Christopher Mullally

The abundant skin commensal, Staphylococcus epidermidis, is the leading cause of late-onset sepsis (LOS) in preterm infants but rarely causes infections in term infants and adults. Staphylococcal virulence mechanisms and the role of the preterm immune responses in driving these life-threatening infections remain poorly understood. Using an ex vivo sepsis model, we challenged whole blood from very preterm infants (30-32 weeks gestational age, GA; n = 8), term infants (> 37 weeks GA; n = 8), and young adults (18-25 years; n = 8) with either live S. epidermidis or S. aureus (~ 10
colony-forming units, CFU/ml) for 90 min. Dual RNA-sequencing (RNA-seq) was performed to simultaneously assess host and pathogen gene expression profiles, identifying common and pathogen-specific responses across cohorts. We found shared immune processes induced in all age groups upon bacterial challenge, including cytokine (IL1A, IL1B, IL6, IFNB1) and chemokine (CCL20, CCL3, CCL7, CXCL2) signalling. Preterm infants also exhibited unique responses, such as increased platelet activation and fibrin clot formation, Wnt signalling, and hypoxia pathways in response to S. epidermidis challenge. Our findings suggest that bacterial gene co-expression, including iron acquisition and heme biosynthesis genes, are also influenced by the hosts developmental age, highlighting the complexity of host-bacterial interactions in the early stages of neonatal sepsis.

Dual RNA-sequencing (dual RNA-seq) holds significant promise for deciphering bacterial virulence mechanisms during systemic infections. However, its application in sepsis research is hindered by technical challenges, including a low bacterial burden in blood and limited sample volumes and RNA yield from vulnerable populations, such as neonates. We developed an optimized protocol [dual RNA isolation from blood (DRIB)] for simultaneous stabilization, isolation and purification of high-quality host leukocyte and bacterial RNA from low-volume whole blood samples (0.5 ml). This protocol is compatible with clinical sample collection workflows and high-throughput RNA sequencing. The feasibility of DRIB for dual RNA-seq was validated using a pilot cohort of clinical adult sepsis samples, enabling the investigation of host–bacterial gene expression during sepsis. The DRIB protocol yielded 2.10–6.91 µg of total RNA per clinical sample in our pilot cohort. Dual-species ribosomal RNA (rRNA) depletion and RNA-seq generated 16.6–24.8 million filtered reads per sample, with 63±7% of reads uniquely mapped to host or bacterial sequences. Host genes accounted for 51–68% (8.4–10.9 million) reads, while 0.5–6.7% (79,496–789,808 reads) mapped to bacterial genomes. Bioinformatic analysis revealed that both shared and individual transcriptional patterns were identified in host and bacterial responses, including pathways related to immune metabolism and metal-ion binding. Our optimized DRIB protocol and RNA-seq pipeline effectively captured both host and bacterial RNA transcription in clinical sepsis samples. Expanding this approach to larger cohorts and varying disease timepoints will provide crucial new insights into host–bacterial gene co-expression dynamics in sepsis progression and outcomes.

Gut microbiome establishment in early life is influenced by maternal, infant, and environmental factors, with disruptions linked to later disease risk. Although infant diet is a major determinant of microbial composition, longitudinal data in breastfed infants, particularly in the context of birth interventions, remain limited. We profiled 698 stool samples from 84 predominantly or exclusively breastfed infants in the BLOSOM cohort, spanning 10 time points from 1 week to 2 years of age using full-length 16S rRNA gene sequencing and targeted qPCR. After an initial volatile period, microbiome composition and diversity stabilized between months 2 and 5. Introduction of solid foods then triggered a marked ecological shift, with significant increases in diversity (p < 1 × 10−11) and broad compositional restructuring. In contrast, weaning had minimal impact on overall microbiota structure but was associated with lower abundances of several Bifidobacterium species, highlighting the sustained bifidogenic effect of human milk. Cesarean delivery was associated with transient reductions in Bacteroides abundance and prevalence, but did not affect Bifidobacterium, likely due to the bifidogenic effects of human milk. Reductions in Bacteroides, however, were not reflected in quantitative analyses, emphasizing the importance of absolute abundance measures. Our results offer novel insight into gut microbiome development under optimal feeding conditions, suggesting that breastfeeding may buffer early-life microbiome perturbations, while diet transitions exert major and lasting effects.

Introduction
Enterococcus faecium clade B has recently been re-classified as Enterococcus lactis. Although E. lactis was previously associated with food products and probiotics, the recent re-classification has prompted the need for the accurate identification of this species and re-interpretation of its disease-causing ability. Since the re-classified E. lactis can currently only be identified by molecular techniques such as whole-genome sequencing, we constructed a MALDI Biotyper® custom database to rapidly identify and differentiate E. lactis causing bacteraemia from E. faecium.

Hypothesis/Gap statement
The re-classification of E. faecium clade B as E. lactis warrants the development of rapid and accurate identification methods to distinguish these species, particularly in clinical settings where E. lactis may be misidentified as E. faecium.

Aim
The aim of this study was to construct a MALDI Biotyper® custom database to rapidly identify and differentiate E. lactis causing bacteraemia from E. faecium.

Methodology
A total of 97 enterococcal isolates, including 38 E. lactis, 51 E. faecium and 8 non-E. faecium non-E. lactis enterococci (E. avium, E. casseliflavus, E. cecorum, E. durans, E. faecalis, E. faecium, E. gallinarum, E. lactis, E. mundtii and E. raffinosus) were investigated. Whole-genome sequence analysis was used to confirm the species of each isolate. A MALDI Biotyper® in-house database was constructed using 29 E. lactis isolates and the ethanol/formic acid/acetonitrile preparation protocol. The in-house database was validated using the 97 enterococcal isolates and the extended direct transfer preparation protocol.

Results
Our in-house database correctly identified all isolates at the species level, including the E. lactis isolates, all of which were misidentified as E. faecium by the BioTyper® MBT Compass reference library (2022). Of the 38 E. lactis isolates, 84.2% (n=32) were identified at the high probable species level (score ≥2.300), while the remaining 15.8% (n=6) were identified at the probable species level (score 2.000–2.299). Similarly, all E. faecium isolates (n=51) were accurately identified, including 84.3% (n=43/51) identified at the high probable species level and 15.7% (n=8/51) identified at the probable species level.

Conclusion
Our study provides a ready-to-use custom MALDI spectral database that can be implemented in clinical diagnostic and research laboratories to accurately identify E. lactis, which is currently misidentified as E. faecium by the standard spectrum database available on commercial platforms.

In forensic science, detecting transfers of physical and biological material is critical for establishing evidence of criminal involvement. Unique bacterial signatures from the reproductive system transfer during unprotected penetrative intercourse offer a novel tool for criminal investigation. Here, we demonstrate this transfer using full-length 16S rRNA gene sequencing and discuss the impact of barrier contraceptives. These microbial signatures can potentially aid in sexual assault casework for perpetrator identification when human male DNA is absent.
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•Transfer of unique and non-unique bacterial taxa during intercourse was observed•Lactobacillus spp. contributed to the most female to male sexome transfer•Despite the use of a condom during intercourse, transfer of the sexome was still observed•Condom/lube use, oral intercourse, circumcision/pubic hair: no impact on microbial diversity
Biological sciences; Microbiome

Neisseria meningitidis, the meningococcus, resides exclusively in humans and causes invasive meningococcal disease (IMD). The population of N. meningitidis is structured into stable clonal complexes by limited horizontal recombination in this naturally transformable species. N. meningitidis is an opportunistic pathogen, with some clonal complexes, such as cc53, effectively acting as commensal colonizers, while other genetic lineages, such as cc11, are rarely colonizers but are over-represented in IMD and are termed hypervirulent. This study examined theoretical evolutionary pathways for pathogenic and commensal lineages by examining the prevalence of horizontally acquired genomic islands (GIs) and loss-of-function (LOF) mutations. Using a collection of 4850 genomes from the BIGSdb database, we identified 82 GIs in the pan-genome of 11 lineages (10 hypervirulent and one commensal lineage). A new computational tool, Phaser, was used to identify frameshift mutations, which were examined for statistically significant association with genetic lineage. Phaser identified a total of 144 frameshift loci of which 105 were shown to have a statistically significant non-random distribution in phase status. The 82 GIs, but not the LOF loci, were associated with genetic lineage and invasiveness using the disease carriage ratio metric. These observations have been integrated into a new model that infers the early events of the evolution of the human adapted meningococcus. These pathways are enriched for GIs that are involved in modulating attachment to the host, growth rate, iron uptake and toxin expression which are proposed to increase competition within the meningococcal population for the limited environmental niche of the human nasopharynx. We surmise that competition for the host mucosal surface with the nasopharyngeal microbiome has led to the selection of isolates with traits that enable access to cell types (non-phagocytic and phagocytic) in the submucosal tissues leading to an increased risk for IMD.

Neisseria meningitidis is the causative agent of invasive meningococcal disease (IMD). A recombinant vaccine called Bexsero® incorporates four subcapsular antigens (fHbp, NHBA, NadA and PorA) which are used to assign a Bexsero® antigen sequence type (BAST) to each meningococcal strain. The vaccine elicits an immune response against combinations of variants of these antigens which have been grouped into specific BAST profiles that have been shown to have different distributions within geographical locations thus potentially affecting the efficacy of the vaccine. In this study, invasive meningococcal disease isolates from the western seaboard of Australia (Western Australia; WA) were compared to those from the south-eastern seaboard (Victoria; VIC) from 2008 to 2012. Whole-genome sequencing (WGS) of 131 meningococci from VIC and 70 meningococci from WA were analysed for MLST, FetA and BAST profiling. Serogroup B predominated in both jurisdictions and a total of 10 MLST clonal complexes (cc) were shared by both states. Isolates belonging to cc22, cc103 and cc1157 were unique to VIC whilst isolates from cc60 and cc212 were unique to WA. Clonal complex 41/44 represented one-third of the meningococcal population in each state but the predominant ST was locally different: ST-6058 in VIC and ST-146 in WA. Of the 108 BAST profiles identified in this collection, only 9 BASTs were simultaneously observed in both states. A significantly larger proportion of isolates in VIC harboured alleles for the NHBA-2 peptide and fHbp-1, antigenic variants predicted to be covered by the Bexsero® vaccine. The estimate for vaccine coverage in WA (47.1% [95% CI: 41.1–53.1%]) was significantly lower than that in VIC (66.4% [95% CI: 62.3–70.5%]). In conclusion, the antigenic structure of meningococci causing invasive disease in two geographically distinct states of Australia differed significantly during the study period which may affect vaccine effectiveness and highlights the need for representative surveillance when predicting potential impact of meningococcal B vaccines.

Enterococci are a diverse group of Gram-positive bacteria that are typically found as commensals in humans, animals, and the environment. Occasionally, they may cause clinically relevant diseases such as endocarditis, septicemia, urinary tract infections, and wound infections. The majority of clinical infections in humans are caused by two species: Enterococcus faecium and Enterococcus faecalis. However, there is an increasing number of clinical infections caused by non-faecium non-faecalis (NFF) enterococci. Although NFF enterococcal species are often overlooked, studies have shown that they may harbor antimicrobial resistance (AMR) genes and virulence factors that are found in E. faecium and E. faecalis. In this review, we present an overview of the NFF enterococci with a particular focus on human clinical manifestations, epidemiology, virulence genes, and AMR genes.

In 2010 a single isolate of a trimethoprim-resistant multilocus sequence type 5, Panton-Valentine leucocidin-positive, community-associated methicillin-resistant
(PVL-positive ST5 CA-MRSA), colloquially named WA121, was identified in northern Western Australia (WA). WA121 now accounts for ~14 % of all WA MRSA infections. To gain an understanding of the genetic composition and phylogenomic structure of WA121 isolates we sequenced the genomes of 155 WA121 isolates collected 2010-2021 and present a detailed genomic description. WA121 was revealed to be a single clonally expanding lineage clearly distinct from sequenced ST5 strains reported outside Australia. WA121 strains were typified by the presence of the distinct PVL phage φSa2wa-st5, the recently described methicillin resistance element SCC
IVo carrying the trimethoprim resistance (
) transposon Tn
, the novel β-lactamase transposon Tn
and the epidermal cell differentiation inhibitor (EDIN-A) plasmid p2010-15611-2. We present evidence that SCC
IVo together with Tn
has horizontally transferred to
and evidence of intragenomic movement of both Tn
and Tn
. We experimentally demonstrate that p2010-15611-2 is capable of horizontal transfer by conjugative mobilization from one of several WA121 isolates also harbouring a pWBG749-like conjugative plasmid. In summary, WA121 is a distinct and clonally expanding Australian PVL-positive CA-MRSA lineage that is increasingly responsible for infections in indigenous communities in northern and western Australia. WA121 harbours a unique complement of mobile genetic elements and is capable of transferring antimicrobial resistance and virulence determinants to other staphylococci.