Genomic Analysis and Surveillance of Respiratory Syncytial Virus (RSV) Using Wastewater-Based Epidemiology (WBE).

Respiratory syncytial virus (RSV) causes severe infections in infants, immunocompromised or elderly individuals resulting in annual epidemics of respiratory disease. Currently, limited clinical surveillance and the lack of predictable seasonal dynamics limits the public health response. Wastewater-based epidemiology (WBE) has recently been used globally as a key metric in determining prevalence of SARS-CoV-2 in the community but its application to other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, applying RT-qPCR and partial G-gene sequencing to track RSV levels and variants in the community. We report increasing detection of RSV in wastewater concomitant with increasing numbers of positive clinical cases. Analysis of wastewater-derived RSV sequences permitted identification of distinct circulating lineages within and between seasons. Altogether, our genomic WBE platform has the potential to complement ongoing global surveillance and aid the management of RSV by informing the timely deployment of pharmaceutical and non-pharmaceutical interventions.

Longitudinal changes in the cystic fibrosis airway microbiota with time and treatment.

BACKGROUND: Whether there is any benefit in integrating culture-independent molecular analysis of the lower airway microbiota of people with cystic fibrosis into clinical care is unclear. This study determined the longitudinal trajectory of the microbiota and if there were microbiota characteristics that corresponded with response to treatment or predicted a future pulmonary exacerbation. METHODS: At least one sputum sample was collected from 149 participants enrolled in this prospective longitudinal multi-centre study and total bacterial density and microbiota community measurements were determined and compared with clinical parameters. RESULTS: In 114 participants with paired samples when clinically stable, ∼8 months apart, the microbiota remained conserved between timepoints, regardless of whether participants received acute intravenous antibiotic treatment or not. In 62 participants, who presented with an acute exacerbation, a decrease in community richness correlated best with patient response to antibiotic treatment. Analysis of baseline samples from 30 participants who exacerbated within 4 months of their stable sample being collected and 72 participants who remained stable throughout the study showed that community characteristics such as lower richness at baseline may be predictive of an exacerbation in addition to several clinical parameters. However, lasso regression analysis indicated that only lung function (p = 0.014) was associated with a future exacerbation. CONCLUSIONS: The airway microbiota remains stable over periods <1 year with modest shifts related to treatment apparent which might provide some additional insights to patient-level measurements.

Improved recovery of SARS-CoV-2 from wastewater through application of RNA and DNA stabilising agents.

Wastewater Based Epidemiology (WBE) has become an integral part of the public health effort to track the levels of SARS-CoV-2 within communities. Detection of SARS-CoV-2 in wastewater can be challenging due to relatively low levels of virus within the sample. The wastewater matrix is also comprised of commercial and domestically derived contaminants, as well as RNases, all of which can adversely affect RT-qPCR analysis. To improve SARS-CoV-2 detection within wastewater samples we investigated both the effect of template dilution (as a means to reduce RT-qPCR inhibition) and sample stabilisation via addition of DNA/RNA Shield™ and/or RNA Later™ (to prevent RNA degradation via RNases) as a means to improve viral fragment detection. Using both methodologies, a significant improvement in SARS-CoV-2 detection from wastewater samples was observed. No adverse effects of stabilising agent addition on downstream Next-Generation Sequencing workflows were detected.

Detection of human adenovirus F41 in wastewater and its relationship to clinical cases of acute hepatitis of unknown aetiology.

As of 8 July 2022, the World Health Organization (WHO) have reported 1010 probable cases of acute hepatitis of unknown aetiology in children worldwide, including approximately 250 cases in the United Kingdom (UK). Clinical presentations have often been severe, with liver transplantation a frequent clinical outcome. Human adenovirus F41 (HAdV-F41) has been detected in most children with acute hepatitis, but its role in the pathogenesis of this infection has yet to be established. Wastewater-based epidemiology (WBE) has become a well-established tool for monitoring the community spread of SARS-CoV-2, as well as other pathogens and chemicals. In this study, we adopted a WBE approach to monitoring levels of HAdV-F40/41 in wastewater before and during an acute hepatitis outbreak in Northern Ireland. We report increasing detection of HAdV-F40/41 in wastewater, concomitant with increasing numbers of clinical cases. Amplicon whole genome sequencing further classified the wastewater-derived HAdV as belonging to the F41 genotype which in turn was homologous to clinically derived sequences. We propose that WBE has the potential to inform community surveillance of HAdV-F41 and can further contribute to the ongoing global discussion supporting HAdV-F41 involvement in acute hepatitis cases.

Antimicrobial resistance in urinary pathogens and culture-independent detection of trimethoprim resistance in urine from patients with urinary tract infection.

BACKGROUND: Although urinary tract infections (UTIs) are extremely common, isolation of causative uropathogens is not always routinely performed, with antibiotics frequently prescribed empirically. This study determined the susceptibility of urinary isolates from two Health and Social Care Trusts (HSCTs) in Northern Ireland to a range of antibiotics commonly used in the treatment of UTIs. Furthermore, we determined if detection of trimethoprim resistance genes (dfrA) could be used as a potential biomarker for rapid detection of phenotypic trimethoprim resistance in urinary pathogens and from urine without culture. METHODS: Susceptibility of E. coli and Klebsiella spp. isolates (n = 124) to trimethoprim, amoxicillin, ceftazidime, ciprofloxacin, co-amoxiclav and nitrofurantoin in addition to susceptibility of Proteus mirabilis (n = 61) and Staphylococcus saprophyticus (n = 17) to trimethoprim was determined by ETEST® and interpreted according to EUCAST breakpoints. PCR was used to detect dfrA genes in bacterial isolates (n = 202) and urine samples(n = 94). RESULTS: Resistance to trimethoprim was observed in 37/124 (29.8%) E. coli and Klebsiella spp. isolates with an MIC90 > 32 mg/L. DfrA genes were detected in 29/37 (78.4%) trimethoprim-resistant isolates. Detection of dfrA was highly sensitive (93.6%) and specific (91.4%) in predicting phenotypic trimethoprim resistance among E. coli and Klebsiella spp. isolates. The dfrA genes analysed were detected using a culture-independent PCR method in 16/94 (17%) urine samples. Phenotypic trimethoprim resistance was apparent in isolates cultured from 15/16 (94%) dfrA-positive urine samples. There was a significant association (P < 0.0001) between the presence of dfrA and trimethoprim resistance in urine samples containing Gram-negative bacteria (Sensitivity = 75%; Specificity = 96.9%; PPV = 93.8%; NPV = 86.1%). CONCLUSIONS: This study demonstrates that molecular detection of dfrA genes is a good indicator of trimethoprim resistance without the need for culture and susceptibility testing.

Phenotypic characteristics of incident and chronic MRSA isolates in cystic fibrosis.

BACKGROUND: Chronic methicillin resistant Staphylococcus aureus (MRSA) in CF is associated with worse outcomes compared to early or intermittent infection. This observation could be related to adaptive bacterial changes such as biofilm formation or anaerobic growth. METHODS: MRSA isolates stored from incident and during chronic (>2 years) infection were included at two study sites. MRSA isolates were characterised by spa-typing, antimicrobial susceptibility testing, biofilm formation and haemolysis under aerobic and anaerobic culture conditions. RESULTS: Paired MRSA isolates from 49 patients were included. Mean age at incident infection was 9.7±1.2 years with mild to moderate lung disease (FEV1 74±4% predicted). Twenty-five subjects showed progression of disease/symptoms after onset of MRSA with significantly increased use of antibiotics. Most isolates belonged to t002 (38%) and t008 (36%) spa-types and 8 patients had a change in spa-type over time. Antimicrobial susceptibility testing showed few differences between incident and late isolates but significantly lower MIC under anaerobic vs. aerobic conditions for vancomycin, fusidic acid, rifampin but higher MIC for trimethoprim-sulfamethoxazole. Biofilm formation and haemolysis did not differ by stage of infection or disease course but both were lower under anaerobic conditions (biofilm p=0.018; haemolysis p=0.002) in multi-variate analyses that included study site, growth condition and stage of infection. CONCLUSIONS: Persistent MRSA infection is frequently associated with clinical decline. Anaerobic growth conditions, which occur in CF airways, affect the expression of virulence factors and antibiotic susceptibility of MRSA more than duration of infection.

Extended-culture and culture-independent molecular analysis of the airway microbiota in cystic fibrosis following CFTR modulation with ivacaftor.

BACKGROUND: Treatment with Ivacaftor provides a significant clinical benefit in people with cystic fibrosis (PWCF) with the class III G551D-CFTR mutation. This study determined the effect of CFTR modulation with ivacaftor on the lung microbiota in PWCF. METHODS: Using both extended-culture and culture-independent molecular methods, we analysed the lower airway microbiota of 14 PWCF, prior to commencing ivacaftor treatment and at the last available visit within the following year. We determined total bacterial and Pseudomonas aeruginosa densities by both culture and qPCR, assessed ecological parameters and community structure and compared these with biomarkers of inflammation and clinical outcomes. RESULTS: Significant improvement in FEV1, BMI, sweat chloride and levels of circulating inflammatory biomarkers were observed POST-ivacaftor treatment. Extended-culture demonstrated a higher density of strict anaerobic bacteria (p = 0.024), richness (p = 1.59*10-4) and diversity (p = 0.003) POST-treatment. No significant difference in fold change was observed by qPCR for either total bacterial 16S rRNA copy number or P. aeruginosa density for oprL copy number with treatment. Culture-independent (MiSeq) analysis revealed a significant increase in richness (p = 0.03) and a trend towards increased diversity (p = 0.07). Moreover, improvement in lung function, richness and diversity displayed an inverse correlation with the main markers of inflammation (p < 0.05). CONCLUSIONS: Following treatment with ivacaftor, significant improvements in clinical parameters were seen. Despite modest changes in overall microbial community composition, there was a shift towards a bacterial ecology associated with less severe CF lung disease. Furthermore, a significant correlation was observed between richness and diversity and levels of circulating inflammatory markers.

Microbial Community Composition in Explanted Cystic Fibrosis and Control Donor Lungs.

To date, investigations of the microbiota in the lungs of people with Cystic Fibrosis (PWCF) have primarily focused on microbial community composition in luminal mucus, with fewer studies observing the microbiota in tissue samples from explanted lung tissue. Here, we analysed both tissue and airway luminal mucus samples extracted from whole explanted lungs of PWCF and unused donor lungs. We determined if the lung microbiota in end-stage CF varied within and between patients, was spatially heterogeneous and related to localized structural damage. Microbial community composition was determined by Illumina MiSeq sequencing and related to the CF-Computed Tomography (CT) score and features of end-stage lung disease on micro-CT. Ninety-eight CF tissue (n=11 patients), 20 CF luminal mucus (n=8 patients) and 33 donor tissue (n=4 patients) samples were analysed. Additionally, we compared 20 paired CF tissue and luminal mucus samples that enabled a direct "geographical" comparison of the microbiota in these two niches. Significant differences in microbial communities were apparent between the 3 groups. However, overlap between the three groups, particularly between CF and donor tissue and CF tissue and CF luminal mucus was also observed. Microbial diversity was lower in CF luminal mucus compared to CF tissue, with dominance higher in luminal mucus. For both CF and donor tissue, intra- and inter-patient variability in ecological parameters was observed. No relationships were observed between ecological parameters and CF-CT score, or features of end-stage lung disease. The end-stage CF lung is characterised by a low diversity microbiota, differing within and between individuals. No clear relationship was observed between regional microbiota variation and structural lung damage.

Multi-Omics Approaches: The Key to Improving Respiratory Health in People With Cystic Fibrosis?

The advent of high-throughput multi-omics technologies has underpinned the expansion in lung microbiome research, increasing our understanding of the nature, complexity and significance of the polymicrobial communities harbored by people with CF (PWCF). Having established that structurally complex microbial communities exist within the airways, the focus of recent research has now widened to investigating the function and dynamics of the resident microbiota during disease as well as in health. With further refinement, multi-omics approaches present the opportunity to untangle the complex interplay between microbe-microbe and microbe-host interactions in the lung and the relationship with respiratory disease progression, offering invaluable opportunities to discover new therapeutic approaches for our management of airway infection in CF.

Influence of azithromycin and allograft rejection on the post-lung transplant microbiota.

BACKGROUND: Alterations in the lung microbiota may drive disease development and progression in patients with chronic respiratory diseases. Following lung transplantation (LTx), azithromycin is used to both treat and prevent chronic lung allograft dysfunction (CLAD). The objective of this study was to determine the association between azithromycin use, CLAD, acute rejection, airway inflammation, and bacterial microbiota composition and structure after LTx. METHODS: Bronchoalveolar lavage samples (n = 219) from 69 LTx recipients (azithromycin, n = 32; placebo, n = 37) from a previously conducted randomized placebo-controlled trial with azithromycin were analyzed. Samples were collected at discharge, 1, and 2 years following randomization and at CLAD diagnosis. Bacterial microbial community composition and structure was determined using 16S ribosomal RNA gene sequencing and associated with clinically important variables. RESULTS: At discharge and following 1 and 2 years of azithromycin therapy, no clear differences in microbial community composition or overall diversity were observed. Moreover, no changes in microbiota composition were observed in CLAD phenotypes. However, acute rejection was associated with a reduction in community diversity (p = 0.0009). Significant correlations were observed between microbiota composition, overall diversity, and levels of inflammatory cytokines in bronchoalveolar lavage, particularly CXCL8. CONCLUSIONS: Chronic azithromycin usage did not disturb the bacterial microbiota. However, acute rejection episodes were associated with bacterial dysbiosis.

Susceptibility of Pseudomonas aeruginosa Recovered from Cystic Fibrosis Patients to Murepavadin and 13 Comparator Antibiotics.

The objective was to determine the in vitro antimicrobial susceptibility of Pseudomonas aeruginosa isolates cultured from cystic fibrosis (CF) patients and explore associations between strain sequence type and susceptibility. Fourteen antibiotics and antibiotic combinations, including the novel antibacterial peptide murepavadin, were tested for activity against 414 Pseudomonas aeruginosa isolates cultured from respiratory samples of CF patients. The complete genomes of the isolates were sequenced, and minimum spanning trees were constructed based on the sequence types (STs). Percentages of resistance according to CLSI 2019 breakpoints were as follows: cefepime, 14%; ceftazidime, 11%; ceftazidime-avibactam, 7%; ceftolozane-tazobactam, 3%; piperacillin-tazobactam, 12%; meropenem, 18%; imipenem, 32%; aztreonam, 23%; ciprofloxacin, 30%; gentamicin, 30%; tobramycin, 12%; amikacin, 18%; and colistin, 4%. Murepavadin MIC50 and MIC90 were 0.12 mg/liter and 2 mg/liter, respectively. There were no apparent clonal clusters associated with resistance, but higher MICs did appear to occur more often in STs with multiple isolates than in single ST isolates. In general, the CF isolates showed a wide genetic distribution. P. aeruginosa CF isolates exhibited the lowest resistance rates against ceftolozane-tazobactam, ceftazidime-avibactam, and colistin. Murepavadin demonstrated the highest activity on a per-weight basis and may therefore become a valuable addition to the currently available antibiotics for treatment of respiratory infection in people with CF.

Electronic cigarette vapour increases virulence and inflammatory potential of respiratory pathogens.

INTRODUCTION: Bacteria have been extensively implicated in the development of smoking related diseases, such as COPD, by either direct infection or bacteria-mediated inflammation. In response to the health risks associated with tobacco exposure, the use of electronic cigarettes (e-cigs) has increased. This study compared the effect of e-cig vapour (ECV) and cigarette smoke (CSE) on the virulence and inflammatory potential of key lung pathogens (Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa). METHODS: Biofilm formation, virulence in the Galleria mellonella infection model, antibiotic susceptibility and IL-8/TNF-α production in A549 cells, were compared between bacteria exposed to ECV, CSE and non-exposed bacteria. RESULTS: Statistically significant increases in biofilm and cytokine secretion were observed following bacterial exposure to either ECV or CSE, compared to non-exposed bacteria; the effect of exposure to ECV on bacterial phenotype and virulence was comparable, and in some cases greater, than that observed following CSE exposure. Treatment of A549 cells with cell signaling pathway inhibitors prior to infection, did not suggest that alternative signaling pathways were being activated following exposure of bacteria to either ECV or CSE. CONCLUSIONS: These findings therefore suggest that ECV and CSE can induce changes in phenotype and virulence of key lung pathogens, which may increase bacterial persistence and inflammatory potential.

Assessment of stability and fluctuations of cultured lower airway bacterial communities in people with cystic fibrosis.

BACKGROUND: Routine clinical culture detects a subset of the cystic fibrosis (CF) airways microbiota based on culture-independent (molecular) methods. This study aimed to determine how extended sputum culture of viable bacteria changes over time in relation to clinical status and predicts exacerbations. METHODS: Sputa from patients at a baseline stable and up to three subsequent time-points were analysed by extended-quantitative culture; aerobe/anaerobe densities, ecological indexes and community structure were assessed together with clinical outcomes. RESULTS: Eighty patients were prospectively recruited. Sputa were successfully collected and cultured at 199/267 (74.5%) study visits. Eighty-two sputa from 25 patients comprised a complete sample-set for longitudinal analyses. Bacterial density, ecological indexes and clinical outcomes were unchanged in 18 patients with three sequential stable visits. Conversely, in 7 patients who had an exacerbation, total bacterial and aerobe densities differed over four study visits (P < .001) with this difference particularly apparent between the baseline visit and completion of acute antibiotic treatment where a decrease in density was observed. Bacterial communities were more similar within than between patients but stable patients had the least variation in community structure over time. Using logistic regression in a further analysis, baseline features in 37 patients without compared to 15 patients with a subsequent exacerbation showed that clinical measures rather than bacterial density or ecological indexes were independent predictors of an exacerbation. CONCLUSIONS: Greater fluctuation in the viable bacterial community during treatment of an exacerbation than between stable visits was observed. Extended-quantitative culture did not provide prognostic information of a future exacerbation.

Activity of hypothiocyanite and lactoferrin (ALX-009) against respiratory cystic fibrosis pathogens in sputum.

Objectives: To determine the antimicrobial activity of ALX-009, a combination of bovine lactoferrin and hypothiocyanite, in sputum against Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc), key pathogens causing infection in the lungs of cystic fibrosis (CF) patients. Methods: The antimicrobial activity of ALX-009 against clinical respiratory P. aeruginosa isolates was determined by time-kill assay. Sputum from CF patients was treated with ALX-009, either alone or in combination with tobramycin, and the effect on P. aeruginosa, Bcc and total sputum density was determined. Results: Time-kill assay indicated that ALX-009 was bactericidal at 24 h against 4/4 P. aeruginosa isolates under aerobic conditions, and against 3/4 isolates under anaerobic conditions. ALX-009 was also bactericidal against P. aeruginosa in sputum samples at 6 h (n = 22/24 samples) and 24 h (n = 14/24 samples), and demonstrated significantly greater activity than tobramycin at both timepoints. Activity against Bcc in sputum samples (n = 9) was also demonstrated, but the magnitude of change in Bcc density was less than for P. aeruginosa. To determine the effect of treating sputum with two doses of ALX-009, similar to current regimens for inhaled antibiotics, aliquots of a further 10 sputum samples positive for P. aeruginosa were treated with one (t = 0 h) or two doses (t = 0 h, t = 12 h) of ALX-009; treatment with two doses resulted in bactericidal activity in 7/10 samples at 34 h compared with only 3/10 samples when treatment was with one dose. Conclusions: ALX-009 demonstrates promise as a novel antimicrobial that could be used to decrease P. aeruginosa density in the lungs of people with CF.

"Pathogen Eradication" and "Emerging Pathogens": Difficult Definitions in Cystic Fibrosis.

Infection is a common complication of cystic fibrosis (CF) airway disease. Current treatment approaches include early intervention with the intent to eradicate pathogens in the hope of delaying the development of chronic infection and the chronic use of aerosolized antibiotics to suppress infection. The use of molecules that help restore CFTR (cystic fibrosis transmembrane conductance regulator) function, modulate pulmonary inflammation, or improve pulmonary clearance may also influence the microbial communities in the airways. As the pipeline of these new entities continues to expand, it is important to define when key pathogens are eradicated from the lungs of CF patients and, equally important, when new pathogens might emerge as a result of these novel therapies.

Criteria required for an acceptable point-of-care test for UTI detection: Obtaining consensus using the Delphi technique.

BACKGROUND: Urinary Tract Infections (UTIs) are common bacterial infections, second only to respiratory tract infections and particularly prevalent within primary care. Conventional detection of UTIs is culture, however, return of results can take between 24 and 72 hours. The introduction of a point of care (POC) test would allow for more timely identification of UTIs, facilitating improved, targeted treatment. This study aimed to obtain consensus on the criteria required for a POC UTI test, to meet patient need within primary care. METHODS: Criteria for consideration were compiled by the research team. These criteria were validated through a two-round Delphi process, utilising an expert panel of healthcare professionals from across Europe and United States of America. Using web-based questionnaires, panellists recorded their level of agreement with each criterion based on a 5-point Likert Scale, with space for comments. Using median response, interquartile range and comments provided, criteria were accepted/rejected/revised depending on pre-agreed cut-off scores. RESULTS: The first round questionnaire presented thirty-three criteria to the panel, of which 22 were accepted. Consensus was not achieved for the remaining 11 criteria. Following response review, one criterion was removed, while after revision, the remaining 10 criteria entered the second round. Of these, four were subsequently accepted, resulting in 26 criteria considered appropriate for a POC test to detect urinary infections. CONCLUSION: This study generated an approved set of criteria for a POC test to detect urinary infections. Criteria acceptance and comments provided by the healthcare professionals also supports the development of a multiplex point of care UTI test.

Antibiotic susceptibility of planktonic- and biofilm-grown staphylococci isolated from implant-associated infections: should MBEC and nature of biofilm formation replace MIC?

PURPOSE: The purpose of this study was to develop an alternative, more clinically relevant approach to susceptibility reporting for implant-associated infections. Using 20 staphylococcal isolates, isolated from clinical implant infections, the majority (85 %) demonstrated biofilm-forming capabilities. A significantly increased minimum biofilm eradication concentration (MBEC) compared to minimum inhibitory concentration (MIC) breakpoint was obtained, with MBEC values greater than 256 µg ml-1 for the majority of bacteria. Such a vast increase was also demonstrated for isolates defined as negligible biofilm formers via crystal violet staining, likely due to the high protein content of biofilms, as confirmed by proteinase-K treatment. METHODOLOGY: This study employed a variety of techniques to assess MIC and MBEC of the isolates tested. In addition, the nature of bacterial biofilm across a range of clinical isolates was investigated using crystal violet staining, sodium metaperiodate and proteinase-K treatment, and PCR analysis.Results/Key findings. Infection of medical implants is associated with increased rates of infection and increased bacterial tolerance to antibiotic strategies. Clinical significance is due to the presence of pathogens attached to biomaterial surfaces enclosed in an extracellular polymeric matrix termed the biofilm. This article highlights the importance of defining the clinical susceptibility of implant-associated infections in vitro using methods that are relevant to the biofilm phenotype in vivo, and highlights how current planktonic-based antimicrobial susceptibility tests are often misleading. CONCLUSION: The use of biofilm-relevant susceptibility tests would improve patient outcomes by enabling correct antimicrobial regimens to be rapidly identified, reducing treatment failure and halting the spread of antimicrobial-resistant strains.

Production of extended-spectrum ß-lactamases and the potential indirect pathogenic role of Prevotella isolates from the cystic fibrosis respiratory microbiota.

Extended-spectrum ß-lactamase (ESBL) production and the prevalence of the ß-lactamase-encoding gene blaTEM were determined in Prevotella isolates (n=50) cultured from the respiratory tract of adults and young people with cystic fibrosis (CF). Time-kill studies were used to investigate the concept of passive antibiotic resistance and to ascertain whether a ß-lactamase-positive Prevotella isolate can protect a recognised CF pathogen from the action of ceftazidime in vitro. The results indicated that approximately three-quarters (38/50; 76%) of Prevotella isolates produced ESBLs. Isolates positive for ESBL production had higher minimum inhibitory concentrations (MICs) of ß-lactam antibiotics compared with isolates negative for production of ESBLs (P<0.001). The blaTEM gene was detected more frequently in CF Prevotella isolates from paediatric patients compared with isolates from adults (P=0.002), with sequence analysis demonstrating that 21/22 (95%) partial blaTEM genes detected were identical to blaTEM-116. Furthermore, a ß-lactamase-positive Prevotella isolate protected Pseudomonas aeruginosa from the antimicrobial effects of ceftazidime (P=0.03). Prevotella isolated from the CF respiratory microbiota produce ESBLs and may influence the pathogenesis of chronic lung infection via indirect methods, including shielding recognised pathogens from the action of ceftazidime.