J. Hatch

Antibiotic resistance in Prevotella species isolated from patients with cystic fibrosis

OBJECTIVES To compare the antimicrobial susceptibility of Prevotella spp. isolated from cystic fibrosis (CF) and non-CF patients and analyse the impact of antibiotic prescribing in the preceding year on resistance amongst CF isolates. METHODS The susceptibility of 80 CF Prevotella isolates to 12 antibiotics was compared with that of 50 Prevotella isolates from invasive infections in people who did not have CF and 27 Prevotella isolates from healthy controls. RESULTS All isolates were susceptible to chloramphenicol, meropenem and piperacillin/tazobactam, with only four isolates resistant to metronidazole. However, resistance to amoxicillin, ceftazidime and tetracycline was apparent in all groups. Significant differences in clindamycin resistance (UK CF, 56%; UK invasive, 10%) and co-amoxiclav non-susceptibility (UK CF, 32%; UK invasive, 12%) were observed between UK CF and UK invasive isolates. The likelihood of non-susceptibility to clindamycin and co-amoxiclav in UK CF isolates was 5.5-fold and 2.5-fold higher relative to that in UK invasive isolates, respectively. Azithromycin MICs were also significantly higher for CF isolates (P < 0.001), which was associated with current prescription of azithromycin. More than 50% of clinical isolates tested in this study were β-lactamase positive. CONCLUSIONS This study profiles antibiotic susceptibility in Prevotella spp. in CF and demonstrates that meropenem, piperacillin/tazobactam, chloramphenicol and metronidazole are likely to be the most effective antibiotics if treatment is indicated.

Mechanisms of reduced susceptibility and genotypic prediction of antibiotic resistance in Prevotella isolated from cystic fibrosis (CF) and non-CF patients

OBJECTIVES To investigate mechanisms of reduced susceptibility to commonly used antibiotics in Prevotella cultured from patients with cystic fibrosis (CF), patients with invasive infection and healthy control subjects and to determine whether genotype can be used to predict phenotypic resistance. METHODS The susceptibility of 157 Prevotella isolates to seven antibiotics was compared, with detection of resistance genes (cfxA-type gene, ermF and tetQ), mutations within the CfxA-type β-lactamase and expression of efflux pumps. RESULTS Prevotella isolates positive for a cfxA-type gene had higher MICs of amoxicillin and ceftazidime compared with isolates negative for this gene (P < 0.001). A mutation within the CfxA-type β-lactamase (Y239D) was associated with ceftazidime resistance (P = 0.011). The UK CF isolates were 5.3-fold, 2.7-fold and 5.7-fold more likely to harbour ermF compared with the US CF, UK invasive and UK healthy control isolates, respectively. Higher concentrations of azithromycin (P < 0.001) and clindamycin (P < 0.001) were also required to inhibit the growth of the ermF-positive isolates compared with ermF-negative isolates. Furthermore, tetQ-positive Prevotella isolates had higher MICs of tetracycline (P = 0.001) and doxycycline (P < 0.001) compared with tetQ-negative isolates. Prevotella spp. were also shown, for the first time, to express resistance nodulation division (RND)-type efflux pumps. CONCLUSIONS This study has demonstrated that Prevotella isolated from various sources harbour a common pool of resistance genes and possess RND-type efflux pumps, which may contribute to tetracycline resistance. The findings indicate that antibiotic resistance is common in Prevotella spp., but the genotypic traits investigated do not reflect phenotypic antibiotic resistance in every instance.

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

Extended-spectrum β-lactamase (ESBL) production and the prevalence of the β-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 β-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 β-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 β-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.

Initial acquisition and succession of the cystic fibrosis lung microbiome is associated with disease progression in infants and preschool children

The cystic fibrosis (CF) lung microbiome has been studied in children and adults; however, little is known about its relationship to early disease progression. To better understand the relationship between the lung microbiome and early respiratory disease, we characterized the lower airways microbiome using bronchoalveolar lavage (BAL) samples obtained from clinically stable CF infants and preschoolers who underwent bronchoscopy and chest computed tomography (CT). Cross-sectional samples suggested a progression of the lower airways microbiome with age, beginning with relatively sterile airways in infancy. By age two, bacterial sequences typically associated with the oral cavity dominated lower airways samples in many CF subjects. The presence of an oral-like lower airways microbiome correlated with a significant increase in bacterial density and inflammation. These early changes occurred in many patients, despite the use of antibiotic prophylaxis in our cohort during the first two years of life. The majority of CF subjects older than four harbored a pathogen dominated airway microbiome, which was associated with a further increase in inflammation and the onset of structural lung disease, despite a negligible increase in bacterial density compared to younger patients with an oral-like airway microbiome. Our findings suggest that changes within the CF lower airways microbiome occur during the first years of life and that distinct microbial signatures are associated with the progression of early CF lung disease.

Prospective study of the ARCHITECTHIV Ag/Ab Combo fourth generation assay to detect HIV infection in sexually transmitted infection clinics.

This prospective, cross-sectional study of HIV testing at two sexually transmitted infection clinics compares testing results from the ARCHITECT HIV Ag/Ab Combo fourth generation assay against the current standard of care in North Carolina (third generation enzyme immunoassay testing with western blot confirmation and reflex nucleic acid amplification testing of pooled seronegative samples). In this setting, the assay reported a sensitivity of 100%, a specificity of 99.9%, and a median turn-around time of 26.1 h.

Anaerobic bacteria cultured from cystic fibrosis airways correlate to milder disease: a multisite study

Anaerobic and aerobic bacteria were quantitated in respiratory samples across three cystic fibrosis (CF) centres using extended culture methods. Subjects aged 1–69 years who were clinically stable provided sputum (n=200) or bronchoalveolar lavage (n=55). 18 anaerobic and 39 aerobic genera were cultured from 59% and 95% of samples, respectively; 16 out of 57 genera had a ≥5% prevalence across centres. Analyses of microbial communities using co-occurrence networks in sputum samples showed groupings of oral, including anaerobic, bacteria, whereas typical CF pathogens formed distinct entities. Pseudomonas was associated with worse nutrition and F508del genotype, whereas anaerobe prevalence was positively associated with pancreatic sufficiency, better nutrition and better lung function. A higher total anaerobe/total aerobe CFU ratio was associated with pancreatic sufficiency and better nutrition. Subjects grouped by factor analysis who had relative dominance of anaerobes over aerobes had milder disease compared with a Pseudomonas-dominated group with similar proportions of subjects that were homozygous for F508del. In summary, anaerobic bacteria occurred at an early age. In sputum-producing subjects anaerobic bacteria were associated with milder disease, suggesting that targeted eradication of anaerobes may not be warranted in sputum-producing CF subjects. Anaerobic bacteria are cultured across all ages, occur as communities and correlate with milder CF disease in adults http://ow.ly/7wQ430khMmE

93 Production of virulence factors by Prevotella isolates belonging to the cystic fibrosis (CF) respiratory microbiota

Objectives The pathogenesis of Prevotella in CF lung infection is not clear. The aim of this study was to determine production of putative virulence factors by CF Prevotella isolates. Methods Prevotella isolates (CF, n=40; non-CF, n=50) were characterised phenotypically/genotypically for extended-spectrum β-lactamase (ESβL) production (Table). The presence of a capsule was determined for CF Prevotella isolates (n = 40) using both light and transmission electron microscopy. Following culture, the haemolytic capacity of cell-free supernatant from CF isolates (n = 40) was ascertained using a semi-quantitative assay. Proteolysis by Prevotella (n = 40) was investigated following growth on agar containing 2% w/v skimmed milk powder. Results ESβL production is summarised in the Table. Twenty-seven of 40 (68%) Prevotella isolates were encapsulated. All isolates (n = 40) exhibited an ability to degrade horse erythrocytes (Range, 2.07–41.95%; Mean, 23.45%) demonstrating the production of haemolysins. Protease activity was identified in 37/40 (93%) isolates. TableESBL productionPhenotypeMethodPositive, n (%)PCFNon-CFESβLCombined disc31/40 (78)27/50 (54)0.036blaTEMPCR & sequencing19/40 (48)7/50 (14)0.001 Conclusion Virulence factor production was common amongst Prevotella spp. CF Prevotella may potentially contribute to treatment failure of CF lung infection with b-lactams (ES3L production), evade the defence mechanism (capsule production) or contribute to host tissue damage (haemolysin/protease production). Funded by DEL NI studentships, HSC R&D, Public Health Agency, NI and the MRC through a US-Ireland Partnership Grant. Non-CF isolates provided by Dr Hall, Anaerobe Reference Unit, Wales.

WS21.6 Comparison of the CF airway microbiome obtained by bronchoscopy vs. sputum

WS21.5 A comparative study of motility and biofilm production in cystic fibrosis (CF) and environmental Pseudomonas aeruginosa (Pa) isolates K.A. Ramsay1, D.W. Reid2,3, S.C. Bell1,2, R.S. Ware1,4, R.E. Watts3, T.J. Kidd1. 1The University of Queensland, Queensland Children’s Medical Research Institute, Brisbane, Australia; 2The Prince Charles Hospital, Thoracic Medicine, Brisbane, Australia; 3QIMR Berghofer Medical Research Institute, Brisbane, Australia; 4The University of Queensland, School of Population Health, Brisbane, Australia

WS2.9 Inflammatory potential and antimicrobial susceptibility of bacteria in the cultivatable CF lung microbiome

WS2.9 Inflammatory potential and antimicrobial susceptibility of bacteria in the cultivatable CF lung microbiome J. Hatch1, J.D. Kim1, S.J. McGrath2, L. McIlreavey2, D.F. Gilpin2, G.M. Lavelle3, M. Murray3, R.C. Boucher1, J.S. Elborn2, N.G. McElvaney3, M. Muhlebach1, M. Tunney2, M.C. Wolfgang1, S.H. Randell1. 1UNC Chapel Hill, Chapel Hill, United States; 2Queen’s University Belfast, Belfast, United Kingdom; 3Royal College of Surgeons, Dublin, Ireland

84 Azithromycin resistance in Prevotella species isolated from CF patients

Objective: The use of chronic azithromycin treatment has been linked with increased macrolide resistance. Although, Prevotella spp. are one of the most common anaerobes detected in CF pulmonary samples, the effect of chronic azithromycin exposure on this genus is unknown. The aims of this study were to i. investigate if resistance is associated with azithromycin prescription ii. compare azithromycin and clindamycin MICs between Prevotella isolates cultured from CF patients and healthy control subjects. Methods: Isolates were grouped according to source and patient prescription of azithromycin: CF (currently prescribed), n = 27; CF (not currently prescribed), n = 30; healthy controls (none), n = 17. Susceptibility was determined by Etest® and MICs compared between groups using the Mann-Whitney test. Results: CF isolates had significantly higher azithromycin and clindamycin MICs compared to the healthy control isolates (P < 0.001). Current prescription of azithromycin was associated with significantly higher azithromycin MICs (P= 0.016) in the CF isolates. CF isolates (not currently prescribed) had significantly higher azithromycin MICs (P= 0.009) compared to isolates from healthy control subjects. Isolates from the 2 CF groups had equal resistance to clindamycin (P= 0.228). Conclusions: CF patients harbour isolates with increased resistance to azithromycin and clindamycin. Azithromycin resistance is associated with current azithromycin prescription. Work supported by a Department of Employment and Learning, NI (DEL) studentship to L. Sherrard and by HSC Research and Development, Public Health Agency, NI and the Medical Research Council through a US-Ireland Partnership Grant.