Polymyxin B and ethylenediaminetetraacetic acid act synergistically against Pseudomonas aeruginosa and Staphylococcus aureus.

Polymyxin B and ethylenediaminetetraacetic acid are antimicrobials possessing antibiofilm activity. They act by displacement and chelation, respectively, of divalent cations in bacterial membranes and may therefore act synergistically when applied in combination. If so, this combination of agents may be useful for the treatment of diseases like cystic fibrosis (CF), in which biofilms are present on the respiratory epithelium. We used checkerboard assays to investigate the synergy between these agents using reference strains Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 6538 in planktonic form. We then determined the efficacy of each agent against biofilms of both species grown on 96-pin lids and proceeded to combination testing against the P. aeruginosa reference strain and 10 clinical isolates from patients with CF. Synergism was observed for planktonic forms of both species and for biofilms of P. aeruginosa. The susceptibility of biofilms of P. aeruginosa clinical isolates to these agents was variable compared to the laboratory reference strain. This combination of agents may be useful in the management of biofilm-associated conditions, particularly those amenable to topical therapies. These results provide a basis upon which the antimicrobial and antibiofilm efficacy of preparations containing these agents may be enhanced.IMPORTANCEBacteria living in biofilms produce a protective matrix which makes them difficult to kill. Patients with severe respiratory disease often have biofilms. Polymyxin B is an antibiotic commonly used in topical medications, such as eye drops and nasal sprays. Ethylenediaminetetraacetic acid (EDTA) is used widely as a preservative in medication but also has antimicrobial properties. It has been hypothesized that Polymyxin B and EDTA could have a synergistic relationship: when used in combination their antimicrobial effect is enhanced. Here, we evaluated the levels at which Polymyxin B and EDTA work together to kill common pathogens Pseudomonas aeruginosa and Staphylococcus aureus. We found that Polymyxin B and EDTA were synergistic. This synergy may be useful in the management of planktonic infection with P. aeruginosa and S. aureus, or biofilm infection with P. aeruginosa. This synergy may be beneficial in the treatment of respiratory biofilms, in which P. aeruginosa biofilms are common.

Current evidence of biofilms in chronic rhinosinusitis- a microbiological perspective.

INTRODUCTION: Chronic rhinosinusitis (CRS) is characterized by inflammation of the paranasal sinus mucosa persisting for more than 12 weeks. This condition is associated with reduced quality-of-life and causes a high direct and indirect economic burden. Several pathogenic factors have been attributed to CRS, including bacterial and fungal biofilms on the sinonasal mucosa. Biofilms are well-established contributors to recalcitrance to treatment in other chronic inflammatory mucosal conditions such as cystic fibrosis and otitis media. AREAS COVERED: This review will present an overview of the role of biofilms in CRS, including the evidence for biofilms being present on the sinonasal mucosa and their implications for disease severity. Furthermore, the interactions between biofilms and host-mediated immune factors are explored. EXPERT OPINION: The eradication of biofilms has been a focus of research shortly after their recognition as a cause of disease. The currently available methodologies for identifying biofilms on mucosal surfaces are not sufficiently well-developed to be used in a clinical setting. A more accurate, cheaper, faster approach for biofilm detection is necessary, and molecular techniques may provide the possibility for this.

The Effect of Amoxicillin with Clavulanate on the Microbiota of Tonsillar Tissue in Disease: a Randomized Control Trial.

Despite antibiotics being the primary medical treatment for recurrent tonsillitis, the impact of antibiotics on the tonsillar microbiome is not well understood. This study aimed to determine the effect of amoxicillin with clavulanate on the composition and quantity of bacteria in the tonsils of children with recurrent tonsillitis. A multicenter randomized clinical trial in Auckland, New Zealand was undertaken between August 1, 2017, and June 30, 2018. Sixty children undergoing tonsillectomy for the indication of recurrent tonsillitis were recruited for this study. Following random allocation, 30 participants were prescribed amoxicillin with clavulanate for the week before surgery. The remaining 30 received no antibiotics. Immediately following surgery, the crypts of the right and left tonsils were swabbed. Bacterial 16S rRNA gene-targeted amplicon sequencing and histological techniques were utilized. In the control group, there were significantly higher relative abundances of Haemophilus, Streptococcus, Neisseria, and Porphyromonas. Members from the genera Fusobacterium and Treponema were found to be significantly more abundant in the antibiotic group. There were no significant differences in the absolute quantities of bacteria between the groups. Microscopic examination found fewer bacterial microcolonies present in the tonsillar crypts of participants in the antibiotic group. Streptococcus pyogenes was not present in these bacterial microcolonies. These results suggest that a single course of antibiotics has a significant impact on the tonsil microbiota composition. The duration of this effect and the effect that the altered microbiome has on the course of the condition need to be determined. IMPORTANCE Several studies have identified the presence of multiple pathogenic bacteria in hyperplastic adenoids and palatine tonsils. However, there are currently no studies that utilize this technology to investigate the effect of oral antibiotics in children with recurrent tonsillitis on the tonsillar microbiome. This is the first study to investigate the effect of antibiotics on the microbiome of tonsillar tissue in children with recurrent tonsillitis using molecular techniques. This study has shown that participants who received amoxicillin with clavulanate immediately before tonsillectomy had a significantly reduced number of bacterial taxa commonly associated with recurrent tonsillitis, as well as the number of bacterial microcolonies observed in the tonsillar crypts. This novel finding suggests that either the effect of antibiotics is not sustained or that they are not an effective treatment for recurrent tonsillitis.

Longitudinal analysis of sinus microbiota post endoscopic surgery in patients with cystic fibrosis and chronic rhinosinusitis: a pilot study.

BACKGROUND: Cystic fibrosis is a debilitating, autosomal recessive disease which results in chronic upper and lower airway infection and inflammation. In this study, four adult patients presenting with cystic fibrosis and chronic rhinosinusitis were recruited. Culture and molecular techniques were employed to evaluate changes in microbial profiles, host gene expression and antimicrobial resistance (AMR) in the upper respiratory tract over time. METHODS: Swab samples from the sinonasal cavity were collected at the time of surgery and at follow-up clinics at regular time intervals for up to 18 months. Nucleic acids were extracted, and DNA amplicon sequencing was applied to describe bacterial and fungal composition. In parallel, RNA was used to evaluate the expression of 17 AMR genes and two inflammatory markers (interleukins 6 and 8) using custom qPCR array cards. Molecular results were compared with routine sinus and sputum culture reports within each patient. RESULTS: Bacterial amplicon sequencing and swab culture reports from the sinonasal cavity were mostly congruent and relatively stable for each patient across time. The predominant species detected in patients P02 and P04 were Pseudomonas aeruginosa, Staphylococcus aureus in patient P03, and a mixture of Enterobacter and S. aureus in patient P01. Fungal profiles were variable and less subject specific than bacterial communities. Increased expressions of interleukins 6 and 8 were observed in all patients throughout the sampling period compared with other measured genes. The most prevalent AMR gene detected was ampC. However, the prevalence of AMR gene expression was low in all patient samples across varying time-points. CONCLUSIONS: We observed a surprising degree of stability of sinonasal microbial composition, and inflammatory and AMR gene expression across all patients post sinus surgery.

Oral antibiotics used in the treatment of chronic rhinosinusitis have limited penetration into the sinonasal mucosa: a randomized trial.

Despite the widespread prescription of antibiotics for patients with chronic rhinosinusitis (CRS), the extent to which drug distribution to the sinonasal mucosa occurs remains largely undefined. Twenty subjects undergoing functional endoscopic sinus surgery (FESS) for CRS were randomized to one of two groups: 1) doxycycline (100 mg daily for seven days) 2) roxithromycin (300 mg daily for seven days). Drug levels were measured using liquid chromatography-tandem mass spectrometry in sinonasal mucus, sinonasal tissues and serum at steady state. Doxycycline concentrations measured in the mucus were significantly lower compared to that in the serum (mean mucus/serum ratio = 0.16, p < 0.001) and the tissue (mean mucus/tissue ratio = 0.18, p < 0.0001). Roxithromycin concentrations in the mucus were also significantly lower compared to that in the serum (mean mucus/serum ratio = 0.37, p = 0.002) and the tissue (mean mucus/tissue ratio = 0.60, p < 0.001). Although the efficacy of doxycycline and roxithromycin in sinonasal mucus in vivo cannot be predicted solely from reported minimum inhibitory concentrations, given the added complexity of bacterial biofilm antimicrobial tolerance, these results suggest that low mucosal penetration of antibiotics may be one of the factors contributing to the limited efficacy of these agents in the treatment of CRS.

Detection and quantification of Staphylococcus in chronic rhinosinusitis.

BACKGROUND: The sinonasal microbiota has been implicated in chronic rhinosinusitis (CRS) pathogenesis, particularly related to the presence of Staphylococcus aureus. Staphylococcus epidermidis is also prevalent within the sinonasal microbiota and may inhibit S. aureus colonization. We investigated polymerase chain reaction (PCR) primer pairs for measuring absolute abundances of S. aureus and S. epidermidis, then compared bacterial community composition and absolute abundances of these species between CRS patients and controls. METHODS: Six candidate Staphylococcus species-specific primer pairs were tested in silico and in vitro against pure bacterial isolates. Quantitative PCR (qPCR) for absolute quantification of S. aureus, S. epidermidis, and overall bacterial load were assessed in 40 CRS (CRS without nasal polyposis [CRSsNP] = 22, CRS with nasal polyposis [CRSwNP] = 18) patients and 14 controls. Amplicon sequencing of the V3-V4 hypervariable regions of the 16S ribosomal RNA (rRNA) bacterial gene were conducted to investigate community composition. RESULTS: Primer pairs targeting the gmk gene of S. aureus and nrd gene from S. epidermidis were the most specific and sensitive primers. S. aureus (CRSsNP = 81.8% occurrence, CRSwNP = 83%, control = 92.9%) and S. epidermidis (CRSsNP = 95.5%, CRSwNP = 100%, control = 92.9%) were very prevalent, as indicated by qPCR results. Both CRSsNP and CRSwNP had significantly (p < 0.05) higher bacterial load when compared with controls (p < 0.05 for both). No significant correlation was observed between S. aureus and S. epidermidis abundances (p > 0.05). CONCLUSION: Bacterial community sequencing detected Staphylococcus-assigned sequences in nearly all patients; however, it could not differentiate between S. aureus and S. epidermidis. Here, we present primer pairs that can distinguish between these species. We report a very high prevalence of S. aureus in both CRS patients and controls.

Comparison of Subtyping Approaches and the Underlying Drivers of Microbial Signatures for Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is a heterogeneous condition characterized by persistent sinus inflammation and microbial dysbiosis. This study aimed to identify clinically relevant subgroups of CRS patients based on distinct microbial signatures, with a comparison to the commonly used phenotypic subgrouping approach. The underlying drivers of these distinct microbial clusters were also investigated, together with associations with epithelial barrier integrity. Sinus biopsy specimens were collected from CRS patients (n = 23) and disease controls (n = 8). The expression of 42 tight junction genes was evaluated using quantitative PCR together with microbiota analysis and immunohistochemistry for measuring mucosal integrity and inflammation. CRS patients clustered into two distinct microbial subgroups using probabilistic modelling Dirichlet (DC) multinomial mixtures. DC1 exhibited significantly reduced bacterial diversity and increased dispersion and was dominated by Pseudomonas, Haemophilus, and Achromobacter DC2 had significantly elevated B cells and incidences of nasal polyps and higher numbers of Anaerococcus, Megasphaera, Prevotella, Atopobium, and Propionibacterium In addition, each DC exhibited distinct tight junction gene and protein expression profiles compared with those of controls. Stratifying CRS patients based on clinical phenotypic subtypes (absence or presence of nasal polyps [CRSsNP or CRSwNP, respectively] or with cystic fibrosis [CRSwCF]) accounted for a larger proportion of the variation in the microbial data set than with DC groupings. However, no significant differences between CRSsNP and CRSwNP cohorts were observed for inflammatory markers, beta-dispersion, and alpha-diversity measures. In conclusion, both approaches used for stratifying CRS patients had benefits and pitfalls, but DC clustering provided greater resolution when studying tight junction impairment. Future studies in CRS should give careful consideration to the patient subtyping approach used.IMPORTANCE Chronic rhinosinusitis (CRS) is a major human health problem that significantly reduces quality of life. While various microbes have been implicated, there is no clear understanding of the role they play in CRS pathogenesis. Another equally important observation made for CRS patients is that the epithelial barrier in the sinonasal cavity is defective. Finding a robust approach to subtype CRS patients would be the first step toward unravelling the pathogenesis of this heterogeneous condition. Previous work has explored stratification based on the clinical presentation of the disease (with or without polyps), inflammatory markers, pathology, or microbial composition. Comparisons between the different stratification approaches used in these studies have not been possible due to the different cohorts, analytical methods, or sample sites used. In this study, two approaches for subtyping CRS patients were compared, and the underlying drivers of the heterogeneity in CRS were also explored.

Inflammatory Endotypes and Microbial Associations in Chronic Rhinosinusitis.

A complex mix of inflammatory and microbial associations underscores the chronic inflammatory condition chronic rhinosinusitis (CRS), and the etiology remains poorly understood. Recent work has begun to delineate between variants (endotypes) of CRS on the basis of inflammatory biomarkers. This study aimed to assess inflammatory patterns in CRS phenotypes, identify putative endotypes of CRS, and to assess inflammatory associations with the sinonasal microbiota. Ten cytokines and six inflammatory cell types were assessed in mucosal biopsies from 93 CRS subjects and 17 controls via cytometric bead array and immunohistochemical techniques. Putative endotypes were identified via cluster analysis of subjects on the basis of inflammatory markers and comorbidities including polyposis, asthma, and aspirin sensitivity. Finally, previously published bacterial data for this cohort were reanalyzed to evaluate associations with inflammatory markers and CRS subtypes. Inflammatory patterns were highly variable within standard CRS phenotypes. Cluster analysis identified eight subject clusters, with strong delineation on the basis of polyposis and asthma, but also subtle distinctions in inflammatory markers. An association was also identified between depletion of several "health-associated" bacterial taxa, reduced bacterial diversity and increased overall bacterial load, with markers of inflammation and clinical severity. This study contributes to ongoing efforts to define distinct endotypes of CRS on the basis of underlying inflammatory processes, and also offers compelling evidence of a link between bacterial community dysbiosis and inflammation in CRS. Further resolving the heterogeneity of CRS is vital to inform clinical management and personalized treatment approaches.

Differentially Regulated Host Proteins Associated with Chronic Rhinosinusitis Are Correlated with the Sinonasal Microbiome.

The chronic inflammatory nature of chronic rhinosinusitis (CRS) makes it a morbid condition for individuals with the disease and one whose pathogenesis is poorly understood. To date, proteomic approaches have been applied successfully in a handful of CRS studies. In this study we use a multifaceted approach, including proteomics (iTRAQ labeling) and microbiome (bacterial 16S rRNA gene sequencing) analyses of middle meatus swabs, as well as immune cell analysis of the underlying tissue, to investigate the host-microbe interaction in individuals with CRS (n = 10) and healthy controls (n = 9). Of the total 606 proteins identified in this study, seven were significantly (p < 0.05) more abundant and 104 were significantly lower in the CRS cohort compared with healthy controls. The majority of detected proteins (82% of proteins identified) were not significantly correlated with disease status. Elevated levels of blood and immune cell proteins in the CRS cohort, together with significantly higher numbers of B-cells and macrophages in the underlying tissue, confirmed the inflammatory status of CRS individuals. Protein PRRC2C and Ras-related protein (RAB14) (two of the seven elevated proteins) showed the biggest fold difference between the healthy and CRS groups. Validation of the elevated levels of these two proteins in CRS samples was provided by immunohistochemistry. Members of the bacterial community in the two study cohorts were not associated with PRRC2C, however members of the genus Moraxella did correlate with RAB14 (p < 0.0001, rho = -0.95), which is a protein involved in the development of basement membrane. In addition, significant correlations between certain members of the CRS bacterial community and 33 lower abundant proteins in the CRS cohort were identified. Members of the genera Streptococcus, Haemophilus and Veillonella were strongly correlated with CRS and were significantly associated with a number of proteins with varying functions. The results from this study reveal a strong association between the host and microbes in the sinonasal cavity. Proteins identified as associated with CRS could be new targets for drug therapies and biomarkers for assessment of treatment efficacy.

Evidence of microbiota dysbiosis in chronic rhinosinusitis.

BACKGROUND: Despite considerable research, the pathogenesis of chronic rhinosinusitis (CRS) remains poorly understood. Potential microbial roles in the etiology or progression of CRS have long been hypothesized, yet few specific associations have been identified. In this study we investigate associations between patterns in resident bacterial communities and clinical variants of CRS. METHODS: Bacterial communities were assessed in 94 patients with extensive bilateral CRS undergoing endoscopic sinus surgery (ESS) and 29 controls undergoing ESS for indications other than CRS. Patients were grouped on the basis of phenotypic variants (with or without polyposis) and clinical parameters, including asthma and cystic fibrosis. Bacterial communities were characterized via 16S rRNA gene amplicon sequencing, and quantified by quantitative polymerase chain reaction. RESULTS: Controls and idiopathic CRS subjects tended to be dominated by members of the genera Corynebacterium and Staphylococcus, together with lower abundances of several other genera, including Streptococcus, Moraxella, and Haemophilus. Aberrant (dysbiotic) bacterial assemblages (with changes in community membership and structure, reduced diversity, and increased bacterial load) and increased inter- and intrasubject variability were more common in subjects with comorbidities such as asthma and cystic fibrosis. Dysbiotic communities were variably dominated by members of the genera Staphylococcus, Streptococcus, Haemophilus, Pseudomonas, Moraxella, or Fusobacterium. CONCLUSION: Bacterial community dysbiosis was more apparent than specific associations with examined phenotypes or endotypes, and may play a role in the pathogenesis or influence the severity of CRS. Reductions in several common core bacterial taxa, increased inter- and intrasubject variability, reduced bacterial diversity, and increased bacterial load characterized aberrant bacterial communities in CRS.