The spread of antimicrobial resistant pathogens, including extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae is a global health threat and can be addressed only through a One Health approach. We aimed to characterize ESBL producing Escherichia coli isolates from World Health Organization Tricycle surveillance using data from whole genome sequencing (WGS) to decipher the potential dynamics of their circulation at the human, poultry and environment interface. WGS was performed on 100 non-duplicate representative ESBL E. coli isolates including 28 isolates from humans, 36 from poultry caeca, and 36 from water samples. Minimum Inhibitory Concentration (MIC) was determined using Vitek 2 Compact. WGS was performed on Illumina NextSeq 2000 platform and open-source bioinformatics pipelines were used to analyze WGS data for genomic characterization including phylogenetic analysis and in silico multi-locus sequence typing and, serotyping and, ESBL gene detection. Most isolates were susceptible to imipenem (98

Corynebacterium diphtheriae, the main aetiological agent of diphtheria, is a re-emerging bacterial pathogen of public health concern, yet remains largely understudied globally. In this study, we analysed the population structure and antimicrobial resistance (AMR) of 210 C. diphtheriae isolates from Victoria, Australia, including 103 historical (1950-1970) and 107 contemporary clinical isolates (2004-2023), using whole-genome sequencing and phenotypic susceptibility testing. The diphtheria toxin gene (tox) was detected in 89 isolates, the majority of which (n=83; 93.3%) were historical. Population structure comprised two primary phylogenetic lineages, Mitis and Gravis, each containing multiple sublineages. Multi-locus sequence type (MLST) analysis revealed a highly diverse population structure with multiple novel MLST profiles and alleles. When placed within a global phylogenetic framework, Australian isolates were broadly distributed, reflecting substantial genetic diversity. Phenotypic susceptibility testing against 11 antimicrobials revealed that several contemporary isolates were resistant to multiple agents, including penicillin and erythromycin, first-line treatments of Corynebacterium infections. Eight contemporary isolates were multidrug-resistant (resistant to ≥3 antimicrobial classes), including five with resistance to both penicillin and erythromycin. Genomic analysis identified multiple genes and mutations conferring resistance among contemporary isolates. In contrast, no AMR phenotypes or genotypes were observed in historical isolates. Analysis of historical genomes provides valuable insights into a period of heightened diphtheria activity in Victoria prior to widespread immunization. Overall, these findings establish a baseline for ongoing genomic surveillance in the face of increasing global outbreaks, support informed empiric treatment strategies and contribute to the knowledge of the global population structure of C. diphtheriae.

This study elucidates potential genetic determinants and mechanisms involved in the synergistic effects of daptomycin (DAP) + fosfomycin (FOF) combination therapy. Among 33 clinically derived DAP-susceptible (S)/DAP-resistant (R) isogenic strain pairs, mutations in the mprF gene occurred in 30/33 DAP-R strains, including polymorphisms of L826F (33%) or T345A/L/I (15%). Strain variants of DAP-S CB1483 serially passaged in vitro for 10 days in DAP +/− FOF identified a key non-synonymous mutation in mprF (L826F) only in the DAP monotherapy arm. Interestingly, passage in FOF alone or DAP + FOF prevented the emergence of this mprF mutation following 10-day passage. This L826F mprF polymorphism, associated with a “gain-in-function” phenotype, exhibited increased amounts of lysyl-phosphatidylglycerol (L-PG) in the cell membrane (CM). Transcriptomics revealed a relatively modest number (~10) of distinct genes that were significantly up- or downregulated (≥2 log fold) in both the DAP-S and DAP-R strain pairs upon DAP + FOF exposures (vs. DAP or FOF alone). Of note, DAP + FOF decreased expression of lrgAB and sdrE and increased the expression level of fosB. In a rabbit infective endocarditis (IE) model, the DAP-R CB185 strain treated with DAP +/− FOF showed significantly reduced lrgB expression in vegetations compared with DAP treatment alone. Overall, these findings indicate that DAP + FOF therapy impacts MRSA through multiple specific mechanisms, enhancing bacterial clearance.

Salmonella enterica serovar Panama, a causative agent of non-typhoidal salmonellosis (NTS), is one of several serovars that causes invasive NTS disease (iNTS) in humans. S. Panama is an understudied pathogen, with its pathobiology poorly understood. It is a predominant iNTS serovar in Australia, a high-income country with high rates of salmonellosis, where S. Panama has been documented to have a high odds ratio (13.9-15.26) for causing iNTS. This study investigates the genomic epidemiology and antimicrobial resistance profiles of all S. Panama isolates recovered in Victoria, Australia, between 2000 and 2021. We examined the infection dynamics of S. Panama in seven isolates, representing the genetic diversity of the study population. Two sub-lineages, encompassed within a previously described Asian lineage, were identified. Multi-drug resistance (resistance to ≥3 drug classes) was detected in 46 (51.7%) Australian isolates. The plasmid-mediated colistin resistance gene, mcr1.1, was detected in one Australian S. Panama isolate, carried by an IncI plasmid previously reported in Salmonella and Escherichia coli isolates collected from poultry in South-East Asia. Examination of the intracellular replication dynamics of S. Panama isolates demonstrated diverse phenotypes. In THP-1 derived macrophages, despite low host cell uptake, S. Panama showed higher replication rates over time compared to S. enterica serovar Typhimurium. However, a causative genotype could not be identified to explain this observed phenotype. This study provides insights into the S. Panama isolates circulating in Australia over two-decades, finding that 78% were linked to international travel suggesting importation in Australia. It shows MDR was common in this iNTS serovar, and colistin resistance reported for the first time. It provides the first data on the host-pathogen interactions of S. Panama in Australia, which will aid our collective understanding of the pathobiology of S. Panama and iNTS serovars more broadly.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes . Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes . More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

The past decade has seen an increase in the prevalence of sequence type (ST) 45 methicillin-resistant Staphylococcus aureus (MRSA), yet the underlying drivers for its emergence and spread remain unclear. To better understand the worldwide dissemination of ST45 S. aureus , we performed phylogenetic analyses of Australian isolates, supplemented with a global population of ST45 S. aureus genomes. Our analyses revealed a distinct lineage of multidrug-resistant ST45 MRSA harbouring qacA , predominantly found in Australia and Singapore. Bayesian inference predicted that the acquisition of qacA occurred in the late 1990s. qacA was integrated into a structurally variable region of the chromosome containing Tn 552 (carrying blaZ ) and Tn 4001 (carrying aac(6 ’ )-aph(2 ” ) ) transposable elements. Using mutagenesis and in vitro assays, we provide phenotypic evidence that qacA confers tolerance to chlorhexidine. These findings collectively suggest both antimicrobial resistance and the carriage of qacA may play a role in the successful establishment of ST45 MRSA.

Invasive Staphylococcus aureus infections are common, causing high mortality, compounded by the propensity of the bacterium to develop drug resistance. S. aureus is an excellent case study of the potential for a bacterium to be commensal, colonizing, latent or disease-causing; these states defined by the interplay between S. aureus and host. This interplay is multidimensional and evolving, exemplified by the spread of S. aureus between humans and other animal reservoirs and the lack of success in vaccine development. In this Review, we examine recent advances in understanding the S. aureus–host interactions that lead to infections. We revisit the primary role of neutrophils in controlling infection, summarizing the discovery of new immune evasion molecules and the discovery of new functions ascribed to well-known virulence factors. We explore the intriguing intersection of bacterial and host metabolism, where crosstalk in both directions can influence immune responses and infection outcomes. This Review also assesses the surprising genomic plasticity of S. aureus, its dualism as a multi-mammalian species commensal and opportunistic pathogen and our developing understanding of the roles of other bacteria in shaping S. aureus colonization. In this Review, Howden and co-workers examine and integrate recent key advances in understanding the mechanisms that Staphylococcus aureus uses to cause infections.

Vancomycin-resistant Enterococcus (VRE) is an increasingly identified cause of human disease with most infections resulting from the vanA and vanB genotypes; less is known about other clinically relevant genotypes. Here we report a genomic exploration of a vanD VRE faecium (VREfm), which arose de novo during a single infectious episode. The genomes of the vancomycin-susceptible E. faecium (VSEfm) recipient and resulting VREfm were subjected to long-read sequencing and closed, with whole genome alignments and cross-mapping used to identified genomic variation. Three key differences were identified. (i) The VREfm chromosome gained a 142.6kb integrative conjugative element (ICE) harbouring the vanD locus. (ii) The native ligase (ddl) was disrupted by an ISEfm1 insertion. (iii) A large 1.74Mb chromosomal inversion of unknown consequence occurred. Alignment and phylogenetic-based comparisons of the VREfm with a global collection of vanD-harbouring genomes identified strong similarities in the 120-160kb genomic region surrounding vanD, suggestive of a common mobile element and integration site, irrespective of the diverse taxonomic, geographic, and host origins of the isolates. This isolate diversity revealed that this putative ICE (and its source) is globally disseminated and is capable of being acquired by different genera. Although the incidence of vanD VREfm is low, understanding its emergence and potential for spread is crucial for the ongoing efforts to reduce antimicrobial resistance.

AbstractStreptococcus dysgalactiaesubsp.equisimilis(SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species,Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE andS. pyogenes.More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were found across species. Assessing potential cross-protection from leadingS. pyogenesvaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

Outcomes of severe bacterial infections are determined by the interplay between host, pathogen, and treatments. While human genomics has provided insights into host factors impacting Staphylococcus aureus infections, comparatively little is known about S. aureus genotypes and disease severity. Building on the hypothesis that bacterial pathoadaptation is a key outcome driver, we developed a genome-wide association study (GWAS) framework to identify adaptive mutations associated with treatment failure and mortality in S. aureus bacteremia (1,358 episodes). Our research highlights the potential of vancomycin-selected mutations and vancomycin minimum inhibitory concentration (MIC) as key explanatory variables to predict infection severity. The contribution of bacterial variation was much lower for clinical outcomes (heritability <5%); however, GWASs allowed us to identify additional, MIC-independent candidate pathogenesis loci. Using supervised machine learning, we were able to quantify the predictive potential of these adaptive signatures. Our statistical genomics framework provides a powerful means to capture adaptive mutations impacting severe bacterial infections.

Among the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus, only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus, the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis (ltaS): translation (rplK), DNA compaction (hup), initiation of DNA replication (dnaA, hup) and purine nucleotide metabolism (prs). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics. IMPORTANCE The opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20–40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli , Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes . Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.

Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa (intracellular toxicity of S. aureus) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.

AbstractStaphylococcus aureusinfections are associated with high mortality rates. Often considered an extracellular pathogen,S. aureuscan persist and replicate within host cells, evading immune responses and causing host cell death. Classical methods for assessingS. aureuscytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform calledInToxSa(IntracellularToxicity ofS. aureus) to quantify intracellula cytotoxicS. aureusphenotypes. Studying a panel of 387S. aureusbacteraemia isolates, and combined with comparative, statistical and functional genomics, our platform identified mutations inS. aureusclinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations inausA,encoding the aureusimine non-ribosomal peptide synthetase, reducedS. aureuscytotoxicity and increased intracellular persistence.InToxSais a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevantS. aureuspathoadaptive mutations that promote intracellular residency.

Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily cytotoxin driven, leading to the suggestion that hypervirulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the cytotoxicity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced cytotoxicity. Interestingly, the majority of isolates that were attenuated in cytotoxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.

Non-typhoidal Salmonella (NTS) is the second most common cause of foodborne bacterial gastroenteritis in Australia with antimicrobial resistance (AMR) increasing in recent years. Whole-genome sequencing (WGS) provides opportunities for in silico detection of AMR determinants. The objectives of this study were two-fold: (1) establish the utility of WGS analyses for inferring phenotypic resistance in NTS, and (2) explore clinically relevant genotypic AMR profiles to third generation cephalosporins (3GC) in NTS lineages. The concordance of 2490 NTS isolates with matched WGS and phenotypic susceptibility data against 13 clinically relevant antimicrobials was explored. In silico serovar prediction and typing was performed on assembled reads and interrogated for known AMR determinants. The surrounding genomic context, plasmid determinants and co-occurring AMR patterns were further investigated for multidrug resistant serovars harbouring bla CMY-2, bla CTX-M-55 or bla CTX-M-65. Our data demonstrated a high correlation between WGS and phenotypic susceptibility testing. Phenotypic-genotypic concordance was observed between 2440/2490 (98.0 %) isolates, with overall sensitivity and specificity rates >98 % and positive and negative predictive values >97 %. The most common AMR determinants were bla TEM-1, sul2, tet(A), strA-strB and floR. Phenotypic resistance to cefotaxime and azithromycin was low and observed in 6.2 % (151/2486) and 0.9 % (16/1834) of the isolates, respectively. Several multi-drug resistant NTS lineages were resistant to 3GC due to different genetic mechanisms including bla CMY-2, bla CTX-M-55 or bla CTX-M-65. This study shows WGS can enhance existing AMR surveillance in NTS datasets routinely produced in public health laboratories to identify emerging AMR in NTS. These approaches will be critical for developing capacity to detect emerging public health threats such as resistance to 3GC.

Topical antibiotic preparations, such as fusidic acid (FA) or mupirocin, are used in the prevention and treatment of superficial skin infections caused by staphylococci. Previous genomic epidemiology work has suggested an association between the widespread use of topical antibiotics and the emergence of methicillin-resistant Staphylococcus aureus in some settings. In this study, we provide experimental proof of coselection for multidrug resistance in S. aureus following exposure to FA or mupirocin. Through targeted mutagenesis and phenotypic analyses, we confirmed that fusC carriage confers resistance to FA and mupA carriage confers high-level resistance to mupirocin in multiple S. aureus genetic backgrounds. In vitro experiments demonstrated that carriage of fusC and mupA confers a competitive advantage in the presence of subinhibitory concentrations of FA and mupirocin, respectively. Further, we used a porcine skin colonization model to show that clinically relevant concentrations of topical antibiotics can coselect for the presence of unrelated antimicrobial resistance determinants, such as mecA, blaZ, and qacA, in fusC- or mupA-harboring S. aureus. These findings provide valuable insights on the role of acquired FA or mupirocin resistance in coselecting for broader antibiotic resistance in S. aureus, prompting a greater need for judicious use of topical antibiotics.

Here, we announce the complete genome sequence of an exfoliative toxin-producing strain of Staphylococcus aureus sequence type 582 (ST582), isolated from a case of staphylococcal scalded-skin syndrome. The genome consists of a single circularized unitig with a total length of 2,792,190 bp carrying 2,699 genes. The genome is the basis for future epidemiological and genomic studies.

The reversal of daptomycin resistance in MRSA to a daptomycin-susceptible phenotype following prolonged passage in selected β-lactams occurs coincident with the accumulation of multiple point mutations in the mprF gene. MprF regulates surface charge by modulating the content and translocation of the positively charged cell membrane phospholipid, lysyl-phosphatidylglycerol (LPG). The precise cell membrane adaptations accompanying such β-lactam-induced mprF perturbations are unknown. This study examined key cell membrane metrics relevant to antimicrobial resistance among three daptomycin-resistant MRSA clinical strains, which became daptomycin-susceptible following prolonged exposure to cloxacillin (‘daptomycin-resensitized’). The causal role of such secondary mprF mutations in mediating daptomycin resensitization was confirmed through allelic exchange strategies. The daptomycin-resensitized strains derived either post-cloxacillin passage or via allelic exchange (vs. their respective daptomycin-resistant strains) showed the following cell membrane changes: (i) enhanced BODIPY-DAP binding; (ii) significant reductions in LPG content, accompanied by significant increases in phosphatidylglycerol content (p < 0.05); (iii) no significant changes in positive cell surface charge; (iv) decreased cell membrane fluidity (p < 0.05); (v) enhanced carotenoid content (p < 0.05); and (vi) lower branched chain fatty acid profiles (antiso- vs. iso-), resulting in increases in saturated fatty acid composition (p < 0.05). Overall, the cell membrane characteristics of the daptomycin-resensitized strains resembled those of parental daptomycin-susceptible strains. Daptomycin resensitization with selected β-lactams results in both definable genetic changes (i.e., mprF mutations) and a number of key cell membrane phenotype modifications, which likely facilitate daptomycin activity.

Enterotoxigenic E. coli (ETEC) is a common cause of diarrhea in children in low- and middle-income countries, and in travelers to these countries. ETEC is also an important cause of morbidity and premature mortality in piglets, calves, goat kids and lambs. The major virulence determinants of ETEC are enterotoxins and colonization factors, which enable the pathogen to colonize the small intestine and deliver enterotoxins, such as the heat-stable enterotoxins, STp and STh, to epithelial cells. Because most ETEC strains are host-specific, there are few convenient animal models to investigate the pathogenesis of ETEC infections or to evaluate specific anti-ETEC interventions, such as drugs and vaccines. An exception is ETEC strains bearing F41 pili, which mediate intestinal colonization of various young animals, including neonatal mice, to cause disease and in some cases death. In this study, we used the archetypal F41-producing bovine ETEC strain, B41 (O101:NM; K99, F41, STp) to validate and further explore the contribution of F41 and STp to bacterial virulence. By using targeted gene deletion and trans-complementation studies, augmented by whole genome sequencing, and in vitro and animal studies of virulence, we established that F41 mediates colonization of the mouse intestine and is essential for bacterial virulence. In addition, we showed for the first time that STp is as important as F41 for virulence. Together, these findings validate the use of neonatal mice to study the pathogenesis of F41-bearing ETEC and to investigate possible specific anti-ETEC interventions including vaccines that target heat-stable enterotoxins.