Microbiology-Sgm最新文献

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.

Flagella are widely distributed appendages in bacteria with well-characterized functions in motility and chemotaxis. They also interact directly with hosts and, due to their ubiquity, are potent immune elicitors for hosts from both the plant and animal kingdoms. Furthermore, flagella have been shown to facilitate attachment for several different bacterial species, including several plant-associated bacteria to plant hosts. We previously demonstrated binding of flagella from Escherichia coli to ionic lipids in plant plasma membranes for horticultural species and Arabidopsis thaliana. As such, flagella could be considered as a generic colonization factor, especially in the early stages of the interactions. Therefore, we tested whether flagella from a genetically related species of plant pathogen, Pectobacterium atrosepticum, mediated binding to its susceptible plant host, potato, in a similar manner to E. coli. Surprisingly, flagella containing the filament flagellin from P. atrosepticum did not confer any binding advantage to potato roots. Furthermore, there was no direct interaction between purified flagella and potato membrane lipids (charged or uncharged). The binding capacity of Pectobacterium to potato is dependent upon the motility function of flagella, as both flagella-deficient and motor-deficient mutants were reduced in their binding to potato roots.

Bacillus velezensis HG88 was isolated from ileal mucosa samples of egg layer hens that were raised without the use of antibiotics. Its cell-free supernatant (CFS) was found to inhibit the growth of Clostridium perfringens, the causative agent of necrotic enteritis in chickens. The inhibitory compound was determined to be proteinaceous due to its susceptibility to protease digestion. The antimicrobial activity was specific towards C. perfringens, as the CFS did not inhibit the growth of Gram-positive or Gram-negative bacteria across nine different species and two yeast fungi. Separation of proteins from the CFS followed by peptide mass fingerprinting and genomic analyses of the strain enabled the identification of a putative antibacterial peptide with an export signal for secretion from the cell. The peptide from B. velezensis HG88, named IP_HG88, has sequence similarity to bacterial SH3 domains that are known to bind to the peptide portion of peptidoglycan. The gene encoding this peptide was cloned, and the peptide was purified from recombinant Escherichia coli as an N-terminal His-tagged peptide. The IP_HG88 with or without the His-tag inhibited the growth of C. perfringens with a minimum bactericidal concentration of ~57.0 or 39.1 µg ml^-1, respectively. The 3D structure of IP_HG88 was also predicted using AlphaFold 2.0.

This study investigates the role of polyamine biosynthesis in the pathogenesis of the bacterial phytopathogen Pseudomonas syringae pv. tomato. Through a comprehensive phenotypic analysis of mutant strains affected in the synthesis of putrescine and spermidine, we reveal a complex interplay between this metabolic pathway and bacterial virulence. Disruption of putrescine synthesis impairs a variety of virulence traits such as motility, biofilm formation, siderophore production, prevention of plant stomatal closure and the functionality of the type III secretion system. This is reversed by reintroducing the deleted genes, but not by the supplementation of culture media with putrescine or apoplastic washing fluids (AWF). Similarly, suppression of spermidine biosynthesis results in a comparable phenotype. However, in this case, the wild-type phenotype is restored by adding spermidine, AWF or expressing the spermidine synthase gene. We conclude that both putrescine and spermidine are important for bacterial virulence and that plant-derived spermidine can partially compensate for bacterial needs. Accordingly, whereas putrescine deficiency leads to a hypovirulent phenotype, spermidine synthesis perturbation does not affect plant colonization. These findings emphasize the critical role of polyamine metabolism in the plant invasion process by bacterial pathogens.

In the absence of the broadly conserved deaminase RidA (Reactive intermediate deaminase A), Salmonella enterica and other organisms accumulate the reactive enamine species 2-aminoacrylate (2AA). Free 2AA, generated from serine by the serine/threonine dehydratase IlvA, reacts with and covalently inactivates a subset of pyridoxal 5'-phosphate-dependent enzymes. The metabolic stress caused by 2AA generates growth defects in S. enterica, including (i) when l-alanine is used as a nitrogen source, (ii) when pyruvate is used as a carbon source or (iii) in the presence of exogenous serine. Although the enzymatic targets of 2AA are consistent between growth conditions, the consequences of 2AA-dependent damage differ depending on the distribution of metabolic flux required in different conditions. Analysing the suppressors of a ridA mutant has furthered our understanding of the RidA stress paradigm and, more generally, how a metabolic network responds to perturbation. Many such suppressors modulate the metabolic network to eliminate 2AA production by IlvA. Here, we describe that eliminating the MetJ transcriptional repressor allows a ridA mutant to grow in the presence of 2AA stress in each of the three conditions. The mechanisms by which a ΔmetJ suppresses a ridA mutant are nuanced and medium-dependent, emphasizing that consequences of 2AA stress differ based on environmental and metabolic context.

Culturomics approaches have advanced microbial research by enabling the high-throughput isolation and characterization of a broader range of bacterial taxa, including some previously considered unculturable. Here, we present the testing and optimization of a protocol for isolating and identifying hundreds of cultivable microbes from field-grown plants. This protocol was tested and optimized using the root microbiomes of field-grown corn and pea plants under varying environmental conditions in ND, USA. By employing dilution-to-extinction culturing and a two-step barcoding PCR strategy targeting the V4 region of the 16S rRNA gene, we identified over 200 unique bacterial isolates. The optimized bioinformatic pipeline, built around the DADA2 package, ensured accurate amplicon sequence variant detection and taxonomy assignment. The resulting bacterial isolates span diverse phylogenetic groups, including plant-associated taxa known for promoting plant growth and mitigating stress. Our findings highlight the value of culturomics in generating microbial collections for synthetic community design and advancing plant-microbe interaction research. The protocol's scalability, cost-effectiveness and robust performance demonstrate its potential for widespread application in agricultural microbiome studies.