1001. Chronic Colonization with Toxigenic Clostridioides difficile Strains Drives Colonic Tumorigenesis in Mice

Background Long-term effects of chronic and/or recurrent C. difficile infections (CDI) are not well understood, and any potential role of CDI in colorectal cancer (CRC) risk is presently unknown. While pursuing efforts to identify novel procarcinogenic microbes, we identified two mucosal slurries from CRC patients (3728T and 3752T) that were tumorigenic in germ-free (GF) ApcMin/+ mice. Surprisingly, both of these CRC patient slurries were positive for C. difficile by 16S rRNA amplicon sequencing. Given the ability of other chronic infections to promote tumorigenesis (e.g., H. pylori), we hypothesized that chronic colonization with C. difficile could promote tumorigenesis in the colon. Methods A consortium of 30 bacterial isolates including a toxigenic tcdA+ tcdB+ C. difficile strain (CIm_3728T) was cultured from GF ApcMin/+ mice gavaged with the 3728T slurry. This consortium was gavaged into additional GF ApcMin/+ mice with or without C. difficile strains CIm_3728T, CIm_3752T (isolated from mice gavaged with the 3752T slurry), or isogenic tcdA/tcdB mutants of the M7404 R027 strain. Single cell RNA sequencing (scRNAseq), high dimensional (HD) flow cytometry, and fluorescence in situ hybridization (FISH) with EUB338 and Cd198 probes were performed on distal colons from mice gavaged with either complex CRC slurries or the 3728T isolates with CIm_3728T. Results C. difficile strains drove tumorigenesis of the 3728T isolate mixture (Fig. 1A,B). Tumorigenesis was associated with early procarcinogenic signaling and spatial changes including induction of Wnt signaling in colonic epithelial progenitor cells by scRNAseq, IL-17 induction in immune cells by HD flow cytometry, and bacterial biofilm invasion deep into epithelial crypts by FISH. Tumorigenesis correlated with chronic colonization with toxigenic strains of C. difficile and was toxin-dependent, as toxin mutant strains (M7404 tcdA-tcdB-) did not induce tumors. Figure 1. C. difficile strains from CRC patients induce distal colonic tumorigenesis in germ-free (GF) ApcMin/+ mice. A consortium of 30 bacteria, including C. difficile, were isolated from mice gavaged with the 3728T human CRC mucosal slurry. These isolates were then gavaged into additional GF ApcMin/+ mice, with or without C. difficile isolates from mice gavaged with the 3728T slurry or 3752T slurry. (A) Colonic tumor numbers in GF ApcMin/+ mice at 10 wk p.i. demonstrate that C. difficile (Cd) drives the tumorigenesis of this 30-member bacterial consortium. (B) Gross tumors can be observed in the colon of a representative mouse gavaged with the 3728T isolates with the CIm_3728T (top) or CIm_3752T (middle) strain of C. difficile but not in a mouse gavaged with the isolates lacking C. difficile (bottom). Conclusion Toxigenic C. difficile strains isolated from human CRC mucosal slurries were pro-carcinogenic in mice, suggesting that C. difficile is a potential driver of CRC. Given the public health burden of C. difficile, further studies are warranted to determine whether C. difficile infections (initial, recurrent, and chronic asymptomatic) increase CRC risk in patients. Disclosures Jada Domingue, PhD, AstraZeneca (Employee) James White, PhD, Personal Genome Diagnostics (Consultant) Patricia J. Simner, PhD, Accelerate Diagnostics (Grant/Research Support)Affinity Biosensors (Grant/Research Support)BD Diagnostics (Consultant, Grant/Research Support)GeneCapture (Consultant)OpGen, Inc (Consultant, Grant/Research Support)Shionogi, Inc (Consultant) Karen C. Carroll, MD, MeMed (Scientific Research Study Investigator)Meridian Diagnostics, Inc. (Grant/Research Support)Pattern Diagnostics (Advisor or Review Panel member)Scanogen, Inc. (Advisor or Review Panel member) Karen C. Carroll, MD, Pattern Diagnostics, Inc. (Individual(s) Involved: Self): Grant/Research Support; Scanogen, Inc. (Individual(s) Involved: Self): Consultant Cynthia L. Sears, MD, Bristol Myers Squibb (Grant/Research Support)Ferring (Advisor or Review Panel member)Janssen (Grant/Research Support)

Alkyl-Quinolones derivatives as potential biomarkers for Pseudomonas aeruginosa infection chronicity in Cystic Fibrosis

In Cystic Fibrosis (CF), a rapid and standardized definition of chronic infection would allow a better management of Pseudomonas aeruginosa (Pa) infections, as well as a quick grouping of patients during clinical trials allowing better comparisons between studies. With this purpose, we compared the metabolic profiles of 44 in vitro cultures of Pa strains isolated from CF patients at different stages of infection in order to identify metabolites differentially synthetized according to these clinical stages. Compounds produced and secreted by each strain in the supernatant of a liquid culture were analysed by metabolomic approaches (UHPLC-DAD-ESI/QTOF, UV and UPLC-Orbitrap, MS). Multivariate analyses showed that first colonization strains could be differentiated from chronic colonization ones, by producing notably more Alkyl-Quinolones (AQs) derivatives. Especially, five AQs were discriminant: HQC5, HQNOC7, HQNOC7:1, db-PQS C9 and HQNOC9:1. However, the production of HHQ was equivalent between strain types. The HHQ/HQNOC9:1 ratio was then found to be significantly different between chronic and primo-colonising strains by using both UV (p = 0.003) and HRMS data (p = 1.5 × 10–5). Our study suggests that some AQ derivatives can be used as biomarkers for an improved management of CF patients as well as a better definition of the clinical stages of Pa infection.

Biofilm Formation in Methicillin-Resistant Staphylococcus aureus Isolated in Cystic Fibrosis Patients Is Strain-Dependent and Differentially Influenced by Antibiotics

Cystic fibrosis (CF) is a genetic disease with lung abnormalities making patients particularly predisposed to pulmonary infections. Staphylococcus aureus is the most frequently identified pathogen, and multidrug-resistant strains (MRSA, methicillin-resistant S. aureus) have been associated with more severe lung dysfunction leading to eradication recommendations. Diverse bacterial traits and adaptive skills, including biofilm formation, may, however, make antimicrobial therapy challenging. In this context, we compared the ability of a collection of genotyped MRSA isolates from CF patients to form biofilm with and without antibiotics (ceftaroline, ceftobiprole, linezolid, trimethoprim, and rifampicin). Our study used standardized approaches not previously applied to CF MRSA, the BioFilm Ring test® (BRT®), the Antibiofilmogram®, and the BioFlux™ 200 system which were adapted for use with the artificial sputum medium (ASM) mimicking conditions more relevant to the CF lung. We included 63 strains of 10 multilocus sequence types (STs) isolated from 35 CF patients, 16 of whom had chronic colonization. The BRT® showed that 27% of the strains isolated in 37% of the patients were strong biofilm producers. The Antibiofilmogram® performed on these strains showed that broad-spectrum cephalosporins had the lowest minimum biofilm inhibitory concentrations (bMIC) on a majority of strains. A focus on four chronically colonized patients with inclusion of successively isolated strains showed that ceftaroline, ceftobiprole, and/or linezolid bMICs may remain below the resistance thresholds over time. Studying the dynamics of biofilm formation by strains isolated 3years apart in one of these patients using BioFlux™ 200 showed that inhibition of biofilm formation was observed for up to 36h of exposure to bMIC and ceftaroline and ceftobiprole had a significantly greater effect than linezolid. This study has brought new insights into the behavior of CF MRSA which has been little studied for its ability to form biofilm. Biofilm formation is a common characteristic of prevalent MRSA clones in CF. Early biofilm formation was strain-dependent, even within a sample, and not only observed during chronic colonization. Ceftaroline and ceftobiprole showed a remarkable activity with a long-lasting inhibitory effect on biofilm formation and a conserved activity on certain strains adapted to the CF lung environment after years of colonization.

Leptospira interrogans biofilm formation in Rattus norvegicus (Norway rats) natural reservoirs

Rattus norvegicus (Norway rat) is the main reservoir host of pathogenic Leptospira, the causative agent of leptospirosis, in urban environments. Pathogenic Leptospira forms biofilms in the environment, possibly contributing for bacterial survival and maintenance. Nonetheless, biofilms have not yet been studied in natural animal reservoirs presenting leptospiral renal carriage. Here, we described biofilm formation by pathogenic Leptospira inside the renal tubules of R. norvegicus naturally infected and captured in an urban slum endemic for leptospirosis. From the 65 rats carrying Leptospira in their kidneys, 24 (37%) presented biofilms inside the renal tubules. The intensity of leptospiral colonization in the renal tubules (OR: 1.00; 95% CI 1.05–1.1) and the type of occlusion pattern of the colonized renal tubules (OR: 3.46; 95% CI 1.20–9.98) were independently associated with the presence of Leptospira biofilm. Our data showed that Leptospira interrogans produce biofilms during renal chronic colonization in rat reservoirs, suggesting a possible role for leptospiral biofilms in the pathogenesis of leptospirosis and bacterial carriage in host reservoirs.

Genome Sequences of Two Pseudomonas aeruginosa Isolates with Defects in Type III Secretion System Gene Expression from a Chronic Ankle Wound Infection

A common feature of microorganisms that cause chronic infections is a stealthy lifestyle that promotes immune avoidance and host tolerance. During chronic colonization of cystic fibrosis (CF) patients, Pseudomonas aeruginosa acquires numerous adaptations that include reduced expression of some factors, such as motility, O antigen, and the T3SS, and increased expression of other traits, such as biofilm formation.

Analysis of Vitamin D Levels on Bronchiectasis Severity

Background: Bronchiectasis is a chronic disease caused by repeated infection and inflammation of the bronchial walls. Vitamin D plays a role secretion of antimicrobial peptide and inhibits release of pro-inflammatory cytokines in the lungs. Vitamin D deficiency is associated with exacerbations, severity and decreased lung function in bronchiectasis. Several studies have found an association between vitamin D levels and bronchiectasis severity. Methods: This study used cross-sectional study design with consecutive sampling method on bronchiectasis patients who enrolled outpatient and inpatient at Wahidin Sudirohusodo hospital in February - May 2020. All research procedures obtained the approval of the Health Research Ethics Commission, Medicine faculty, Hasanuddin University Makassar. Bronchiectasis severity was assessed based on the FACED score (FEV1, Aged, chronic Colonization by Pseudomonas aeuroginosa, radiological Extension of the disease, Dyspnea). Levels of vitamin D serum {25 (OH) D} were checked using the ELISA method. Results: The study subjects were 44 patients, consisting of 61.4% male and 38.6% female. Most of the bronchiectasis patients in this study were mild (77.3%) based on the FACED score, 15.9% moderate and 6.8% severe. As many as 77.3% of patients had vitamin D deficiency and insufficiency as much as 9.1%. All patients with moderate-severe FACED scores had vitamin D deficiency. The correlation between vitamin D levels and FACED scores showed a positive significant with p-value 0.04. Conclusion: Low vitamin D levels are a risk factor for aggravating bronchiectasis severity and have a positive significant correlation between the two.

PREDICTING FACTORS OF DECANNULATION IN CHILDREN WITH TRACHEOSTOMY

Objectives: Our aim was to determine the treatable causes to increase the chance of decannulation success. For this purpose we evaluated the differences between the patients who succesfully decannulated and the patients who still has tracheostomy. Metods: A retrospective cohort study was conducted based on medical records of all pediatric patients with tracheostomy in a single centre. Results: Decannulation was successfully achieved in 59 patients (34.5%) of total 171 patients with tracheostomy between the years 2012-2019. Median duration of tracheostomy was 41.5 and 12 months in patients who remained with tracheostomy and decannulated respectively. Neurological disorders were higher in patients remained with tracheostomy, congenital heart disease and airway abnormalities were higher in decannulated patients. Presence of bacterial colonization (3.8-fold), history of invasive respiratory support following tracheostomy (2.9-fold), and having any neurological disorder and/or comorbidity (5.2-fold) were significantly associated lower rates of decannulation. Almost 33 % of patients had bacterial colonization and colonization rates were higher in patients who needed invasive respiratory support following tracheostomy placement (p<0.001), patients with feeding/swallowing problems (p=0.005) and neurological disorders(0.002). There was significant correlation between duration of tracheostomy and bacterial colonization rates (p=0.008). But after analysing with logistic regression only having a neurological disorder was associated with bacterial colonization (OR= 2.9; 95% Cl: 1.15-7.47 p=0.024). Conclusion: While conducting decanulation assessment, the presence of colonization should be considered. Future prospective researchs are necessary in order to determine the role of chronic colonization on decannulation success.

Impact of Pseudomonas aeruginosa Infection on Patients with Chronic Inflammatory Airway Diseases

Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous and opportunistic microorganism and is considered one of the most significant pathogens that produce chronic colonization and infection of the lower respiratory tract, especially in people with chronic inflammatory airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and bronchiectasis. From a microbiological viewpoint, the presence and persistence of P. aeruginosa over time are characterized by adaptation within the host that precludes any rapid, devastating injury to the host. Moreover, this microorganism usually develops antibiotic resistance, which is accelerated in chronic infections especially in those situations where the frequent use of antimicrobials facilitates the selection of “hypermutator P. aeruginosa strain”. This phenomenon has been observed in people with bronchiectasis, CF, and the “exacerbator” COPD phenotype. From a clinical point of view, a chronic bronchial infection of P. aeruginosa has been related to more severity and poor prognosis in people with CF, bronchiectasis, and probably in COPD, but little is known on the effect of this microorganism infection in people with asthma. The relationship between the impact and treatment of P. aeruginosa infection in people with airway diseases emerges as an important future challenge and it is the most important objective of this review.

Role of Stenotrophomonas maltophilia isolation in patients with non-CF bronchiectasis

Introduction Stenotrophomonas maltophilia is a bacteria whose role in patients with cystic fibrosis (CF) bronchiectasis has been previously studied; little is known about its role in non-CF bronchiectasis. Materials and methods Aim of our study is to investigate the risk factors for S. maltophilia acquisition and its clinical impact on bronchiectasis patients. A retrospective observational cohort study enrolling patients attending the Bronchiectasis Clinic at the Royal Infirmary of Edinburgh, Scotland, UK. A total of 167 bronchiectasis patients undergoing intravenous (IV) antibiotic therapy were selected and divided according to single or chronic S. maltophilia isolation in sputum. The risk factors and prognostic impact were studied. Results Single isolation was independently associated with lower baseline % predicted forced expiratory volume in 1 s [odds ratio (OR) 0.98; 95% confidence interval (CI) 0.970–1.044; P = 0.025] and with less radiological involvement (OR 0.379; 95% CI 0.175–0.819; P = 0.01). Chronic isolation was associated with the number of IV antibiotic courses in the year before and after the first isolation (OR 1.2; 95% CI 1.053–1.398; P = 0.007) and with the absence of Pseudomonas aeruginosa colonization (OR 0.207; 95% CI 0.056–0.764; P = 0.02). In the chronic isolation group, there were more exacerbations and more need of IV antibiotics in the year after the first isolation. Conclusions Poor lung function is the main independent risk factor for single isolation of S. maltophilia. For chronic colonization, the main independent risk factor is the number of IV antibiotic courses and the absence of P. aeruginosa chronic colonization. Only when chronically present, S. maltophilia had a clinical impact with more exacerbations.