of target DNA, with under 0.01% of the sequenced reads being from the target pathogens. Second, host DNA depletion followed by Illumina sequencing was not found to produce enrichment in these very low-load samples. Third, we tried a selective long-read approach with the new adaptive sequencing from Oxford Nanopore Technologies, which also did not improve enrichment sufficiently to provide genomic information. Finally, target enrichment using a novel pan-genome set of custom SureSelect probes targeting CT, NG, TP and MG followed by Illumina sequencing was successful. We produced whole genomes from 64% of CT-positive samples, from 36% of NG-positive samples and 60% of TP-positive samples. Additionally, we enriched MG DNA to gain partial genomes from 60% of samples. This is the first publication to date to utilize a pan-genome STI panel in target enrichment. Target enrichment, though costly, proved essential for obtaining genomic data from clinical samples. These data can be utilized to examine circulating strains and genotypic resistance and guide public health strategies.

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.

Carbapenem-resistant Acinetobacter baumannii is recognized by the World Health Organization (WHO) as one of the top priority pathogens. Despite its public health importance, genomic data of clinical isolates from Malaysia remain scarce. In this study, whole-genome sequencing was performed on 126 A. baumannii isolates collected from the main tertiary hospital in the state of Terengganu, Malaysia, over a 10-year period (2011–2020). Antimicrobial susceptibilities determined for 20 antibiotics belonging to 8 classes showed that 77.0% (n=97/126) of the isolates were categorized as multidrug resistant (MDR), with all MDR isolates being carbapenem resistant. Multilocus sequence typing analysis categorized the Terengganu A. baumannii clinical isolates into 34 Pasteur and 44 Oxford sequence types (STs), with ST2Pasteur of the Global Clone 2 lineage identified as the dominant ST (n=76/126; 60.3%). The ST2Pasteur isolates could be subdivided into six Oxford STs with the majority being ST195Oxford (n=35) and ST208Oxford (n=17). Various antimicrobial resistance genes were identified with the bla OXA-23-encoded carbapenemase being the predominant acquired carbapenemase gene (n=90/126; 71.4%). Plasmid-encoded rep genes were identified in nearly all (n=122/126; 96.8%) of the isolates with the majority being Rep_3 family (n=121). Various virulence factors were identified, highlighting the pathogenic nature of this bacterium. Only 14/126 (11.1%) of the isolates were positive for the carriage of CRISPR-Cas arrays with none of the prevalent ST2Pasteur isolates harbouring them. This study provided a genomic snapshot of the A. baumannii isolates obtained from a single tertiary healthcare centre in Malaysia over a 10-year period and showed the predominance of a single closely related ST2Pasteur lineage, indicating the entrenchment of this clone in the hospital.

Acinetobacter baumannii is a nosocomial pathogen associated with various infections, including urinary tract infections (UTIs). In the course of an infection, A. baumannii is known to rapidly become resistant to antibiotic therapy, but much less is known about possible adaptation without antibiotic pressure. Through a retrospective study, we investigated within-host genetic diversity during a subclinical 5-year UTI in an animal–patient after withdrawal of colistin treatment. We conducted whole-genome sequencing and phenotypic assays on 17 clonally related isolates from the Sequence Type 25 lineage. Phylogenomic analysis revealed their proximity with animal and human strains from the same country suggesting zoonotic transmission (France). In this case study, the clonally related strains presented variations in genome sizes and nucleotide sequences. Over the course of the infection, A. baumannii underwent genome reduction through insertion sequence (IS) recombination, phage excision or plasmid curing. Alongside this global genome reduction, we observed an expansion of IS17, initially located on the endogenous large plasmid. Genetic variations were mainly located in biofilm formation and metabolism genes. We observed repeated variations affecting three biofilm genes and two adhesion operons associated with weak biofilm-forming capacity. Conversely, only two metabolic genes were recurrently affected, and phenotypic assays indicated a rather stable metabolism profile between the isolates suggesting minor adaptations to its host. Lastly, an overall decreased antibiotic resistance – expected in the absence of antibiotic treatment – contrasted with a conserved colistin resistance due to a pmrB mutation among the isolates.

Assessing individual and population health in endangered wildlife poses unique challenges due to the lack of an adequate baseline and ethical constraints on invasive sampling. For endangered bats, minimally invasive samples like guano can often be the ethical and technical limit for studies of pathogens and the microbiome. In this study, we use multiplex metabarcode sequencing to describe the faecal microbiome and parasites of 56 Indiana bats (Myotis sodalis). We show evidence of a high prevalence of Eimeria spp. protozoan parasite and characterize associations between infection and changes to the faecal microbiome. We identify a strong and significant enrichment of Clostridium species in Eimeria-positive bats, including isolates related to Clostridium perfringens.

Penaeus vannamei singly enveloped nuclear polyhedrosis virus (PvSNPV), also known as Baculovirus penaei (BP), is the first viral pathogen of penaeid shrimp described in 1974. Although PvSNPV was discovered almost 50 years ago, the complete genome sequence has not been elucidated until now. We detected the virus in a quarantine stock of P. vannamei shrimp by light microscopy of faecal samples and by PCR screening of broodstock. Subsequently, next-generation sequencing was deployed to determine the complete genome sequence of PvSNPV. The PvSNPV genome is a circular, double-stranded DNA molecule of 119 883 bp in length encoding 101 ORFs. The deduced aa sequences from 28 ORFs were homologous to 28 core proteins from all identified nudiviruses. Phylogenetic analyses based on deduced aa sequences of the core genes and orthologous genes revealed that PvSNPV clusters with Penaeus monodon nudivirus. Therefore, we propose to rename BP/PvSNPV as P. vannamei nudivirus and re-assign the virus to the family Nudiviridae instead of Baculoviridae.

Edwardsiella ictaluri continues to pose a significant risk to the health and production of striped catfish (Pangasianodon hypophthalmus) in Vietnam. Whilst recent advances in genomic sequencing provide an insight into the global genomic diversity of this important fish pathogen, genome-wide analysis of Vietnamese isolates recovered over time is lacking. In this study, we used a whole-genome sequencing approach to compare the genomes of 31 E. ictaluri isolates recovered over a 20-year period (2001–2021) and performed comparative genomic analysis to explore temporal changes in genome diversity, population structure and mechanisms driving pathogenesis and antimicrobial resistance. Our findings revealed an open pan-genome with 4148 genes and a core genome (3 060 genes) accounting for over two-thirds of the genome. Moreover, we found the genomes sequenced to classify into two distinct lineages and estimated the ancestral origin of these lineages within Vietnam to date back to the 1950s. Plasmids were highly prevalent in Vietnamese E. ictaluri, with isolates harbouring up to four plasmids within their genome. Further, a diverse mobilome was observed with nine different plasmid types detected across the genome collection. Exploration of putative plasmids revealed a diverse set of antimicrobial resistance genes (ARGs) against key antibiotics used in Vietnamese aquaculture and virulence genes associated with protein secretion systems. Correlation analysis revealed the total number of ARGs detected in genomes to increase with isolate recovery time. Whilst the number of virulence genes remained relatively stable, temporal variation was noted in several virulence factors related to motility and immune system modulation. Findings from this study highlight the need for continued genomic surveillance to monitor changes in antimicrobial resistance and pathogenesis, to help inform the development of disease control and management strategies.

The combined application of furazolidone and vancomycin has previously been shown to be synergistic against Gram-negative pathogens, with great therapeutic promise. However, the emergence and mechanism of resistance to this antibiotic combination have not been characterized. To fill this gap, we here selected Escherichia coli progeny for growth on the furazolidone–vancomycin combination at the concentration where the parent was sensitive. We show that selected clones were associated with increased resistance to neither, only one drug, or both furazolidone and vancomycin, but in all cases were associated with a decrease in the growth inhibition synergy. Using whole-genome sequencing, we identified various gene mutations in the resistant mutants. We further investigated the mechanism behind the most frequently arising mutations, those in the riboflavin biosynthesis genes ribB and ribE, that represent novel mutations causing furazolidone resistance and diminished vancomycin–furazolidone synergy. It was found that these ribB/ribE mutations act predominantly by decreasing the activity of the NfsA and NfsB nitroreductases. The emergence of the ribB/ribE mutations imposes a significant fitness cost on bacterial growth. Surprisingly, supplementing the medium with riboflavin, which compensates for the affected riboflavin biosynthesis pathway, could restore the normal growth of the ribB/ribE mutants while having no effects on the furazolidone resistance phenotype. Searching the ribB/ribE mutations in the public sequencing database detects the presence of the furazolidone-resistance-conferring ribE mutations (TKAG131–134 deletion or duplication) in clinical isolates from different countries. Hypotheses explaining why these ribE mutations were found in clinical isolates despite having poor fitness were further discussed.

Whole-genome analysis has provided insights into the evolution of Chlamydia trachomatis and, recently, into circulating strains that cause lymphogranuloma venereum (LGV). A large LGV outbreak of a new ompA-genotype, L2b, was first reported in Europe in the early 2000s, primarily affecting men who have sex with men (MSM), and then expanded globally. More recent work shows that this outbreak is diversifying into variants of described ompA-genotypes, with the same L2b genomic backbone. This study extends the investigation of LGV cases to Argentina and Finland. In 2017, an LGV outbreak was described in Argentina characterized by distinct genomic features shown by both ompA-genotyping and Multi-Locus Sequence Typing (MLST) analysis. We have obtained whole-genome sequences from cultured isolates and clinical samples via SureSelect (Agilent) target enrichment. Based on ompA and phylogenetic analyses, we describe further diversity within the ompA-genotype L2b clade, illustrating the transmission dynamics in Argentina and Finland. A key finding is that of a novel clade of Argentinian samples, characterized by a proposed new ompA-genotype L4. Additionally, we present the genome sequence of a non-LGV strain associated with anorectal proctitis. These findings contribute to the investigation of LGV evolution, particularly with the presence of the novel L4 lineage, and provide insights into genomic diversity and transmission dynamics of C. trachomatis.