A new model of endotracheal tube biofilm identifies combinations of matrix-degrading enzymes and antimicrobials able to eradicate biofilms of pathogens that cause ventilator-associated pneumonia.

Ventilator-associated pneumonia is defined as pneumonia that develops in a patient who has been on mechanical ventilation for more than 48 hours through an endotracheal tube. It is caused by biofilm formation on the indwelling tube, which introduces pathogenic microbes such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans into the patient's lower airways. Currently, there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates the biofilm formation of these pathogens in a host-mimicking environment and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilated airway mucus medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-scanning electron microscopy were employed to characterize the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved the biofilm eradication of all pathogens. Our in vitro endotracheal tube model informs on fundamental microbiology in the ventilator-associated pneumonia context and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants.

Mutations in the Staphylococcus aureus Global Regulator CodY Confer Tolerance to an Interspecies Redox-Active Antimicrobial.

Bacteria often exist in multispecies communities where interactions among different species can modify individual fitness and behavior. Although many competitive interactions have been characterized, molecular adaptations that can counter this antagonism and preserve or increase fitness remain underexplored. Here, we characterize the adaptation of Staphylococcus aureus to pyocyanin, a redox-active interspecies antimicrobial produced by Pseudomonas aeruginosa, a co-infecting pathogen frequently isolated from wound and chronic lung infections with S. aureus. Using experimental evolution, we identified mutations in a conserved global transcriptional regulator, CodY, that confer tolerance to pyocyanin and thereby enhance survival of S. aureus. The transcriptional response of a pyocyanin tolerant CodY mutant to pyocyanin indicated a two-pronged defensive response compared to the wild type. Firstly, the CodY mutant strongly suppressed metabolism, by downregulating pathways associated with core metabolism, especially translation-associated genes, upon exposure to pyocyanin. Metabolic suppression via ATP depletion was sufficient to provide comparable protection against pyocyanin to the wild-type strain. Secondly, while both the wild-type and CodY mutant strains upregulated oxidative stress response pathways, the CodY mutant overexpressed multiple stress response genes compared to the wild type. We determined that catalase overexpression was critical to pyocyanin tolerance as its absence eliminated tolerance in the CodY mutant and overexpression of catalase was sufficient to impart tolerance to the wild-type strain. Together, these results suggest that both transcriptional responses likely contribute to pyocyanin tolerance in the CodY mutant. Our data thus provide new mechanistic insight into adaptation toward interbacterial antagonism via altered regulation that facilitates multifaceted protective cellular responses.

Multi-species biofilms of environmental microbiota isolated from fruit packing facilities promoted tolerance of Listeria monocytogenes to benzalkonium chloride.

Listeria monocytogenes may survive and persist in food processing environments due to formation of complex multi-species biofilms of environmental microbiota that co-exists in these environments. This study aimed to determine the effect of selected environmental microbiota on biofilm formation and tolerance of L. monocytogenes to benzalkonium chloride in formed biofilms. The studied microbiota included bacterial families previously shown to co-occur with L. monocytogenes in tree fruit packing facilities, including Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae, and Flavobacteriaceae. Biofilm formation ability and the effect of formed biofilms on the tolerance of L. monocytogenes to benzalkonium chloride was measured in single- and multi-family assemblages. Biofilms were grown statically on polystyrene pegs submerged in a R2A broth. Biofilm formation was quantified using a crystal violet assay, spread-plating, confocal laser scanning microscopy, and its composition was assessed using amplicon sequencing. The concentration of L. monocytogenes in biofilms was determined using the most probable number method. Biofilms were exposed to the sanitizer benzalkonium chloride, and the death kinetics of L. monocytogenes were quantified using a most probable number method. A total of 8, 8, 6, and 3 strains of Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae, and Flavobacteriaceae, respectively, were isolated from the environmental microbiota of tree fruit packing facilities and were used in this study. Biofilms formed by Pseudomonadaceae, Xanthomonadaceae, and all multi-family assemblages had significantly higher concentration of bacteria, as well as L. monocytogenes, compared to biofilms formed by L. monocytogenes alone. Furthermore, multi-family assemblage biofilms increased the tolerance of L. monocytogenes to benzalkonium chloride compared to L. monocytogenes mono-species biofilms and planktonic multi-family assemblages. These findings suggest that L. monocytogenes control strategies should focus not only on assessing the efficacy of sanitizers against L. monocytogenes, but also against biofilm-forming microorganisms that reside in the food processing built environment, such as Pseudomonadaceae or Xanthomonadaceae.

Bacterial stress response, physiological metabolism and antimicrobial tolerance and the control strategies / 生物工程学报

Overuse of antibiotics in medical care and animal husbandry has led to the development of bacterial antimicrobial resistance, causing increasingly more health concern. In addition to genetic mutations and the formation of resistance, the various stresses bacteria encountered in the natural environment trigger their stress responses, which not only protect them from these stresses, but also change their tolerance to antimicrobials. The emergence of antimicrobial tolerance will inevitably affect the physiological metabolism of bacteria. However, bacteria can restore their sensitivity to drugs by regulating their own metabolism. This article reviews recent studies on the relationship between bacterial stress responses or the physiological metabolism and antimicrobial tolerance, intending to take more effective measures to control the occurrence and spread of antimicrobial resistance.

Biopelículas microbianas y su impacto en áreas médicas: fisiopatología, diagnóstico y tratamiento / Microbial biofilms and their impact on medical areas: physiopathology, diagnosis and treatment

Resumen Las biopelículas son comunidades de microorganismos que crecen agregados y rodeados por una matriz extracelular que ellos mismos producen, la cual favorece la adhesión covalente sobre superficies inertes y vivas; además, les ayuda a desarrollar alta tolerancia a las moléculas con actividad antimicrobiana. Por otra parte, las biopelículas se asocian con infecciones crónicas y persistentes que impactan de manera negativa en distintas áreas médicas. Además, generan altos costos a los sistemas de salud y a los pacientes cada año, porque son difíciles de tratar con antimicrobianos convencionales; adicionalmente, generan altas tasas de morbilidad y mortalidad. El objetivo de esta revisión es presentar información extensa y actualizada sobre el origen, la biosíntesis y la fisiopatología de las biopelículas, así como sobre su relación con infecciones crónicas, el diagnóstico, los tratamientos antimicrobianos actuales con actividad antibiopelícula y las perspectivas sobre la búsqueda de nuevos tratamientos. Estos últimos aún representan una importante área de investigación.

Abstract Biofilms are communities of microorganisms that grow aggregated and surrounded by an extracellular matrix, which they produce and favors them to adhere covalently to inert and living surfaces; it also helps them to develop high tolerance to molecules with antimicrobial activity. Moreover, biofilms are associated with chronic and persistent infections, which negatively impact different medical areas since they generate high costs to health care systems and patients every year because they are difficult to treat with conventional antimicrobial drugs. Additionally, they generate high rates of morbidity and mortality. The objective of this review was to present extensive and up-to-date information on the origin, biosynthesis, and pathophysiology of biofilms. Also, its relationship with chronic infections, diagnosis, current antimicrobial treatments with antibiotic activity, and perspectives on the search for new treatments, since the latter still represent an important area of research.