Microbiology-Sgm最新文献

Quantitative reverse transcription PCR (RT-qPCR) is a popular and reliable tool for monitoring fluctuations in functional bacterial gene expression. A necessary step of the qRT-qPCR process is the use of a reference gene, which acts to distinguish between technical bias and true biological variation. Many reference genes have been defined for bacterial species; however, few studies have validated their stability across strain types and environmental test conditions. In this study of Pseudomonas aeruginosa, the expression consistency of seven commonly used reference genes (rpoD, proC, rpoS, 16S, algD, gyrA and ampC) was assessed in P. aeruginosa laboratory (PAO1) and clinical (LESB65) isolates grown in Lysogeny broth, synthetic cystic fibrosis (CF) media 2 (SCFM2) and CF lung media (CFLM) at various growth time points (2, 6, 24 and 72 h). The stability of the reference genes was then ranked using the RefFinder programme, and three differentially ranked (rpoS, 16S and ampC) were used to interpret the expression of a Pseudomonas virulence-related gene (exoS). The results showed that 16S was the only reference gene that was quantifiably expressed by both P. aeruginosa strains grown in all media types at all growth times. Furthermore, analysing the expression of exoS with different reference genes significantly influenced the calculated expression of exoS in SCFM2 and CFLM. This study has identified a suitable reference gene for RT-qPCR with P. aeruginosa grown in complex respiratory-mimicking media. The results presented here also highlight the importance of validating reference gene expression under the chosen experimental conditions and increase our understanding of how pathogen biology can fluctuate across diverse conditions. Such knowledge is paramount for the development of novel therapeutics, including antimicrobials and anti-virulence agents.

There is an urgent need to identify new chemical compounds with novel modes of action to help manage the antimicrobial resistance crisis. Fungi are prolific producers of secondary metabolites, including those with antimicrobial properties, and contain biosynthetic gene clusters that awaken only under certain growth conditions. In recent years, a wealth of novel fungal biosynthetic pathways and compounds have been identified, suggesting fungi remain a viable source for developing new antimicrobials. The International Collection of Microorganisms from Plants (ICMP) contains thousands of fungi and bacteria primarily sourced from Aotearoa New Zealand. Here, we report the results of our efforts to screen 32 fungal ICMP isolates for activity against Escherichia coli, a leading cause of deaths attributable to antimicrobial resistance. We used a 'one strain-many compounds' approach, growing the ICMP isolates on seven different media with different pH and various carbon and nitrogen sources. We also tested the isolates for activity at various ages. Our results indicate that several of the tested fungi possess anti-E. coli activity and are suitable for further study. Our results also provide further strong evidence for the impact of media on both fungal growth and bioactivity.

Single-copy chromosomal integration systems are essential tools for stable gene expression in bacteria, minimizing variability associated with plasmid-based systems. The Tn7 transposon-based system is widely used for this purpose, and one important application is the generation of reporter systems, such as the bioluminescent luxCDABE operon (lux). However, current Tn7-lux vectors exhibit undesirable background expression due to cryptic promoter activity near the antibiotic resistance cassette. Here, we report the construction of an improved vector, pTn7-lux-B0015, incorporating a strong synthetic terminator upstream of the lux operon. This modification effectively eliminated basal luminescence in the absence of a promoter and enhanced the dynamic range and responsiveness of the reporter. Using a Xanthomonas citri type III secretion system promoter as a model, we demonstrate that pTn7-lux-B0015 enables more accurate detection of gene expression under relevant growth conditions. This vector provides a valuable tool for the development of precise and tunable bioluminescent reporters in bacterial systems.

Campylobacter, a bacterium commonly found in the chicken gut, is the leading cause of bacterial foodborne gastroenteritis globally. Despite various interventions aimed at controlling Campylobacter in the food chain, such as enhanced biosecurity measures, improved hygiene practices and farm-level controls, reducing its prevalence remains a significant challenge. While the European Union's (EU) 2006 ban on antimicrobials as growth promoters was primarily intended to control antimicrobial resistance, its impact on Campylobacter load has been limited. The emergence of antibiotic-resistant Campylobacter has created a requirement to develop alternative methods to improve food safety, enhance performance and mitigate pathogenic bacteria. This study explored the potential of Maranta arundinacea (arrowroot) extract as a prospective dietary supplement for both humans and chickens. The investigation focused on its safety, its ability to reduce Campylobacter in T84 intestinal epithelial cells and its anti-inflammatory properties. Results showed that 4% and 25% concentrations of arrowroot extract were non-cytotoxic to human T84 cells and significantly reduced bacterial growth in Campylobacter jejuni strains. Additionally, the extract inhibited the growth of Campylobacter coli strains and Escherichia coli, with statistical significance observed against E. coli at the 25% concentration. These results suggest that arrowroot extract could be a promising natural alternative for addressing antibiotic resistance and enhancing food safety.

Glutamine is the most abundant amino acid in the human body, playing a crucial role in numerous cellular processes. Notably for enteric bacteria, glutamine is abundant in the intestines where it helps to maintain gut health of the host, therefore presenting itself as an accessible nutrient. Campylobacter jejuni, a largely non-saccharolytic organism, favours just a few amino acids for growth, and glutamine is particularly efficient as a nitrogen source. Despite this, a glutamine transporter has not been conclusively identified in this important human pathogen. By measuring the global transcriptomic response of C. jejuni to replete glutamine conditions, we identified several candidate transporters, ultimately characterising Cj0903, here named glutamine uptake transporter A, as the major glutamine transporter belonging to the alanine or glycine:cation symporter family. We show that this transporter is ubiquitous in thermotolerant Campylobacter, demonstrating a conserved ability to utilise exogenous glutamine. In contrast, the ammonium transporter Amt was only present in a subset of C. jejuni, and we confirmed that amt negative isolates do not effectively utilise ammonium as a nitrogen source.

Candida species infection of vascular and urinary catheters is a growing clinical concern. By understanding how biomaterial physicochemical surface properties affect fungal behaviour, catheters could be designed to mechanically discourage infection as a form of 'mechanoprophylaxis'. In this study, silicone surfaces were synthesized with 'stiff' or 'soft' mechanical properties and the subsequent adherence, proliferation and biofilm production of Candida albicans, Candida parapsilosis and Nakaseomyces glabratus isolates on these surfaces were analysed. Candida significantly bound more, proliferated more and produced more biofilm on softer silicone surfaces. Importantly, the observed differences in fungal adhesion and biofilm formation between catheter surface types persisted when surfaces were pre-coated with host serum proteins. This study demonstrated that catheter synthesis parameters can affect physical properties and subsequent susceptibility to fungal colonization. These data lay important groundwork in exploiting mechanical design to decrease the ability of Candida to colonize devices and thus prevent medical device infections.

Fluorescent pseudomonads catabolize purines via uric acid and allantoin, a pathway whose end-product is glyoxylate. In this work, we show that in Pseudomonas aeruginosa strain PAO1, the ORFs PA1498-PA1502 encode a pathway that converts the resulting glyoxylate into pyruvate. The expression of this cluster of ORFs was stimulated in the presence of allantoin, and mutants containing transposon insertions in the cluster were unable to grow on allantoin as a sole carbon source. The likely operonic structure of the cluster is elucidated. We also show that the purified proteins encoded by PA1502 and PA1500 have glyoxylate carboligase (Gcl) and tartronate semialdehyde (TSA) reductase (GlxR) activity, respectively, in vitro. Gcl condenses two molecules of glyoxylate to yield TSA, which is then reduced by GlxR to yield d-glycerate. GlxR displayed much greater specificity (k _cat/K_M) for Gcl-derived TSA than it did for the TSA tautomer, hydroxypyruvate. This is relevant because TSA can potentially spontaneously tautomerize to yield hydroxypyruvate at neutral pH. However, kinetic and [¹H]-NMR evidence indicate that PA1501 (which encodes a putative hydroxypyruvate isomerase, Hyi) increases the rate of the Gcl-catalysed reaction, possibly by minimizing the impact of this unwanted tautomerization. Finally, we use X-ray crystallography to show that apo-GlxR is a configurationally flexible enzyme that can adopt two distinct tetrameric assemblies in vitro.

Acinetobacter baumannii is associated with severe hospital-acquired, multi-drug-resistant infections worldwide, causing significant mortality and morbidity in intensive care patients or those under prolonged hospitalization. Multiple studies have recently shown that a proportion of circulating clinical isolates establish a transient multiplication niche inside phagocytic and non-phagocytic eukaryotic cells. We have previously demonstrated that the A. baumannii ABC141 strain invades human endothelial and epithelial cells, where it efficiently multiplies without induction of cytotoxicity. Here, we show that ABC141 adhesion, invasion and intracellular multiplication depend on the growth stage, being most efficient in the exponential growth phase. To define the gene expression signature most favourable to an intracellular lifestyle, a transcriptomic comparison was carried out between exponentially grown ABC141 and cultures in the stationary phase. Although most of the pathways identified reflected growth-related metabolic changes, we observed an up-regulation of the twin-arginine translocation (Tat) export system. Analysis of a mutant strain lacking the tatABC operon revealed that this export system is required only for adhesion to host cells, but not for invasion or intracellular multiplication. These data highlight a new role for the Tat export pathway in A. baumannii pathogenesis.

The human body hosts a complex and dynamic microbial community that is crucial for maintaining health. While bacteria dominate this system, fungal communities, collectively called the mycobiome, are increasingly recognized as vital contributors. However, fungi remain understudied due to challenges in culturing many species, limiting our understanding of their roles, interactions and effects on human biology. Advances in next-generation sequencing have transformed mycobiome research, revealing fungal diversity and its impact on health and disease. This review examines the mycobiome's composition and function across major body sites, including the gut, mouth, lungs, reproductive tract and skin. It also explores connections between fungal imbalances (dysbiosis) and diseases such as neurological disorders, cancer and post-COVID-19 complications. Despite progress, challenges persist, including the need for better culture-independent diagnostic tools and standardized research methods. Combining culturomics and metagenomics could help overcome these limitations and identify new treatment targets. By summarizing current knowledge and highlighting research gaps, this review aims to guide future studies on the mycobiome's role in human health.

The Staphylococcus aureus type VIIb secretion system (T7SSb) is a multiprotein secretion system that secretes toxins with antibacterial activity, but which is also required for full virulence in animal models of infection. S. aureus strains carry one of four T7SSb locus types, named essC1 to essC4, each of which encodes a characteristic LXG-family substrate at the T7SS locus. In essC2 strains, this LXG-domain protein is EsxX, which has a glycine zipper sequence in its C-terminus and has potent antibacterial, membrane-depolarizing activity. In this work, we recognize conserved features of the essC2 and essC3 systems, identifying the LXG protein SAR0287 as structurally and functionally similar to EsxX. Using a zebrafish larval hindbrain ventricle infection model, we demonstrate that the T7SSb of essC2 and essC3 representative strains contributes to bacterial replication and zebrafish mortality. However, there is no significant loss of virulence in the model system if EsxX or SAR0287 is absent. These findings indicate that there is no discernible role for either toxin in this virulence model.