Barbara Waddell

Enteropathogenic Escherichia coli Tir translocation and pedestal formation requires membrane cholesterol in the absence of bundle‐forming pili

Enteropathogenic Escherichia coli (EPEC) is a significant cause of paediatric diarrhoea worldwide. Virulence requires adherence to intestinal epithelial cells, mediated in part through type IV bundle‐forming pili (BFP), and the EPEC protein Tir. Tir is inserted into the enterocyte plasma membrane (PM), resulting in the formation of actin‐rich pedestals. Tir is translocated by the type III secretion system (TTSS), through a pore comprised of EPEC proteins inserted into the PM. Here, we demonstrate that in the absence of BFP, EPEC adherence, effector translocation and pedestal formation are dependent on lipid rafts. Lipid raft disruption using methyl‐β‐cyclodextrin (MβCD) decreased adherence by an EPEC BFP‐deficient strain from 85% to 1%. Translocation of the effectors Tir and EspF was blocked by MβCD treatment, although the TTSS pore still formed. MβCD treatment after Tir delivery decreased pedestal formation by EPEC from 40% to 5%, but not by the related pathogen E. coli O157:H7 which uses a different Tir‐based mechanism. In contrast, EPEC expressing the BFP can circumvent the requirement for membrane cholesterol. This suggests that lipid rafts play a role in virulence of this medically important pathogen.

Amino Acid Residues within Enterohemorrhagic Escherichia coli O157:H7 Tir Involved in Phosphorylation, α-Actinin Recruitment, and Nck-Independent Pedestal Formation

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) adherence to epithelial cells results in the formation of actin pedestals. Pedestal formation requires the bacterial protein Tir, which is inserted into the epithelial cell plasma membrane by the type III secretion system. EPEC and EHEC use different Tir-based mechanisms for pedestal formation, and the EPEC Tir residues required have been well described. In contrast, little is known about the regions of EHEC O157:H7 Tir that are essential for pedestal formation. Additionally, EHEC O157:H7 Tir is serine/threonine phosphorylated, although the residues involved and their role in pedestal formation are not known. In this study, we describe two regions within the carboxy terminus of EHEC O157:H7 Tir that are required for phosphorylation and pedestal formation. Serines 436 and 437 are substrates for protein kinase A phosphorylation, although this is not required to form pedestals. Using a series of internal deletion mutants, we found that amino acids 454 to 463 are required for efficient pedestal formation. Deleting this region resulted in a significant decrease in the recruitment of both filamentous actin and the actin binding protein α-actinin. As α-actinin binds directly to the EHEC O157:H7 amino terminus, these data suggest that its recruitment is dependent on pedestal formation.

Epidemic Pseudomonas aeruginosa infection in patients with cystic fibrosis is not a risk factor for poor clinical Outcomes following lung transplantation

BACKGROUND Epidemic strains of Pseudomonas aeruginosa (ePA) causing infection in cystic fibrosis (CF) have been commonly identified from clinics around the world. ePA disproportionally impacts CF patient pre-transplant outcomes manifesting in increased exacerbation frequency, worsened treatment burden and increased rate of lung function decline, and disproportionally leads to death and/or transplantation. As other CF factors such as pre-transplant infection with multi-resistant organisms, and isolation of P. aeruginosa in the post transplant graft, may impact post-transplant outcomes, we sought to determine if infection with ePA similarly adversely impact post-transplant outcomes. METHODS Between 1991-2014, 53 CF patients from our center received lung transplants. Bacterial strain typing was performed retrospectively on isolates collected prior to transplantation. Comprehensive chart reviews were performed to obtain baseline patient characteristics and post-transplant outcomes. RESULTS Of the 53 transplanted patients, 57% of patients were infected with ePA prior to transplant; the other 43% of patients had unique strains of P. aeruginosa. Mean age at transplant was 29.0years for ePA and 33.3years for unique (p=0.04). There were no differences in overall survival (HR=0.75, 95% CI 0.31-1.79), bronchiolitis obliterans syndrome (BOS) free survival (HR 1.43, 95% CI 0.54-4.84) or all other assessed outcomes including exacerbation frequency, chronic renal failure, acute cellular rejections, Aspergillus infection, airway stenosis, and post-transplant lymphoproliferative disorder. CONCLUSION Unlike pre-transplant outcomes, CF patients infected with ePA do not experience worse post-transplant outcomes than those infected with unique strains. Therefore, lung transplantation should be considered for all patients with P. aeruginosa infection and end stage lung disease, irrespective of infection with ePA.

Long-term clinical outcomes of 'Prairie Epidemic Strain' Pseudomonas aeruginosa infection in adults with cystic fibrosis.

Rationale Epidemic Pseudomonas aeruginosa (PA) plays an important role in cystic fibrosis (CF) lung disease. A novel strain, the ‘Prairie Epidemic Strain’ (PES), has been identified in up to 30% of patients in Prairie-based Canadian CF centres. Objective To determine the incidence, prevalence and long-term clinical impact of PES infection. Methods A cohort of adults with CF was followed from 1980 to 2014 where bacteria isolated from clinical encounters were prospectively collected. Strain typing was performed using pulse-field gel electrophoresis and multilocus sequence typing. Patients were divided into one of four cohorts: no PA, transient PA, chronic PA with unique strains and chronic PES. Proportional Cox hazard and linear mixed models were used to assess for CF-associated respiratory death or transplantation, and rates of %FEV1 and body mass index (BMI) decline. Results 274 patients (51.7% male) were analysed: 44––no PA, 29––transient PA, 137––unique PA, 64––PES. A total of 92 patients (33.6%) died or underwent lung transplantation (2423.0 patient-years). PES infection was associated with greater risk of respiratory death or lung transplant compared with the no PA group (aHR, 3.94 (95% CI 1.18 to 13.1); p=0.03) and unique PA group (aHR, 1.75 (95% CI 1.05 to 2.92) p=0.03). Rate of lung function decline (%FEV1 predicted) was greatest in the PES group (1.73%/year (95% CI 1.63% to 1.82%); p<0.001). BMI improved over time but at an attenuated rate in the PES group (p=0.001). Conclusions Infection with PES was associated with increased patient morbidity through three decades and manifested in an increased risk of respiratory death and/or lung transplantation.

Development and Validation of a PCR Assay To Detect the Prairie Epidemic Strain of Pseudomonas aeruginosa from Patients with Cystic Fibrosis

ABSTRACT The monitoring of epidemic Pseudomonas aeruginosa is important for cystic fibrosis (CF) infection control. The prairie epidemic strain (PES) is common in western Canadian CF clinics. Using whole-genome sequencing, we identified a novel genomic island and developed a PCR assay for PES. Against a collection of 186 P. aeruginosa isolates, the assay had 98% sensitivity and 100% specificity.

Prevalence and Outcomes of Achromobacter Species Infections in Adults with Cystic Fibrosis: a North American Cohort Study

ABSTRACT Achromobacter species are increasingly being detected in cystic fibrosis (CF) patients, with an unclear epidemiology and impact. We studied a cohort of patients attending a Canadian adult CF clinic who had positive sputum cultures for Achromobacter species in the period from 1984 to 2013. Infection was categorized as transient or persistent (≥50% positive cultures for 1 year). Those with persistent infection were matched 2:1 with age-, sex-, and time-matched controls without a history of Achromobacter infection, and mixed-effects models were used to assess pulmonary exacerbation (PEx) frequency and lung function decline. Isolates from a biobank were retrospectively assessed, identified to the species level by nrdA sequencing, and genotyped using pulsed-field gel electrophoresis (PFGE). Thirty-four patients (11% of those in our clinic), with a median age of 24 years (interquartile range [IQR], 20.3 to 29.8 years), developed Achromobacter infection. Ten patients (29%) developed persistent infection. Persistence did not denote permanence, as most patients ultimately cleared infection, often after years. Patients were more likely to experience PEx at incident isolation than at prior or subsequent visits (odds ratio [OR], 2.7 [95% confidence interval {CI}, 1.2 to 6.7]; P = 0.03). Following persistent infection, there was no difference in annual lung function decline (−1.08% [95% CI, −2.73 to 0.57%] versus −2.74% [95% CI, −4.02 to 1.46%]; P = 0.12) or the odds of PEx (OR, 1.21 [95% CI, 0.45 to 3.28]; P = 0.70). Differential virulence among Achromobacter species was not observed, and no cases of transmission occurred. We demonstrated that incident Achromobacter infection was associated with a greater risk of PEx; however, neither transient nor chronic infection was associated with a worsened long-term prognosis. Large, multicenter studies are needed to clarify the clinical impact, natural history, and transmissibility of Achromobacter.

65 Clinical outcomes of chronic “Prairie Epidemic Strain” Pseudomonas aeruginosa infection in adults with cystic fibrosis

Objectives Transmissible Pseudomonas aeruginosa (PA) strains have been described in CF and may be associated with a poorer prognosis. The “Prairie Epidemic Strain” (PES) has been recently identified in up to 30% of patients at prairie-based CF centres, however, its clinical impact remains to be determined. Methods A cohort study of adults with cystic fibrosis from 1981–2014 was conducted and all PA isolates from clinical visits were prospectively collected. PA strain typing at clinic enrolment, and most recent was conducted by PFGE. Patients were divided into one of four cohorts: no PA, transient PA, unique chronic PA, and chronic PES. Random effects and proportional Cox hazard models were conducted for outcome of death, transplantation and FEV1% decline. Results 204 patients (54% male) with CF were analysed: 38 no PA, 20 transient PA, 103 unique PA, 43 PES. Baseline FEV1% was lowest in the chronic PES group (p = 0.002). Overall rate of FEV1% decline was –1.19%/year (95% CI: –1.41 to –0.97, p<0.001); the chronic PES group had the greatest rate of decline at –1.24%/year (p<0.001). There were a total of 42 deaths and 37 transplants through 1862 and 2050 follow-up years, respectively. The age-adjusted hazard ratio (HR) for death was not different for the PA groups compared to the no PA group. Relative to the no PA group, risk of transplant was increased in those with chronic PES (HR 9.13, CI 1.29–69, p = 0.032) compared to the no PA group. Conclusion Chronic PES infection is significantly associated with a greater rate of FEV1% decline and risk of lung transplantation but not with increased risk of death compared to no PA infection.

Epidemiology and natural history of Pseudomonas aeruginosa airway infections in non-cystic fibrosis bronchiectasis

The natural history and epidemiology of Pseudomonas aeruginosa infections in non-cystic fibrosis (non-CF) bronchiectasis is not well understood. As such it was our intention to determine the evolution of airway infection and the transmission potential of P. aeruginosa in patients with non-CF bronchiectasis. A longitudinal cohort study was conducted from 1986–2011 using a biobank of prospectively collected isolates from patients with non-CF bronchiectasis. Patients included were ≥18 years old and had ≥2 positive P. aeruginosa cultures over a minimum 6-month period. All isolates obtained at first and most recent clinical encounters, as well as during exacerbations, that were morphologically distinct on MacConkey agar were genotyped by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 203 isolates from 39 patients were analysed. These were compared to a large collection of globally epidemic and local CF strains, as well as non-CF isolates. We identified four patterns of infection in non-CF bronchiectasis including: 1) persistence of a single strain (n=26; 67%); 2) strain displacement (n=8; 20%); 3) temporary disruption (n=3; 8%); and 4) chaotic airway infection (n=2; 5%). Patterns of infection were not significant predictors of rates of lung function decline or progression to end-stage disease and acquisition of new strains did not associate with the occurrence of exacerbations. Rarely, non-CF bronchiectasis strains with similar pulsotypes were observed in CF and non-CF controls, but no CF epidemic strains were observed. While rare shared strains were observed in non-CF bronchiectasis, whole-genome sequencing refuted patient–patient transmission. We observed a higher incidence of strain-displacement in our patient cohort compared to those observed in CF studies, although this did not impact on outcomes. Pseudomonas aeruginosa demonstrates distinct infection patterns in non-cystic fibrosis bronchiectasis http://ow.ly/PnvA30jvZDi

Group B streptococcus (GBS) is an important pathogen in human disease- but what about in cystic fibrosis?

BackgroundGroup B Streptococcus (GBS) is a common commensal capable of causing severe invasive infections. Most GBS infections occur in neonates (often as pneumonia). GBS can also cause infection in adults with diabetes and other immunological impairments but rarely leads to pneumonia in adults. GBS has occasionally been found in the sputum of Cystic Fibrosis (CF) patients, an inherited condition known for progressive lung disease. However, the epidemiology and clinical significance of GBS in CF are not understood.MethodsWe retrospectively reviewed a large single-centre adult CF population with an associated comprehensive, prospectively collected bacterial biobank beginning in 1978. We identified all individuals with GBS isolated from their sputum on at least one occasion. The primary outcome was risk of pulmonary exacerbation (PEx) at the time of the first GBS isolate compared to the preceding visit. Secondary outcomes included determining: prevalence of GBS infection in a CF population, whether GBS infections where transient or persistent, whether GBS strains were shared among patients, change in % predicted FEV1 at the time of GBS isolate compared to the preceding visit, PEx frequency after the first GBS isolate, change in % predicted FEV1 after the first GBS isolate, and complications of GBS infection.ResultsGBS was uncommon, infecting 3.5% (11/318) adults within our cohort. Only three individuals developed persistent GBS infection, all lasting > 12 months. There were no shared GBS strains among patients. PEx risk was not increased at initial GBS isolation (RR 5.0, CI 0.69–36.1, p=0.10). In the two years preceding initial GBS isolation compared to the two following years, there was no difference in PEx frequency (median 2, range 0–4 vs 1, range 0 to 5, respectively, p=0.42) or lung function decline, as measured by % predicted FEV1, (median −1.0%, range −19 to 7% vs median −6.0%, range −18 to 22%, p=0.86). There were no invasive GBS infections.ConclusionIn adults with CF, GBS is uncommon and is generally a transient colonizer of the lower airways. Despite the presence of structural lung disease and impaired innate immunity in CF, incident GBS infection did not increase PEx risk, PEx frequency, rate of lung function decline, or other adverse clinical outcomes.

64 Infection control (IC) knowledge, beliefs and behaviours (KBB) amongst those with epidemic Pseudomonas aeruginosa (ePA)

Objective ePA strains are common in CF, and are associated with worse outcomes. Factors associated with ePA are unclear, and evidence based IC is lacking. Methods Patients attending the Calgary Adult CF clinic completed a survey on IC KBB using a 7-point Likert scale. Chronic infection (CI) was defined using the Leeds criteria. For patients who had received lung transplantation (LTx), pre-LTx cultures were used to determine CI. PA strain typing was performed using PFGE. ePA strains were defined as strains shared by ≥3 individuals or established strains including: LES (Liverpool Epidemic Strain) and PES (Prairie Epidemic Strain). Results 144 of 169 patients (85%) participated. Median age was 30.3 years (45% males), and 18% were post LTx. CI was observed with PA 69%, MSSA 47%, MRSA 6%, NTM 10%, Bcc 3%. 30 patients with ePA were identified (28 PES, 2 LES). Those with ePA were no more likely to have other co-infections, but were less likely to have MSSA (OR 0.29). Patients with ePA did not differ with respect to beliefs regarding transmission potential of specific pathogens, circumstances that may lead to transmission or strategies to avoid infection. However, patients with ePA were more likely to associate with others outside of (p = 0.03) and at clinic (p = 0.05). Patients with ePA were more likely to have attended a CF camp (OR 8.32), and have a personal history of past (but not current) involvement with fundraising (OR 1.83). Conclusion Infections with ePA are closely linked to past behaviours and exposures, now routinely discouraged. In contrast, current beliefs and practices with the exception of direct patient–patient contact do not associate with risk of ePA.