S. aureus biofilm properties correlate with immune B cell subset frequencies and severity of chronic rhinosinusitis.

Staphylococcus aureus mucosal biofilms are associated with recalcitrant chronic rhinosinusitis (CRS). However, S. aureus colonisation of sinus mucosa is frequent in the absence of mucosal inflammation. This questions the relevance of S. aureus biofilms in CRS etiopathogenesis. This study aimed to investigate whether strain-level variation in in vitro-grown S. aureus biofilm properties relates to CRS disease severity, in vitro toxicity, and immune B cell responses in sinonasal tissue from CRS patients and non-CRS controls. S. aureus clinical isolates, tissue samples, and matched clinical datasets were collected from CRS patients with nasal polyps (CRSwNP), CRS without nasal polyps (CRSsNP), and controls. B cell responses in tissue samples were characterised by FACS. S. aureus biofilms were established in vitro, followed by measuring their properties of metabolic activity, biomass, colony-forming units, and exoprotein production. S. aureus virulence was evaluated using whole-genome sequencing, mass spectrometry and application of S. aureus biofilm exoproteins to air-liquid interface cultures of primary human nasal epithelial cells (HNEC-ALI). In vitro S. aureus biofilm properties were correlated with increased CRS severity scores, infiltration of antibody-secreting cells and loss of regulatory B cells in tissue samples. Biofilm exoproteins from S. aureus with high biofilm metabolic activity had enriched virulence genes and proteins, and negatively affected the barrier function of HNEC-ALI cultures. These findings support the notion of strain-level variation in S. aureus biofilms to be critical in the pathophysiology of CRS.

Staphylococcus aureus biofilm properties and chronic rhinosinusitis severity scores correlate positively with total CD4+ T-cell frequencies and inversely with its Th1, Th17 and regulatory cell frequencies.

Chronic rhinosinusitis (CRS) represents chronic inflammation of the sinus mucosa characterised by dysfunction of the sinuses' natural defence mechanisms and induction of different inflammatory pathways ranging from a Th1 to a Th2 predominant polarisation. Recalcitrant CRS is associated with Staphylococcus aureus dominant mucosal biofilms; however, S. aureus colonisation of the sinonasal mucosa has also been observed in healthy individuals challenging the significance of S. aureus in CRS pathogenesis. We aimed to investigate the relationship between CRS key inflammatory markers, S. aureus biofilm properties/virulence genes and the severity of the disease. Tissue samples were collected during endoscopic sinus surgery from the ethmoid sinuses of CRS patients with (CRSwNP) and without (CRSsNP) nasal polyps and controls (n = 59). CD3+ T-cell subset frequencies and key inflammatory markers of CD4+ helper T cells were determined using FACS analysis. Sinonasal S. aureus clinical isolates were isolated (n = 26), sequenced and grown into biofilm in vitro, followed by determining their properties, including metabolic activity, biomass, colony-forming units and exoprotein production. Disease severity was assessed using Lund-Mackay radiologic scores, Lund-Kennedy endoscopic scores and SNOT22 quality of life scores. Our results showed that S. aureus biofilm properties and CRS severity scores correlated positively with total CD4+ T-cell frequencies but looking into CD4+ T-cell subsets showed an inverse correlation with Th1 and Th17 cell frequencies. CD4+ T-cell frequencies were higher in patients harbouring lukF.PV-positive S. aureus while its regulatory and Th17 cell subset frequencies were lower in patients carrying sea- and sarT/U-positive S. aureus. Recalcitrant CRS is characterised by increased S. aureus biofilm properties in relation to increased total CD4+ helper T-cell frequencies and reduced frequencies of its Th1, Th17 and regulatory T-cell subsets. These findings offer insights into the pathophysiology of CRS and could lead to the development of more targeted therapies.

Colloidal silver against macrophage infections and biofilms of atypical mycobacteria.

Skin and soft tissue infection (SSTI) caused by atypical mycobacteria such as Mycobacterium abscessus and Mycobacterium avium intracellulare complex (MAIC) have increased in recent years. Current therapeutic options are limited, and hence new and better therapies are urgently required. Colloidal Silver (CS) has been identified for its widespread antibacterial properties and silver-impregnated dressings have been used for SSTIs caused by various pathogens. The efficacy of Green Synthesized Colloidal Silver (GSCS) was investigated for bacterial growth inhibition (BGI) using a microdilution method and minimum biofilm eradication concentration (MBEC) using resazurin assay and confocal scanning laser microscopy (CSLM) of M. abscessus (n = 5) and MAIC (n = 5). The antibacterial effect of GSCS against M. abscessus infected macrophages was also evaluated. The in vitro cytotoxicity of GSCS on a human keratinocyte cell line (HaCaT) and neonatal foreskin fibroblasts was analyzed by the crystal violet proliferation assay. Average BGI and MBEC of GSCS varied between 0.7 and 22 ppm for M. abscessus and MAIC. The concentration of 3 ppm reduced M. abscessus-infection in macrophages significantly. GSCS was not cytotoxic to HaCaT and neonatal foreskin fibroblast cells at concentrations < 3 ppm up to 2 h exposure time. GSCS therefore, has the potential for topical application against atypical mycobacterial SSTI.

Wound healing in endoscopic sinus surgery: Phase 1 clinical trial evaluating the role of Chitogel with adjuvants.

OBJECTIVES: This study aimed to determine the safety and efficacy of Chitogel, with and without Deferiprone (Def) and Gallium Protoporphyrin (GaPP), as a promoter of wound healing to improve surgical outcomes after endoscopic sinus susgery. DESIGN: A double-blinded, randomised control human clinical trial was conducted in patients undergoing ESS as a treatment for chronic rhinosinusitis. Participants underwent functional ESS or FESS with drill out as required and were randomised to receive test product Chitogel, Chitogel in combination with Def or Def-GaPP versus no packing (control). SETTING: Ostial stenosis and persistent inflammation are the main reasons for revision endoscopic sinus surgery (ESS). Post-operative (PO) dressings can improve PO wound healing and patient outcomes after ESS. PARTICIPANTS: Eighty two patients were included in this study with 79 patients completing the study with 40 undergoing full house FESS and 39 FESS plus frontal drillout. MAIN OUTCOME MEASURES: Patients were followed up at 2, 6 and 12 weeks PO, and outcome scores such as SNOT-22, VAS and LKS, pre and post-surgery (12 weeks) were compared. RESULTS: Seventy nine patients completed the study, there was a significant reduction in SNOT-22 score and improvement of VAS at 12 weeks in patients treated with Chitogel compared to control (p < .05). In those patients, the mean ostium area for the Chitogel and the Chitogel + Def + GaPP groups was higher across all three sinuses compared to the no-treatment control group, without statistical significance. Sphenoid sinus ostium was significantly more patent in patients treated with Chitogel compared to the control at the 12-week time point (p < .05). CONCLUSION: Chitogel as a PO dressing after ESS results in the best patient-reported symptom scores and objective measurements. The combination of Def and GaPP to Chitogel though proving safe, had no effect on the ostium patency or mucosal healing.

Functional mgrA Influences Genetic Changes within a Staphylococcus aureus Cell Population over Time.

Prolonged survival in the host-bacteria microenvironment drives the selection of alternative cell types in Staphylococcus aureus, permitting quasi-dormant sub-populations to develop. These facilitate antibiotic tolerance, long-term growth, and relapse of infection. Small Colony Variants (SCV) are an important cell type associated with persistent infection but are difficult to study in vitro due to the instability of the phenotype and reversion to the normal cell type. We have previously reported that under conditions of growth in continuous culture over a prolonged culture time, SCVs dominated a heterogenous population of cell types and these SCVs harbored a mutation in the DNA binding domain of the gene for the transcription factor, mgrA. To investigate this specific cell type further, S. aureus WCH-SK2-ΔmgrA itself was assessed with continuous culture. Compared to the wild type, the mgrA mutant strain required fewer generations to select for SCVs. There was an increased rate of mutagenesis within the ΔmgrA strain compared to the wild type, which we postulate is the mechanism explaining the increased emergence of SCV selection. The mgrA derived SCVs had impeded metabolism, altered MIC to specific antibiotics and an increased biofilm formation compared to non-SCV strain. Whole genomic sequencing detected single nucleotide polymorphisms (SNP) in phosphoglucosamine mutase glmM and tyrosine recombinase xerC. In addition, several genomic rearrangements were detected which affected genes involved in important functions such as antibiotic and toxic metal resistance and pathogenicity. Thus, we propose a direct link between mgrA and the SCV phenotype. IMPORTANCE Within a bacterial population, a stochastically generated heterogeneity of phenotypes allows continual survival against current and future stressors. The generation of a sub-population of quasi-dormant Small Colony Variants (SCV) in Staphylococcus aureus is such a mechanism, allowing for persistent or relapse of infection despite initial intervention seemingly clearing the infection. The use of continuous culture under clinically relevant conditions has allowed us to introduce time to the growth system and selects SCV within the population. This study provides valuable insights into the generation of SCV which are not addressed in standard laboratory generated models and reveals new pathways for understanding persistent S. aureus infection which can potentially be targeted in future treatments of persistent S. aureus infection.

Association between mucosal barrier disruption by Pseudomonas aeruginosa exoproteins and asthma in patients with chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a common chronic respiratory condition, frequently associated with asthma and affecting the majority of cystic fibrosis (CF) patients. Pseudomonas aeruginosa infections and biofilms have been implicated in recalcitrant CRS. One of the mechanisms of action for bacteria in CRS and CF is mucosal barrier disruption by secreted products that contribute to the inflammation. However, the role of biofilm and planktonic forms of P. aeruginosa in this process is not known. The aim is to determine the effect of P. aeruginosa exoproteins isolated from CF and non-CF CRS patients on the mucosal barrier. METHODS: Exoproteins from 40 P. aeruginosa isolates were collected in planktonic and biofilm forms and applied to air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs). Mucosal barrier integrity was evaluated by transepithelial electrical resistance (TEER), passage of FITC-dextrans and immunofluorescence of tight junction proteins. Cytotoxicity assays were performed to measure cell viability, and IL-6 ELISA was carried out to evaluate pro-inflammatory effects. RESULTS: Planktonic exoproteins from 20/40 (50%) clinical isolates had a significant detrimental effect on the barrier and significantly increased IL-6 production. Barrier disruption was characterized by a reduced TEER, increased permeability of FITC-dextrans and discontinuous immunolocalization of tight junction proteins and was significantly more prevalent in isolates harvested from patients with comorbid asthma (P < .05). CONCLUSION: Exoproteins from planktonic P. aeruginosa clinical isolates from asthmatic CRS patients have detrimental effects on the mucosal barrier and induce IL-6 production potentially contributing to the mucosal inflammation in CRS patients.

The international sinonasal microbiome study: A multicentre, multinational characterization of sinonasal bacterial ecology.

The sinonasal microbiome remains poorly defined, with our current knowledge based on a few cohort studies whose findings are inconsistent. Furthermore, the variability of the sinus microbiome across geographical divides remains unexplored. We characterize the sinonasal microbiome and its geographical variations in both health and disease using 16S rRNA gene sequencing of 410 individuals from across the world. Although the sinus microbial ecology is highly variable between individuals, we identify a core microbiome comprised of Corynebacterium, Staphylococcus, Streptococcus, Haemophilus and Moraxella species in both healthy and chronic rhinosinusitis (CRS) cohorts. Corynebacterium (mean relative abundance = 44.02%) and Staphylococcus (mean relative abundance = 27.34%) appear particularly dominant in the majority of patients sampled. Amongst patients suffering from CRS with nasal polyps, a statistically significant reduction in relative abundance of Corynebacterium (40.29% vs 50.43%; P = .02) was identified. Despite some measured differences in microbiome composition and diversity between some of the participating centres in our cohort, these differences would not alter the general pattern of core organisms described. Nevertheless, atypical or unusual organisms reported in short-read amplicon sequencing studies and that are not part of the core microbiome should be interpreted with caution. The delineation of the sinonasal microbiome and standardized methodology described within our study will enable further characterization and translational application of the sinus microbiota.

Sirtuin-1 Controls Poly (I:C)-Dependent Matrix Metalloproteinase 9 Activation in Primary Human Nasal Epithelial Cells.

Matrix metalloproteinase (MMP)-9 is thought to be involved in the etiopathogenesis of chronic rhinosinusitis (CRS) with nasal polyps and cleaves collagen IV, causing hyperpermeability of the basement membrane within mucosal tissue. It is known that MMP-9 expression is negatively affected by sirtuin (SIRT)-1 in human monocytotic cells, retinal endothelial cells, and epithelial carcinoma cells. However, it is unknown which factors affect MMP-9 expression and activity in human nasal epithelial cells (HNECs). To examine factors affecting MMP-9 expression and activity in HNECs, HNECs were stimulated with Toll-like receptor (TLR) agonists, followed by quantitative PCR, immunofluorescence, and zymography to examine MMP-9 expression and activity. MMP-9 expression was evaluated in sinonasal tissue of control subjects without CRS, and patients with CRS without nasal polyps and those with CRS with nasal polyps, in relation to the expression of SIRT1 using a tissue microarray. The effect of SIRT1 stimulation/inhibition on MMP-9 expression in HNECs was also tested. TLR3 agonists increased MMP-9 mRNA expression (473 fold, P = 0.0198) and activity (20.4-fold, P < 0.05). SIRT1 activation or inhibition reciprocally affected MMP-9 expression in the presence of TLR3 agonists. MMP-9 and SIRT1 expression within the epithelial layer of sinonasal tissue was inversely correlated only in patients with CRS but not in control subjects. TLR3 agonists increased MMP-9 expression and activity in HNECs, and the effect was abolished in the presence of SIRT1 activation. SIRT1 and MMP-9 expression was inversely correlated in CRS tissue, supporting SIRT1 as a possible therapeutic target for nasal polyp formation.

Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum-evidence from a mathematical model.

The hetero-dimeric CoA-transferase CtfA/B is believed to be crucial for the metabolic transition from acidogenesis to solventogenesis in Clostridium acetobutylicum as part of the industrial-relevant acetone-butanol-ethanol (ABE) fermentation. Here, the enzyme is assumed to mediate re-assimilation of acetate and butyrate during a pH-induced metabolic shift and to faciliate the first step of acetone formation from acetoacetyl-CoA. However, recent investigations using phosphate-limited continuous cultures have questioned this common dogma. To address the emerging experimental discrepancies, we investigated the mutant strain Cac-ctfA398s::CT using chemostat cultures. As a consequence of this mutation, the cells are unable to express functional ctfA and are thus lacking CoA-transferase activity. A mathematical model of the pH-induced metabolic shift, which was recently developed for the wild type, is used to analyse the observed behaviour of the mutant strain with a focus on re-assimilation activities for the two produced acids. Our theoretical analysis reveals that the ctfA mutant still re-assimilates butyrate, but not acetate. Based upon this finding, we conclude that C. acetobutylicum possesses a CoA-tranferase-independent butyrate uptake mechanism that is activated by decreasing pH levels. Furthermore, we observe that butanol formation is not inhibited under our experimental conditions, as suggested by previous batch culture experiments. In concordance with recent batch experiments, acetone formation is abolished in chemostat cultures using the ctfa mutant.

Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker.

Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms.

Chitogel with deferiprone following endoscopic sinus surgery: improved wound healing and microbiome.

Background: Adhesion formation, sinus ostial narrowing, and presence of pathogenic bacteria are associated with poor outcomes following endoscopic sinus surgery (ESS) for chronic rhinosinusitis. Chitogel has been shown to improve wound healing, restore a healthier microbiome, and reduce post-operative infections post ESS. Deferiprone has antibacterial properties and has been shown to reduce adhesion formation. The aim of the study was to assess whether the addition of low concentration deferiprone to Chitogel further improves surgical outcomes following ESS compared with Chitogel alone. Methods: In this double-blinded trial, 45 patients undergoing ESS were prospectively recruited. At the end of the surgery, patients were randomised to receive Chitogel alone, Chitogel with 1 mM of deferiprone, or Chitogel with 5 mM of deferiprone to one side of the sinuses (allowing the other side to serve as control). Patients underwent routine follow-ups with symptom questionnaires and nasoendoscopies performed at 2, 6, and 12 weeks post-operatively. Sinus ostial measurements, microbiology, and microbiome swabs from bilateral middle meatuses were collected intraoperatively and at 12 weeks post-operatively. Results: A significant improvement in the endoscopic appearance of the sinuses and frontal ostial patency was noted at 12 weeks post-operatively (p < 0.05) in all three treatment groups compared with the control. There was no significant difference noted between patients who received Chitogel alone and those who received Chitogel with 1 or 5 mM deferiprone. Conclusion: Chitogel alone, Chitogel with 1 mM deferiprone, and Chitogel with 5 mM deferiprone used following ESS led to a significant improvement in endoscopic appearance of the sinuses and frontal ostial preservation at 12 weeks post-operatively. No significant difference was found with the addition of deferiprone to Chitogel.

Deferiprone-Gallium-Protoporphyrin Chitogel Decreases Pseudomonas aeruginosa Biofilm Infection without Impairing Wound Healing.

Pseudomonas aeruginosa is one of the most common pathogens encountered in clinical wound infections. Clinical studies have shown that P. aeruginosa infection results in a larger wound area, inhibiting healing, and a high prevalence of antimicrobial resistance. Hydroxypyridinone-derived iron chelator Deferiprone (Def) and heme analogue Gallium-Protoporphyrin (GaPP) in a chitosan-dextran hydrogel (Chitogel) have previously been demonstrated to be effective against PAO1 and clinical isolates of P. aeruginosa in vitro. Moreover, this combination of these two agents has been shown to improve sinus surgery outcomes by quickly reducing bleeding and preventing adhesions. In this study, the efficacy of Def-GaPP Chitogel was investigated in a P. aeruginosa biofilm-infected wound murine model over 6 days. Two concentrations of Def-GaPP Chitogel were investigated: Def-GaPP high dose (10 mM Def + 500 µg/mL GaPP) and Def-GaPP low dose (5 mM Def + 200 µg/mL GaPP). The high-dose Def-GaPP treatment reduced bacterial burden in vivo from day 2, without delaying wound closure. Additionally, Def-GaPP treatment decreased wound inflammation, as demonstrated by reduced neutrophil infiltration and increased anti-inflammatory M2 macrophage presence within the wound bed to drive wound healing progression. Def-GaPP Chitogel treatment shows promising potential in reducing P. aeruginosa cutaneous infection with positive effects observed in the progression of wound healing.

Increased antibiotic resistance of Pseudomonas aeruginosa isolates from chronic rhinosinusitis patients grown in anaerobic conditions.

Introduction: In chronic rhinosinusitis (CRS), the congestion and blockage of the nose can cause anaerobic conditions within the sinus cavities which may promote the expression of virulence and antibiotic resistance genes in invading pathogens. Pseudomonas aeruginosa is a facultative anaerobic bacteria and causes severe recalcitrant CRS. In this study, we aimed to evaluate the antimicrobial resistance of P. aeruginosa isolates of CRS patients in planktonic and biofilm form grown in aerobic and anaerobic conditions. Methods: P. aeruginosa clinical isolates of CRS patients (n = 25) were grown in planktonic and biofilm form in aerobic and anaerobic conditions. Minimum inhibitory concentrations (MIC) of planktonic forms and minimum biofilm eradication concentrations (MBEC) were determined. Additionally, metabolic activity by fluorescein diacetate assay, biofilm biomass by crystal violet assay and eDNA concentration were assessed in both conditions. Results: P. aeruginosa planktonic cells grown in anaerobic condition exhibited increased gentamicin resistance (p < .01), whereas P. aeruginosa biofilms grown in anaerobic condition displayed significantly increased MBEC values for gentamicin (p < .0001) and levofloxacin (p < .001). The metabolic activity of anaerobic biofilms was significantly higher compared with aerobic biofilms (p < .0001). However, the biofilm biomass of isolates grown in aerobic conditions was higher than anaerobic conditions (p < .5). Conclusion: P. aeruginosa isolates from CRS patients grown in anaerobic conditions showed significantly increased resistance to antibiotics with an increased metabolic activity but decreased biofilm biomass. Level of Evidence: NA.

S. aureus biofilm metabolic activity correlates positively with patients' eosinophil frequencies and disease severity in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a persistent inflammation of the sinus mucosa. Recalcitrant CRS patients are unresponsive to medical and surgical interventions and often present with nasal polyps, tissue eosinophilia, and Staphylococcus aureus dominant mucosal biofilms. However, S. aureus sinonasal mucosal colonisation occurs in the absence of inflammation, questioning the role of S. aureus in CRS pathogenesis. Here, we aimed to investigate the relationship between S. aureus biofilm metabolic activity and virulence genes, innate immune cells, and disease severity in CRS. Biospecimens, including sinonasal tissue and nasal swabs, and clinical datasets, including disease severity scores, were obtained from CRS patients and non-CRS controls. S. aureus isolates were grown into biofilms in vitro, characterised, and sequenced. The patients' innate immune response was evaluated using flow cytometry. S. aureus was isolated in 6/19 (31.58%) controls and 23/53 (43.40%) CRS patients of 72 recruited patients. We found increased S. aureus biofilm metabolic activity in relation to increased eosinophil cell frequencies and disease severity in recalcitrant CRS cases. Mast cell frequencies were higher in tissue samples of patients carrying S. aureus harbouring lukF.PV, sea, and fnbB genes. Patients with S. aureus harbouring lukF.PV and sdrE genes had more severe disease. This offers insights into the pathophysiology of CRS and could lead to the development of more targeted therapies.

Deferiprone-gallium-protoporphyrin (IX): A promising treatment modality against Mycobacterium abscessus.

Non-Tuberculous Mycobacterial Pulmonary Disease (NTM-PD) caused by Mycobacterium abscessus is a frequent complication in patients with cystic fibrosis (CF) that worsens lung function over time. Currently, there is no cure for NTM-PD, hence new therapies are urgently required. Disrupting bacterial iron uptake pathways using gallium-protoporphyrin (IX) (GaPP), a heme analog, has been proposed as a novel antibacterial approach to tackle multi-drug resistant M. abscessus. However, the antibacterial activity of GaPP has been tested only in iron-deficient media, which cannot accurately mirror the potential activity in vivo. Herein, we investigated the potential synergistic activity between GaPP and the iron-chelating agent deferiprone (Def) in regular media against M. abscessus-infected macrophages. The safety of the treatment was assessed in vitro using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in Nuli-1 and THP-1 cell lines. Def-GaPP had synergistic activity against M. abscessus-infected macrophages where 10 mM-12.5 mg/L of Def-GaPP reduced the viability by up to 0.9 log10. Furthermore, Def-GaPP showed no cytotoxicity to Nuli-1 and THP-1 cell lines at the effective antibacterial concentrations (10 mM-12.5 mg/L) of Def- GaPP. These data encourage future investigation of Def-GaPP as a novel antimicrobial against NTM-PD.

The intra-host evolutionary landscape and pathoadaptation of persistent Staphylococcus aureus in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a common chronic sinonasal mucosal inflammation associated with Staphylococcus aureus biofilm and relapsing infections. This study aimed to determine rates of S. aureus persistence and pathoadaptation in CRS patients by investigating the genomic relatedness and antibiotic resistance/tolerance in longitudinally collected S. aureus clinical isolates. A total of 68 S. aureus paired isolates (34 pairs) were sourced from 34 CRS patients at least 6 months apart. Isolates were grown into 48 h biofilms and tested for tolerance to antibiotics. A hybrid sequencing strategy was used to obtain high-quality reference-grade assemblies of all isolates. Single nucleotide variants (SNV) divergence in the core genome and sequence type clustering were used to analyse the relatedness of the isolate pairs. Single nucleotide and structural genome variations, plasmid similarity, and plasmid copy numbers between pairs were examined. Our analysis revealed that 41 % (14/34 pairs) of S. aureus isolates were persistent, while 59 % (20/34 pairs) were non-persistent. Persistent isolates showed episode-specific mutational changes over time with a bias towards events in genes involved in adhesion to the host and mobile genetic elements such as plasmids, prophages, and insertion sequences. Furthermore, a significant increase in the copy number of conserved plasmids of persistent strains was observed. This was accompanied by a significant increase in biofilm tolerance against all tested antibiotics, which was linked to a significant increase in biofilm biomass over time, indicating a potential biofilm pathoadaptive process in persistent isolates. In conclusion, our study provides important insights into the mutational changes during S. aureus persistence in CRS patients highlighting potential pathoadaptive mechanisms in S. aureus persistent isolates culminating in increased biofilm biomass.

The bacteriology of diabetic foot ulcers and infections and incidence of Staphylococcus aureus Small Colony Variants.

Introduction. Uninfected diabetes-related foot ulcer (DFU) progression to diabetes-related foot infection (DFI) is a prevalent complication for patients with diabetes. DFI often progresses to osteomyelitis (DFI-OM). Active (growing) Staphylococcus aureus is the most common pathogen in these infections. There is relapse in 40-60 % of cases even when the initial treatment at the DFI stage apparently clears infection.Hypothesis. S. aureus adopts the quasi-dormant Small Colony Variant (SCV) state during DFU and consequently infection, and when present in DFI cases also permits survival in non-diseased tissues as a reservoir to cause relapse.Aim. The aim of this study was to investigate the bacterial factors that facilitate persistent infections.Methodology. People with diabetes were recruited from two tertiary hospitals. Clinical and bacterial data was taken from 153 patients with diabetes (51 from a control group with no ulcer or infection) and samples taken from 102 patients with foot complications to identify bacterial species and their variant colony types, and then compare the bacterial composition in those with uninfected DFU, DFI and those with DFI-OM, of whom samples were taken both from wounds (DFI-OM/W) and bone (DFI-OM/B). Intracellular, extracellular and proximal 'healthy' bone were examined.Results. S. aureus was identified as the most prevalent pathogen in diabetes-related foot pathologies (25 % of all samples). For patients where disease progressed from DFU to DFI-OM, S. aureus was isolated as a diversity of colony types, with increasing numbers of SCVs present. Intracellular (bone) SCVs were found, and even within uninfected bone SCVs were present. Wounds of 24 % of patients with uninfected DFU contained active S. aureus. All patients with a DFI with a wound but not bone infection had previously had S. aureus isolated from an infection (including amputation), representing a relapse.Conclusion. The presence of S. aureus SCVs in recalcitrant pathologies highlights their importance in persistent infections through the colonization of reservoirs, such as bone. The survival of these cells in intracellular bone is an important clinical finding supporting in vitro data. Also, there seems to be a link between the genetics of S. aureus found in deeper infections compared to those only found in DFU.

Targeted mutagenesis of the Clostridium acetobutylicum acetone-butanol-ethanol fermentation pathway.

The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone-butanol-ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to ethanol and butanol formation under the standard batch culture conditions employed in this study. All phenotypic changes observed could be reversed by genetic complementation, with exception of those seen for the ptb mutant. This mutant produced around 100 mM ethanol, no acetone and very little (7 mM) butanol. The genome of the ptb mutant was therefore re-sequenced, together with its parent strain (ATCC 824 wild type), and shown to possess a frameshift mutation in the thl gene, which perfectly explained the observed phenotype. This finding reinforces the need for mutant complementation and Southern Blot analysis (to confirm single ClosTron insertions), which should be obligatory in all further ClosTron applications.