Microbiology-Sgm最新文献

Resumption of the planktonic phase of the Bacillus cereus cell cycle necessitates degradation of certain morphological structures and physiological features that confer metabolic dormancy and multi-factorial resistance properties to the spore form of the bacterium. Depolymerization of the peptidoglycan cortex, which is crucial to maintenance of spore dormancy, constitutes a major germination event and is conducted by a complement of spore cortex lytic enzymes that are active only during spore germination. This work reports on the structure and function of the major cortex lytic enzymes in B. cereus spores, revealing insight to their location, individual contributions to germination when triggered by different routes and regions of the SleB protein that are important for mediating interactions with its peptidoglycan substrate. The effect of null mutations to lipoproteins of the YlaJ/YhcN family on spore properties is also characterized, revealing parallels with prior observations concerning YlaJ's influence on SleB activity during germination. Finally, a structural model of a putative SleB-YpeB-YlaJ complex is presented. The model, which was subject to an initial validation by evolutionary covariance analysis and site-directed mutagenesis, reveals how the SleB protein might be held in an inactive state courtesy of its interactions with YpeB and YlaJ during spore dormancy, potentially shedding light on a long-standing puzzle in spore germination.

Actinobacteria are important for industrial production of antibiotics, fine chemicals and food and a source of new compounds for drug discovery. Their central metabolism is regulated by a conserved protein GarA that is unique to the Actinobacteria and has been studied in Mycobacterium tuberculosis and Corynebacterium glutamicum. GarA regulates the TCA cycle and glutamate metabolism by direct binding to enzymes to modulate their activity on glutamate and alpha-ketoglutarate. Given the importance of the TCA cycle in the synthesis of acyl-CoA precursors for antibiotic biosynthesis, and increasing evidence for the role of nitrogen regulators in control of secondary metabolism, we hypothesized that engineering GarA could be used to enhance production of valuable metabolites. His₆-tagged GarA was introduced into Saccharopolyspora erythraea, an overproducer of the polyketide antibiotic erythromycin. Phosphorylation of GarA was detected at the N-terminal ETTS motif, suggesting that it is regulated by protein kinases like in M. tuberculosis. GarA expression was observed at all growth stages, and a truncated form lacking the phosphorylation site accumulated during late fermentation. Engineered S. erythraea expressing phosphoablative GarA produced twofold more erythromycin, both in standard fermentation broth and in minimal medium. To investigate the mechanism for the increased titre, the engineered strain was characterized for transcription of erythromycin biosynthetic genes, as well as its ability to metabolize glutamate and its intracellular and extracellular aa content. The observed alterations in aa metabolism are consistent with the role of GarA as a TCA cycle regulator that may influence precursor supply for polyketide biosynthesis.

Gram-positive bacteria commonly employ autoinducing peptide (AIP) signal molecules to co-ordinate gene expression at the population level. This primer provides a basic overview of agr-dependent quorum sensing systems and outlines how AIPs are produced and sensed, what they control and the importance of agr for both inter-bacterial and host-pathogen interactions.

In this study, the liquid-liquid co-culture method was applied using faecal samples and specific bacterial species as growth-supporting bacteria. We aimed to isolate new, difficult-to-culture bacterial species using metabolites produced by supportive bacteria to promote the growth of small bacteria selected using filter treatment. This study aimed to identify the supporting bacteria and their metabolites that promote the growth of these isolates. Analysis of the 16S rRNA gene sequences of the isolates obtained by co-culture revealed that they were Waltera spp., Roseburia spp. and Phascolarctobacterium faecium. Roseburia spp. and Waltera spp. were isolated from several faecal samples, suggesting that they were specifically isolated using this culture method. We focused on Waltera spp. isolated from several faecal samples with unique shapes, from long to short or thin cells. The growth of Waltera spp. was not promoted by co-culture on the agar medium, suggesting that growth was only promoted by liquid-liquid co-culture. The growth of the selected small-sized Waltera spp. was promoted by co-culture, whereas the growth of the unfiltered long-cell Waltera sp. strain was suppressed by co-culture. The selected small Waltera spp. did not grow when the supporting bacterial supernatant was added, suggesting that the supporting bacteria and Waltera spp. had a symbiotic relationship through the continuous exchange of metabolites. Co-cultured supporting bacteria (diluted faecal samples) with selected small-sized Waltera spp. were predominantly Bacteroides thetaiotaomicron and Escherichia coli, compared with monoculture diluted faecal samples. We further confirmed the growth of filtered Waltera spp. by co-culturing them with B. thetaiotaomicron and E. coli. Additionally, when B. thetaiotaomicron and E. coli were co-cultured with the selected small Waltera spp., some nutrients and metabolites were reduced. Decreased metabolites were added to the medium, and selected small-sized Waltera spp. were cultured, but Waltera spp. did not grow. Therefore, it was again strongly suggested that continuous co-culturing with the supporting bacteria was important for the growth of Waltera spp. The liquid-liquid co-culture method used in this study can be used to isolate new and unique bacterial species from any environment, not just the gut microbiome. Furthermore, this co-culture method helped identify supporting bacteria and understand metabolite variations.

Stakeholder mapping is a process that involves identifying, characterizing and visualizing the connections between stakeholders. It is important to understand the main influencers and those with an interest in or affected by a certain project or field. Stakeholders can have diverse perspectives and priorities, some complementary and others conflicting, and it is important to develop effective engagement strategies to facilitate progress with the desired impact. Here, we present the first such analysis for the bioaerosol research field. This study presents data from a survey and outcomes from workshops attended by researchers with an interest in bioaerosols. Bioaerosols are airborne particles from biological origin that can have diverse and serious effects on public health. Stakeholders were identified and analysed in terms of sector, engagement type, interest and influence scores, expertise and the nature of the relationships between them. Limitations of this study are discussed with suggestions for improvements. The workshops included discussions on missing stakeholders, skills and knowledge gaps, the uses of stakeholder mapping and how to facilitate skill sharing, collaboration and effective progress in the future. We share this to provide a simple but successful approach for researchers with no previous practical experience in stakeholder mapping to modify, use and realize similar benefits within their own fields.

Proteus mirabilis is a frequent cause of catheter-associated urinary tract infection and often exhibits high tolerance to chlorhexidine (CHD), a biocide used widely in healthcare settings. We previously demonstrated that inactivation of the smvR repressor (leading to overexpression of the smvA efflux system), truncation of the MltA-interacting protein MipA and aspects of lipopolysaccharide (LPS) structure modulate CHD susceptibility in this organism. However, the prevalence of these mechanisms among P. mirabilis clinical isolates, the conditions under which they can be acquired and their impact on susceptibility to other cationic biocides require further study. Through phenotypic and genomic analysis of a panel of 78 P. mirabilis clinical isolates, we have confirmed that deleterious mutations in smvR commonly arise in P. mirabilis and are significantly associated with reduced susceptibility to CHD and other cationic biocides. Mutations in mipA were also associated with CHD tolerance. Conversely, mutations in smvA and the rppA response regulator (which governs lipid A modifications that alter LPS surface charge) were associated with increased susceptibility to several biocides. Several isolates harbouring smvR mutations displayed incongruous phenotypes, exhibiting relatively modest CHD tolerance, which could not be accounted for by co-occurring mutations in smvA and rppA or defects in LPS (as assessed by polymyxin B susceptibility). Further analysis of these isolates revealed mutations in the LPS core biosynthesis gene waaG, leading to LPS truncation from the inner core region. Directed evolution experiments further reinforced the importance of smvR inactivation in biocide adaptation in P. mirabilis and demonstrated that relevant mutations can be selected for by exposure to CHD concentrations up to four times lower than the minimum inhibitory concentration. Taken together, these results expand our understanding of mechanisms underlying tolerance to cationic biocides in this species and provide evidence for common mechanisms of cationic biocide tolerance.

The Klebsiella pneumoniae species complex (KpSC) comprises five closely related bacterial species, namely Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola, Klebsiella quasivariicola and Klebsiella africana. The KpSC is ubiquitous in the environment and is also an important human pathogen, particularly associated with healthcare-associated infections. The accurate detection and differentiation of the KpSC is challenging owing to the close phenotypic and genotypic identity (93-95% average nucleotide identity) shared between these members. Current diagnostic assays either fail to detect and identify all KpSC members or misidentify some KpSC members as K. pneumoniae sensu stricto. It is currently estimated that ~20% of human infections are caused by members of the KpSC other than K. pneumoniae. This leads to underreporting of some KpSC members in both clinical and environmental settings, which impacts our understanding of the importance of each species. Furthermore, it limits our understanding of the global and local epidemiological impact of some members of the KpSC. In this study, a rapid multiplex real-time PCR assay (KpSC-ID) was designed and developed to detect all KpSC members while simultaneously identifying the predominant human pathogens K. pneumoniae, K. quasipneumoniae and K. variicola. Assay performance was verified in silico using a panel of over 1,000 publicly available genome sequences and experimentally validated using a panel of genomic DNA extracted from 54 Enterobacteriaceae. The assay displayed excellent specificity against over 1,000 genome sequences tested in silico. During in vitro validation, the pan-KpSC assay detected each (29/29) KpSC species and strains tested. For the species-specific assays, 100% specificity was demonstrated in the K. pneumoniae, K. quasipneumoniae and K. variicola assays, respectively. Sensitivity of 10 genomic equivalents was demonstrated for each assay. Ultimately, the diagnostic assay developed in this study can improve our understanding of the significance of KpSC members, which is important when investigating their routes of transmission and epidemiology.

The improvement in next-generation sequencing technologies has reduced the costs of sequencing significantly. However, library preparation costs for amplicon sequencing have remained largely unchanged - which is ultimately the cost-limiting step in processing large numbers of microbiome samples. Acoustic liquid handlers can transfer volumes as low as 2.5 nl and have been used to miniaturize several different molecular and cellular assays, including single-step PCR amplicon library preparations. However, there are no current methods available for a two-step library preparation process using an acoustic liquid handler. In this study, we tested the efficiency of an acoustic liquid handler to automate the PCRs and library quantification while also incorporating automated library bead cleanup. We compared the material usage and costs for library preparation and sequencing results of this automated method to the standard, manual method. The automated protocol was able to reduce both PCR reaction volumes fivefold and increased efficiency for library preparation by ~32% without affecting bacterial community compositions. The associated increase in the efficiency of our automated method will allow for greater throughput in sequencing hundreds of microbiome samples without affecting the quality of those sequences.

Latilactobacillus sakei, a lactic acid bacterium in diverse environments such as fermented foods, meat and the human gastrointestinal tract, exhibits significant genetic diversity and niche-specific adaptations. This study conducts a comprehensive comparative genomic analysis of 29 complete L. sakei genomes to uncover the genetic mechanisms underlying these adaptations. Phylogenetic analysis divided the species into three distinct clades that did not correlate with the source of isolation and did not suggest any niche-specific evolutionary direction. The pan-genome analysis revealed a substantial core genome alongside a diverse genetic repertoire, indicating both high genetic conservation and adaptability. Predicted growth rates based on codon use bias analysis suggest that L. sakei strains have an overall faster growth rate and may be able to efficiently dominate in competitive environments. Plasmid analysis revealed a variety of plasmids carrying genes essential for carbohydrate metabolism, enhancing L. sakei's ability to thrive in various fermentation substrates. It was also found that the number of genes belonging to the GH1 family amongst sugar metabolism-related genes present on chromosomes and plasmids varies between strains and that AA1, which is involved in alcohol oxidation, has been acquired from plasmids. blast analysis revealed that some strains have environmental adaptation gene clusters of cell surface polysaccharides that may mediate attachment to food and mucosa. The knowledge gleaned from this study lays a solid foundation for future research aimed at harnessing the genetic traits of L. sakei strains for industrial and health-related applications.

Antimicrobial resistance (AMR) is an escalating global health threat, with the greatest risk observed in low- to middle-income countries, particularly in the global south. The World Health Organization advocates for a One Health approach to address AMR, promoting collaboration across sectors, including in agriculture. This study aims to enhance understanding of antimicrobial use and stewardship in livestock within pastoralist communities in northern Kenya, where there is limited information. The study employed a qualitative approach, using semi-structured interviews to gather data on farming practices and antibiotic use. Interviews were conducted by trained volunteers proficient in Swahili and Ma (a Maasai language), across four pastoralist communities in northern Kenya in December 2023. The data were then thematically analysed by four researchers. Fifty-one individuals participated in the study. Thematic analysis revealed several key insights, including the widespread misuse of antibiotics, often used on intuition and without professional support. A notable barrier to appropriate use was the lack of veterinary advice, with many participants relying on agrovets or past experience for guidance. Cross-use of antibiotics, such as administering animal antibiotics to humans, was also observed. Awareness of AMR was limited, and leftover antibiotics were often saved or shared across communities. The findings from this study underscore the critical need for targeted education and training within these communities.