Microbiology-Sgm最新文献

Staphylococcus aureus has evolved a complex regulatory network to coordinate expression of virulence factors, including cytolytic toxins, with host environmental signals. Central to this network are two-component systems (TCSs), in which a histidine kinase senses an external signal and activates a response regulator via phosphorylation, leading to changes in gene expression. Using a comprehensive screen of transposon mutants in each of the non-essential histidine kinase and response regulator genes in S. aureus, we demonstrate that 11 of these 16 systems regulate cytotoxicity. Further characterization of the phosphate-sensing PhoPR system revealed that PhoP affects cytotoxicity in a manner mediated through the Agr quorum-sensing system. Notably, we found that unphosphorylated PhoP is an activator of Agr activity, whilst phosphorylated PhoP also acts as a weak activator of Agr activity in high-phosphate environments but as a repressor in low-phosphate environments. Furthermore, overexpression of PhoP resulted in upregulation of α-type phenol-soluble modulins, which may also contribute to the cytotoxicity phenotype observed in the phoP mutant. Overall, we have demonstrated that phosphate sensing through PhoPR is a novel regulator of cytotoxicity in S. aureus. Moreover, our study challenges the canonical model of TCSs as simple on/off systems and highlights the importance of unphosphorylated response regulators in gene regulation.

The emergence of a new clade of emm89 group A Streptococcus (GAS) (clade 3) has been described in several countries. Strains in this clade have been reported to have genomic characteristics that lead to increased expression of virulence factors and may confer a selective advantage over previous emm89 strains. To investigate whether clade 3 GAS is present in the emm89 GAS population of Queensland, Australia, all emm89 GAS isolates received by the Queensland Public Health Microbiology Reference Laboratory since emm typing began in the early 2000s underwent genomic sequencing and analysis. Analysis of sequences from 293 emm89 GAS isolates demonstrated the presence of distinct genomic groups in the Queensland emm89 GAS population. Unlike emm89 GAS populations described in the UK and USA, which were mostly ST101 and ST407, there were a relatively high number of ST142 and ST812 strains in the Queensland emm89 GAS population. However, the majority of Queensland isolates belonged to clade 3, with 80% (n=233) of emm89 GAS isolated from 2006 onwards belonging to this clade. All Queensland clade 3 isolates had the reported genomic features associated with higher virulence potential including increased streptolysin O production and an acapsular phenotype. Clade 3, which has emerged to become the dominant clade of emm89 GAS in Europe and the USA, is now also the dominant clade in Queensland.

Common beans (Phaseolus vulgaris L) are an important staple crop valued for their high protein content and dietary benefits. However, Fusarium wilt, caused by Fusarium solani, is responsible for up to an 84% yield loss in bean production. This study aimed to isolate and evaluate novel Pseudomonas fluorescens from Lake Bogoria as potential biocontrol agents against F. solani. Using serial dilution, 30 bacterial isolates were obtained; 10 showed varied mycelial inhibition rates (5.95-42.86%) through dual culture and confrontation assays. Molecular identification using 16S rDNA confirmed that two isolates were Pseudomonas fluorescens strains. Antibiosis gene screening revealed the presence of 2,4-diacetylphloroglucinol, pyrrolnitrin, pyoluteorin and hydrogen cyanide. Enzyme assays demonstrated chitinase (1.33-3,160 U ml^-1) and chitosanase (12.67-29.00 mm) production, indicating antifungal capabilities. In vivo pot experiments with isolate TW17⁺ showed reduced wilt symptoms <20.0% and disease incidence (8.0-35.0%). These findings highlight the potential of soda lake-derived Pseudomonas fluorescens as an effective biocontrol agent against F. solani, with additional benefits for common bean growth and yield improvement.

Competition assays are a mainstay of modern microbiology, offering a simple and cost-effective means to quantify microbe-microbe interactions in vitro. Here, we demonstrate a key weakness of this method that arises when competing microbes interact via toxins, such as those secreted via the type VI secretion system (T6SS). Time-lapse microscopy reveals that T6SS-armed Acinetobacter baylyi bacteria can maintain lethal T6SS activity against E. coli target cells, even under selective conditions intended to eliminate A. baylyi. Further, this residual killing creates a density- and T6SS-dependent bias in the apparent recovery of E. coli, leading to a misreporting of competition outcomes where target survival is low. We also show that incubating A. baylyi/E. coli co-cultures in liquid antibiotic prior to selective plating can substantially correct this bias. Our findings demonstrate the need for caution when using selective plating as part of T6SS competition assays, or assays involving other toxin-producing bacteria.

Chronic rhinosinusitis (CRS) is a prevalent and life-altering disease characterized by the persistent inflammation of the sinuses lasting longer than 3 months. Pseudomonas aeruginosa (PA) is a prominent biofilm-forming bacterium that colonizes the sinuses of up to 9% of CRS patients. PA in biofilm exhibits a great resistance to antibiotics and has proven difficult to remove from the sinus mucosa of CRS patients. Influenza A virus (IAV) is the second most common virus detected colonizing CRS patients' sinuses, with previous studies finding that IAV-induced inflammation of human cells causes the dispersal of bacteria encased in biofilms, leading to increased disease exacerbations. Yet, the two pathogens and the effect that co-infection with them has on primary human nasal epithelial cells (HNECs) have to be assessed. In this study, we show that co-infection of HNECs with both clinical and laboratory isolates of PA and with IAV causes no significant change in PA biofilm biomass and no significant change in the production of PA virulence factors. We also show that co-infected HNECs exhibit lower IL-6 response when compared to HNECs infected with IAV alone, suggesting a novel finding where PA is dampening IL-6 response once co-infection occurs.

Cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger regulating many physiological processes in bacteria. In the oral commensal species Streptococcus mitis, c-di-AMP is involved in regulating metabolism, growth, colony morphology, chain length, biofilm formation and DNA stress tolerance. However, no c-di-AMP-regulated effector proteins have yet been characterized in S. mitis. In this study, we first show that a ΔcdaA mutant, unable to produce c-di-AMP, grows slowly under low environmental potassium conditions. Growth of the cdaA mutant was not restored by reintroducing cdaA in the original locus (KBcdaA). Whole-genome sequencing of multiple KBcdaA isolates revealed secondary mutations in a putative potassium transporter. The mutations were predicted to result in the truncation of the protein or the alteration of a conserved glycine residue essential for selective potassium uptake, disrupting protein function. A Δpde2 mutant overproducing c-di-AMP survived poorly under high environmental sodium concentrations. We then characterized the potassium transporter regulator protein TrkA. Biochemical analyses of the purified recombinant TrkA protein revealed that it specifically binds c-di-AMP with high affinity in vitro. Using deletion mutants of trkA, we demonstrate that TrkA is essential for growth under low environmental potassium conditions. Ultra-high-performance liquid chromatography coupled to tandem mass spectrometry revealed lower c-di-AMP concentration in the ΔtrkA mutant compared to the WT. This was not due to transcriptional regulation of the expression of the c-di-AMP turnover proteins CdaA, Pde1 or Pde2. C-di-AMP production is not affected by the extracellular potassium concentrations under the conditions tested. We also demonstrate a potential role of TrkA in UV stress tolerance but do not characterize the mechanism in this study.

Non-encapsulated Streptococcus pneumoniae (NESp) represent up to 19% of circulating pneumococci and exhibit high rates of genetic exchange and antimicrobial resistance. Saliva is increasingly used as a pneumococcal carriage study specimen, and we recently developed a qPCR assay to enhance carriage surveillance and characterization of NESp in saliva. Previous work has established that pneumococci remain viable in unsupplemented saliva for extended periods under various conditions. However, these findings may not be applicable to NESp. Therefore, to ensure the robustness of NESp detection in saliva-based carriage studies, we evaluated the impact of transport and storage conditions of saliva samples on NESp detection. Six NESp strains from two clinically relevant NESp null-capsule clades (NCCs), NCC1 (carrying pspK) and NCC2 (carrying aliC and aliD), were spiked into pneumococcus (lytA)-negative saliva and incubated through various temperatures and freeze-thaw conditions. Endpoints were processed using either culture enrichment (CE) and DNA extraction (CE-DNA), or an extraction-free method without CE, before testing for lytA using qPCR. Detection stability was assessed using linear regression modelling over temperature, time and freeze-thaws. Following CE-DNA, detection of NESp remained stable for ≤24 or ≤72 h when stored at room temperature or 4 °C, respectively, and over two freeze-thaw cycles (-80 °C), with glycerol supplementation providing slight benefits. Stability of detection when using CE-DNA depended on NCC; detection of NCC2 strains was lower and less stable than NCC1. Compared to CE-DNA, extraction-free detection was more stable, with no significant loss over 72 h at room temperature and over three freeze-thaw cycles, and negligible differences in detection between NCC1 and NCC2 strains. Additionally, extraction-free detection of NCC1, and less so NCC2, increased over the first 24 h when stored at 20-30 °C, suggesting growth of the NESp strains in saliva. Testing of ΔaliCaliD and ΔpspK mutants revealed that these genes increased in vitro viability when cultured in broth but did not significantly alter competitive fitness during saliva CE. The NCC1 NESp strains tested exhibited similar stability patterns in unsupplemented saliva as encapsulated pneumococci. However, the NCC2 strains tested here were less resilient during CE, likely due to competition with other oral microbes. Therefore, recovery of NCC2 NESp may be impacted by transport and storage conditions, leading to an underestimation of carriage prevalence when tested using CE-based methods. For the reliable carriage surveillance of NESp, samples should be stored at 4 °C soon after collection and at -80 °C within 72 h. Methods which directly detect DNA without CE may provide a less biassed accounting of NCC2 strains.

Honey bee (Apis mellifera) larvae are susceptible to the bacterial pathogen Paenibacillus larvae, which causes severe damage to bee colonies. Antibiotic treatment requires veterinary supervision in the USA, is not used in many parts of the world, perpetuates problems associated with antibiotic resistance and may necessitate residual testing in bee products. There is interest in using bacteriophages to treat infected colonies (bacteriophage therapy), and several trials are promising. Nevertheless, the safety of using biological agents in the environment must be scrutinized. In this study, we analysed the ability of P. larvae to evolve resistance to several different bacteriophages. We found that bacteriophage resistance rapidly developed in culture but often results in growth defects. Mutations in the bacteriophage-resistant isolates are concentrated in genes encoding potential surface receptors but are also observed in genes controlling general cellular functions and in two cases - lysogeny. Testing one of these isolates in bee larvae, we found it to have reduced virulence compared to the parental P. larvae strain. We also found that bacteriophages are likely able to counteract resistance evolution. This work suggests that while bacteriophage resistance may arise, its impact will likely be mitigated by reduced pathogenicity and secondary bacteriophage mutations that overcome resistance.

Rare actinomycetes are increasingly recognised as a valuable yet underexplored source of bioactive compounds with significant biomedical potential. While it is well established that bacteria have evolved adaptive mechanisms to withstand environmental stressors, such as variations in temperature, salinity or pH, our understanding of how these abiotic parameters influence bacterial metabolism remains limited. This has important implications not only for laboratory cultivation but also for predicting microbial behaviour in natural ecosystems. In this study, we investigated the effect of temperature on specialized metabolite production by the genus Rhodococcus. Seven strains isolated from marine sediments in three regions - Scotland, the sub-Arctic and Antarctica - were cultured at 20, 25 and 30 °C. Strain-specific growth curves were generated to normalize metabolite extraction at equivalent growth stages, resulting in a total of 54 Rhodococcus metabolite extracts. Liquid chromatography-high-resolution mass spectrometry analysis combined with molecular networking revealed that lower cultivation temperatures reduced bacterial biomass and delayed the onset of the stationary phase, and strain Rhodococcus KRD197 exhibited temperature shifts in metabolism that were associated with alterations in carbohydrate and fatty acid metabolism, potentially linked to osmotic regulation and cell membrane adaptation. These findings highlight the impact of temperature on Rhodococcus-specialized metabolism and support the potential of rare actinomycetes from extreme environments for expanding chemistry from these understudied genera.

Bacterial pathogens employ a diverse array of virulence factors to colonize and subsequently elicit disease in their host. These factors are often subject to extensive regulation at the transcriptional level to ensure that their expression is timely. Although many pathogens use bespoke transcription factors that primarily target virulence genes, global transcription factors also sometimes play a role in controlling these genes. Enteroaggregative Escherichia coli (EAEC) is a significant cause of watery and mucoid diarrhoea globally. The organism colonizes the small intestine before producing toxins that elicit disease, using a multitude of virulence factors that are encoded both chromosomally and on virulence plasmids. In this work, we have studied the cAMP receptor protein (CRP), a well-characterized bacterial global transcription factor, focusing on its role in the pathogenicity of the prototype EAEC strain 042. We show that, although most functional CRP binding sites on the chromosome are conserved between E. coli K-12 and 042, CRP has been co-opted to couple the expression of some virulence genes to the nutritional state of the cell. We report novel mechanisms for CRP-dependent regulation of genes whose products contribute to the maturation of a bacterial antibiotic, export of a polysaccharide capsule and production of a putative adhesin.