Effect of GT-Peptide 10 and Triethyl Citrate on P. acnes Biofilm Formation, Viability, and Dispersion.

BACKGROUND: P. acnes biofilms are emerging topics in acne vulgaris pathogenesis and may be responsible for antibiotic tolerance.
OBJECTIVE: To investigate the efficacy of GT peptide 10 either alone or in combination with triethyl citrate (TEC) in in vitro model of P. acnes biofilm.
METHODS: Six-day-old P. acnes biofilms were treated with various concentrations of these substances and biofilm dispersion and cell viability were monitored.
RESULTS: A 24-hour exposure of preformed biofilms to a combination of GT peptide 10/TEC led to killing of up to 92% of bacterial cells inside the biofilm. Neither the single substance nor the combination of both substances affected the biofilm integrity or resulted in biofilm dispersal.
CONCLUSIONS: A combination of GT peptide 10/TEC shows antibacterial effects in in vitro model of P. acnes biofilm.

J Drugs Dermatol. 2016;15(6):778-781.

Transcriptomic analysis of Propionibacterium acnes biofilms in vitro.

Propionibacterium acnes is a well-known commensal of the human skin connected to acne vulgaris and joint infections. It is extensively studied in planktonic cultures in the laboratory settings but occurs naturally in biofilms. In this study we have developed an in vitro biofilm model of P. acnes and studied growth features, matrix composition, matrix penetration by fluorescent-labeled antibiotics as well as gene expression. Antibiotic susceptibility of biofilms was studied and could be enhanced by increased glucose concentrations. Biofilm cells were characterized by up-regulated stress-induced genes and up-regulation of genes coding for the potential virulence-associated CAMP factors. P. acnes can generate persister cells showing a reversible tolerance to 50 fold MIC of common antibiotics.

Cyclic di-AMP homeostasis in bacillus subtilis: both lack and high level accumulation of the nucleotide are detrimental for cell growth.

The genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled.

Propionibacterium (Cutibacterium) granulosum Extracellular DNase BmdE Targeting Propionibacterium (Cutibacterium) acnes Biofilm Matrix, a Novel Inter-Species Competition Mechanism.

Acne vulgaris is the most common dermatological disorder worldwide affecting more than 80% of adolescents and young adults with a global prevalence of 231 million cases in 2019. The involvement of the skin microbiome disbalance in the pathophysiology of acne is recognized, especially regarding the relative abundance and diversity of Propionibacterium acnes a well-known dominant human skin commensal. Biofilms, where bacteria are embedded into a protective polymeric extracellular matrix, are the most prevalent life style for microorganisms. P. acnes and its biofilm-forming ability is believed to be a contributing factor in the development of acne vulgaris, the persistence of the opportunistic pathogen and antibiotic therapy failures. Degradation of the extracellular matrix is one of the strategies used by bacteria to disperse the biofilm of competitors. In this study, we report the identification of an endogenous extracellular nuclease, BmdE, secreted by Propionibacterium granulosum able to degrade P. acnes biofilm both in vivo and in vitro. This, to our knowledge, may represent a novel competitive mechanism between two closely related species in the skin. Antibiotics targeting P. acnes have been the mainstay in acne treatment. Extensive and long-term use of antibiotics has led to the selection and spread of resistant bacteria. The extracellular DNase BmdE may represent a new bio-therapeutical strategy to combat P. acnes biofilm in acne vulgaris.

Evaluation of the Biofire Filmarray Pneumonia panel plus for lower respiratory tract infections.

Background: Standard diagnostic methods for lower respiratory tract infections are currently too slow and insensitive to guide early clinical decisions concerning treatment and isolation. Syndrome-specific, diagnostic panels have potential to provide information about aetiology quickly. Available panels have been of limited use in lower respiratory tract infections due to slow turn-around-time, lack of quantification of important pathogens and lack of detection of resistance genes.Materials/methods: We evaluated the newly developed Biofire® Filmarray® Pneumonia Panel plus (Biomérieux). Eighty-eight consecutive lower respiratory tract samples were analyzed by both standard microbiological methods, as requested by the referring clinician, and by the panel. The agreement with standard methods, empirical treatment coverage and possible impact on isolation practices were assessed by comparing the results from standard diagnostic methods with the panel results in relation to clinical data and information of antimicrobial therapy.Results: Both qualitative and semi-quantitative results from the panel generally displayed good agreement with standard methods and by combining methods, a possible aetiology was detected in 73% of patients. Due to the panel approach, the panel detected viruses more frequently. In 25% of the 60 patients assessed for empirical treatment coverage, a pathogen not covered by current therapy was detected and in 30% of in-house patients the panel results were found to potentially influence clinical decisions related to isolation care.Conclusions: The new diagnostic panel shows promise in improving aetiological diagnostics of lower respiratory tract infections. Correctly applied it has potential to offer support in clinical decision-making within hours of sampling.

Determination of the Gene Regulatory Network of a Genome-Reduced Bacterium Highlights Alternative Regulation Independent of Transcription Factors.

Here, we determined the relative importance of different transcriptional mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae, by employing an array of experimental techniques under multiple genetic and environmental perturbations. Of the 143 genes tested (21% of the bacterium's annotated proteins), only 55% showed an altered phenotype, highlighting the robustness of biological systems. We identified nine transcription factors (TFs) and their targets, representing 43% of the genome, and 16 regulators that indirectly affect transcription. Only 20% of transcriptional regulation is mediated by canonical TFs when responding to perturbations. Using a Random Forest, we quantified the non-redundant contribution of different mechanisms such as supercoiling, metabolic control, RNA degradation, and chromosome topology to transcriptional changes. Model-predicted gene changes correlate well with experimental data in 95% of the tested perturbations, explaining up to 70% of the total variance when also considering noise. This analysis highlights the importance of considering non-TF-mediated regulation when engineering bacteria.

Transcription in Mycoplasma pneumoniae: analysis of the promoters of the ackA and ldh genes.

The nucleotide sequences that control transcription initiation and regulation in Mycoplasma pneumoniae are poorly understood. Moreover, only few regulatory events have been reported for M. pneumoniae. We have studied changes in the global protein synthesis pattern in M. pneumoniae in response to the presence of glycerol. The ackA and ldh genes, encoding acetate kinase and lactate dehydrogenase, respectively, were controlled in a carbon source-dependent manner. While the ackA gene was strongly expressed in the presence of glucose, transcription of ldh was induced by glycerol. The promoters of both genes were mapped by primer extension analysis. Molecular analysis of transcription regulatory mechanisms in M. pneumoniae has so far not been possible due to the lack of appropriate reporter systems that can be used to study the activity of promoter fragments and their mutant derivatives in vivo. Recently, a reporter system has been developed which allows cloning of promoter fragments in front of a promoterless lacZ gene and inserting this construct into the genome of M. pneumoniae. To study the requirements of M. pneumoniae RNA polymerase for promoter recognition, a series of fusions of deletion and mutant variants of the ldh promoter was constructed and analyzed in vivo. While mutations affecting the -10 region strongly interfered with gene expression, the -35 region seems to be of minor importance in M. pneumoniae.