Artificial dry surface biofilm (DSB) models for testing the efficacy of cleaning and disinfection

Dry surface biofilms (DSB) harbouring pathogens are widespread in healthcare settings, difficult to detect and resistant to cleaning and disinfection interventions. Here, we describe a practical test protocol to palliate the lack of standard efficacy test methods for DSB. Staphylococcus aureus DSB were produced over a 12‐day period, grown with or without the presence of organic matter, and their composition and viability were evaluated. Disinfectant treatment was conducted with a modified ASTM2967‐15 test and reduction in viability, transferability, and biofilm regrowth post treatment were measured. Dry surface biofilms produced over a 12‐day period had a similar carbohydrates, proteins and DNA content, regardless the presence or absence of organic matter. The combination of sodium hypochlorite (1,000 ppm) and a microfiber cloth was only effective against DSB in the absence of organic load. With the increasing concerns of the uncontrolled presence of DSB in healthcare settings, the development of effective interventions is paramount. We propose that our DSB model in the presence of organic load is appropriate for the testing of biocidal products, while the use of three parameters, log10 reduction, transferability and regrowth, provides an accurate and practical measurement of product efficacy

Biotic Interactions Shape the Ecological Distributions of Staphylococcus Species.

Many metagenomic sequencing studies have observed the presence of closely related bacterial species or genotypes in the same microbiome. Previous attempts to explain these patterns of microdiversity have focused on the abiotic environment, but few have considered how biotic interactions could drive patterns of microbiome diversity. We dissected the patterns, processes, and mechanisms shaping the ecological distributions of three closely related Staphylococcus species in cheese rind biofilms. Paradoxically, the most abundant species (S. equorum) is the slowest colonizer and weakest competitor based on growth and competition assays in the laboratory. Through in vitro community reconstructions, we determined that biotic interactions with neighboring fungi help resolve this paradox. Species-specific stimulation of the poor competitor by fungi of the genus Scopulariopsis allows S. equorum to dominate communities in vitro as it does in situ Results of comparative genomic and transcriptomic experiments indicate that iron utilization pathways, including a homolog of the S. aureus staphyloferrin B siderophore operon pathway, are potential molecular mechanisms underlying Staphylococcus-Scopulariopsis interactions. Our integrated approach demonstrates that fungi can structure the ecological distributions of closely related bacterial species, and the data highlight the importance of bacterium-fungus interactions in attempts to design and manipulate microbiomes.ImportanceDecades of culture-based studies and more recent metagenomic studies have demonstrated that bacterial species in agriculture, medicine, industry, and nature are unevenly distributed across time and space. The ecological processes and molecular mechanisms that shape these distributions are not well understood because it is challenging to connect in situ patterns of diversity with mechanistic in vitro studies in the laboratory. Using tractable cheese rind biofilms and a focus on coagulase-negative staphylococcus (CNS) species, we demonstrate that fungi can mediate the ecological distributions of closely related bacterial species. One of the Staphylococcus species studied, S. saprophyticus, is a common cause of urinary tract infections. By identifying processes that control the abundance of undesirable CNS species, cheese producers will have more precise control on the safety and quality of their products. More generally, Staphylococcus species frequently co-occur with fungi in mammalian microbiomes, and similar bacterium-fungus interactions may structure bacterial diversity in these systems

It's a trap! The development of a versatile drain biofilm model and its susceptibility to disinfection

Background Pathogens in drain biofilms pose a significant risk for hospital-acquired infection. However, the evidence of product effectiveness in controlling drain biofilm and pathogen dissemination are scarce. A novel in-vitro biofilm model was developed to address the need for a robust, reproduceable and simple testing methodology for disinfection efficacy against a complex drain biofilm. Methods Identical complex drain biofilms were established simultaneously over 8 days, mimicking a sink trap. Reproducibility of their composition was confirmed by next-generation sequencing. The efficacy of sodium hypochlorite 1000 ppm (NaOCl), sodium dichloroisocyanurate 1000 ppm (NaDCC), non-ionic surfactant (NIS) and peracetic acid 4000 ppm (PAA) was explored, simulating normal sink usage conditions. Bacterial viability and recovery following a series of 15-min treatments were measured in three distinct parts of the drain. Results The drain biofilm consisted of 119 mixed species of Gram-positive and -negative bacteria. NaOCl produced a >4 log10 reduction in viability in the drain front section alone, while PAA achieved a >4 log10 reduction in viability in all of the drain sections following three 15-min doses and prevented biofilm regrowth for >4 days. NIS and NaDCC failed to control the biofilm in any drain sections. Conclusions Drains are one source of microbial pathogens in healthcare settings. Microbial biofilms are notoriously difficult to eradicate with conventional chemical biocidal products. The development of this reproducible in-vitro drain biofilm model enabled understanding of the impact of biocidal products on biofilm spatial composition and viability in different parts of the drain. Keyword

Review of decontamination protocols for shared non-critical objects in 35 policies of UK NHS acute care organizations

Background Decontamination of non-critical objects shared by patients is key in reducing hospital-acquired infections (HAIs), but it is a complex process that needs precise guidance from UK National Health Service (NHS) acute care organizations (ACOs). Aim To review the indications given by NHS ACOs' policies regarding the decontamination of shared non-critical devices. Methods Detailed lists of decontamination protocols for shared non-critical objects were retrieved from cleaning, disinfection and decontamination policies of 35 NHS ACOs. Three parameters were considered for each object: decontamination method, decontamination frequency, and person responsible for decontamination. Findings In total, 1279 decontamination protocols regarding 283 different shared non-critical objects were retrieved. Of these, 689 (54%) did not indicate the person responsible for decontamination, and only 425 (33%) were complete, giving indications for all three parameters analysed. Only 2.5% (32/1279) of decontamination protocols were complete and identical in two policies. In policies where cleaning represented the major decontamination method, chemical disinfection was rarely mentioned and vice versa. General agreement among policies was found for four main decontamination methods (detergent and water, detergent wipes, disinfectant wipes, and use of disposable items), two decontamination frequencies (between events and daily) and two responsible person designations (nurses and domestic staff). Conclusions Decontamination protocol policies for shared non-critical objects had some similarities but did not concur on how each individual object should be decontaminated. The lack of clear indications regarding the person responsible for the decontamination process put at risk the ability of policies to serve as guidance

Monoclonal Antibodies Opsonize <i>Burkholderia</i> spp. and Reduce Intracellular Actin Tail Formation in a Macrophage Infection Assay

Melioidosis is difficult to treat successfully due to the causal bacterium being resistant to many classes of antibiotics, therefore limiting available therapeutic options. New and improved therapies are urgently required to treat this disease.</jats:p

Persister Escherichia coli Cells Have a Lower Intracellular pH than Susceptible Cells but Maintain Their pH in Response to Antibiotic Treatment

This is the final version. Available from the American Society for Microbiology via the DOI in this record. Persister and viable but non-culturable (VBNC) cells are two clonal subpopulations that can survive multidrug exposure via a plethora of putative molecular mechanisms. Here, we combine microfluidics, time-lapse microscopy, and a plasmid-encoded fluorescent pH reporter to measure the dynamics of the intracellular pH of individual persister, VBNC, and susceptible Escherichia coli cells in response to ampicillin treatment. We found that even before antibiotic exposure, persisters have a lower intracellular pH than those of VBNC and susceptible cells. We then investigated the molecular mechanisms underlying the observed differential pH regulation in persister E. coli cells and found that this is linked to the activity of the enzyme tryptophanase, which is encoded by tnaA. In fact, in a ΔtnaA strain, we found no difference in intracellular pH between persister, VBNC, and susceptible E. coli cells. Whole-genome transcriptomic analysis revealed that, besides downregulating tryptophan metabolism, the ΔtnaA strain downregulated key pH homeostasis pathways, including the response to pH, oxidation reduction, and several carboxylic acid catabolism processes, compared to levels of expression in the parental strain. Our study sheds light on pH homeostasis, proving that the regulation of intracellular pH is not homogeneous within a clonal population, with a subset of cells displaying a differential pH regulation to perform dedicated functions, including survival after antibiotic treatment.Biotechnology and Biological Sciences Research Council (BBSRC)Wellcome TrustMedical Research Council (MRC)The Royal SocietyThe Gordon and Betty Moore FoundationMarie Skłodowska‐CurieLeverhulme TrustUnited Kingdom Ministry of DefenseUniversity of Exete

Broad-spectrum in vitro activity of macrophage infectivity potentiator inhibitors against Gram-negative bacteria and Leishmania major

Background The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip. Objectives In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. Methods Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. Results Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. Conclusions These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications

Macrophage infectivity potentiator protein, a peptidyl prolyl cis-trans isomerase, essential for Coxiella burnetii growth and pathogenesis

This is the final version. Available from Public Library of Science via the DOI in this record. All relevant data are within the manuscript, supporting files and the MS dataset has been deposited into the PRIDE ProteomeXchange Consortium repository, the dataset identifier is PXD036679.Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available human vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyse the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria. To date the role of the Mip protein in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. The pipecolic acid derived compounds, SF235 and AN296, which have shown utility in targeting other Mip proteins from pathogenic bacteria, demonstrate inhibitory activities against CbMip. These compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain in axenic culture. Comparative proteomics, in the presence of AN296, revealed alterations in stress responses with H2O2 sensitivity assays validating that Mip inhibition increases the sensitivity of C. burnetii to oxidative stress. In addition, SF235 and AN296 were effective in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for replication and that the development of more potent inhibitors against CbMip is warranted and offer potential as novel therapeutics against this pathogen.Defence Science and Technology Laboratory (DSTL)NHMRCNorth Atlantic Treaty Organization (NATO)German Research Foundation (DFG)UK Ministry of DefenceThe Federal Ministry of Education and ResearchDMTC Limited (Australia