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92 Prevalence, colonization patterns and antibiotic susceptibility of Burkholderia cepacia complex isolates from a Spanish cystic fibrosis unit
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Posters / Journal of Cystic Fibrosis 15 (2016) S51–S120
identification, manual and automated biochemical galleries, molecular method (PCR/sequencing) and MALDI-TOF VITEK MS (bioMerieux) ´ coupled with the SARAMIS database (AnagnosTec). Results: In 2011–2012, the use of the MALDI TOF MS technology increased progressively up to 80%, whereas the use of other technics decreased dramatically. Concerning the minor CF bacteria, the MALDI TOF appears to be convenient for their identification even if the small number of strains makes it difficult to perform the comparison. However, the MALDI TOF MS identified at least one strain of each species encountered in 2011–2012 except for the species Pseudomonas luteola identified only by biochemical gallery. Concerning the epidemiology of major CF pathogens, we observed a decrease of the rate of P. aeruginosa from 80.8% in 2008–2009 to 77.6% in 2011–2012, whereas the number of Burkholderia strains increased from 3.8 to 5.6%. Conclusion: Due to its rapidity, its simplicity and its efficiency as well as its low cost per analysis, the MALDI-TOF MS technology led to a decreased use of other methods, and a modification in the identification of P. aeruginosa and Burkholderia sp strains.
before patient’s death. Comprehensive comparative analysis of genomic sequences was performed. Results: While isolates MF16 and FFH2055 were genetically virtually identical, isolate 467 was revealed as their genomic derivative. 70% of total 1,225 point mutations detected in isolate 467 were AT→GC transitions, indicating deficiency in mismatch repair (MMR). Genes encoding MMR proteins contained missense mutations, localized in conserved domains of both MutS (628N→D) and MutL (317S→P). These mutations have not been reported among Argentinean B. contaminans isolates so far. Isolate 467 was found to have its mutation frequency ~100-fold higher than isolates FFH2055 and MF16. The distribution of mutations with respect to their localization (intragenic/intergenic) and protein effect (synonymous/nonsynonymous) corresponded to a random model. Hypermutability was preserved in isolates from 2 last years of chronic infection. Conclusion: Our results document low levels of purifying selection in CF sputum. Hypermutability thus enables rapid evolution that is not detrimental to B. contaminans during chronic infection.
90 Chronic infection with Achromobacter xylosoxidans in cystic fibrosis patients
92 Prevalence, colonization patterns and antibiotic susceptibility of Burkholderia cepacia complex isolates from a Spanish cystic fibrosis unit J. De Dios Caballero1 , L. Mart´ınez1 , M. Tato1 , M.I. Morosini1 , M. Cobo1 , ´ 1 . 1 Hospital Universitario Ramon y Cajal, R. del Campo1 , R. Canton Microbiolog´ıa, Madrid, Spain
V. D’Alessandro1 , M.F. Gil1 , M. Bettiol1 , B. Gatti1 , M.J. Palau1 , F. Renter´ıa1 , G. Diez1 . 1 Childrens Hospital ‘Sor Mar´ıa Ludovica’, La Plata, Argentina Objectives: To describe the occurrence of chronic infection with A. xylosoxidans and to investigate the antimicrobial sensitivity of isolates along a 6 year period. Methods: Retrospective microbiological analysis of respiratory samples obtained from CF patients between 2009–2014 was undertaken. Every patient showing A. xylosoxidans infection was classified as either “chronic” or “sporadic”. Chronic infection was defined as the persistence of three positive cultures within a six-month period. Sporadic infection when less than three cultures were positive in a year. In chronic patients we assessed age of acquisition of the infection, co-infection (defined as sputum culture positive for more than one microorganism) and antimicrobial susceptibility profile. Results: A total of 3120 sputum samples were obtained from 160 patients. 72 isolates of A. xylosoxidans were recovered from 16 patients. Among these, 4 (25%) were catalogued as chronically infected. The age of colonized were 2, 5 and 9 (two occurrences) years. All were co-infected, two with methicillin resistant S. aureus, one with P. aeruginosa and H. influenzae and other with B. cepacia complex. When comparing patient isolations, we found that the last isolate of A. xylosoxidans was more resistant than the initial one, showing resistance with ciprofloxacin, piperacillin-tazobactam and trimethoprim-sulfamethoxazole. Conclusion: We found in our cohort a 10% of prevalence of A.xylosoxidans and 2.5% of chronic infection. Although it seems to become more resistant with time, it remains to be determined whether or not is the same bacterial strain, (probably due to repeated use of antibiotics to treat respiratory infections). 91 Genomic evolution of Burkholderia contaminans ST872 during chronic CF infection J. Nunvar1 , R. Bloodworth2 , J. Degrossi3 , S. Cardona2 , P. Drevinek1 . 1 2nd Faculty of Medicine, Charles University in Prague, Department of Medical Microbiology, Prague, Czech Republic; 2 Faculty of Science, University of Manitoba, Department of Microbiology, Manitoba, Canada; 3 University of Buenos Aires, Buenos Aires, Argentina Objectives: To gain insight into long-term within-patient evolution of Burkholderia contaminans, a recently described B. cepacia complex species, we examined the nature and extent of genomic variability between longitudinal isolates. Methods: The genomic sequences of three B. contaminans isolates (multilocus sequence type 872) were determined. One sputum isolate (FFH2055) was obtained at the beginning of the chronic infection in a female Argentinean patient; other two (467 and MF16) were recovered from sputum and blood, respectively six years later, shortly
Objectives: To study the prevalence, antibiotic susceptibility and colonization patterns of Burkholderia cepacia complex (BCC) species isolated in our Hospital between 2010–2015. Methods: All Burkholderia spp. strains isolated from cystic fibrosis (CF)-patients were recovered for the study. Susceptibility testing was performed by automated microdilution (MicroScan). Clonality of the isolates was assessed by Pulse Field Gel Electrophoresis (PFGE) with SpeI. Identification of BCC species was performed by the sequencing of the recA gene. Results: A total of 313 CF-patients attended to our hospital in the study period, 55 strains of Burkholderia spp. being isolated in 14 of them (4.5% prevalence). B. contaminans (Bcon), B. vietnamiensis (Bv), B. cepacia (Bcep), B. stabilis (Bs) and B. multivorans (Bm) were seen in 36% (n = 5), 29% (n = 4), 14% (n = 2), 7% (n = 1), and 7% of the patients, respectively, being 1 patient (7%) colonized by B. gladioli (Bg). The most active antibiotics against BCC species were meropenem, minocycline and co-trimoxazole, being susceptible 76%, 65% and 59% of the isolates, respectively. PFGE patterns showed that each patient harbored his own clone but a possible case of Bcep cross-transmission was identified. Chronic colonization was found in 7 patients (50%) due to Bv (n = 2), Bs (n = 1), Bm (n = 1), Bcep (n = 1), Bcont (n = 1) and Bg (n = 1). Conclusion: BCC prevalence in our institution was higher than the one reported in other European countries but cross-transmission was not a frequent event. Bcon was the most prevalent species followed by Bv, and no B. cenocepacia was found. BCC chronic colonization was frequent but no species-specific association was seen. 93 The use of MALDI-TOF MS for the identification of non-fermenting Gram-negative bacilli isolated from cystic fibrosis patients L. O’Brien1 , G. Edwards1 , A. Hardy1 , M. Smith1 , H. Green2 , P.J. Barry2 , A.M. Jones2 , M. Cullen3 . 1 Public Health England, Microbiology, Manchester, United Kingdom; 2 Manchester Adult Cystic Fibrosis Centre, Manchester, United Kingdom; 3 University Hospital of South Manchester, Manchester, United Kingdom Objectives: Matrix-Assisted Laser Desorption/Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF MS) is emerging as a technology to rapidly identify bacterial pathogens in the medical microbiology laboratory. We aimed to audit the introduction of MALDI-TOF analysis for identification of non-fermenting Gram-negative bacilli (GNB) to microbiology diagnostics in a large adult CF centre. Methods: The Bruker microflex™ series MALDI Biotyper 3.1 instrument was used to perform MALDI-TOF MS in conjunction with Bruker flex
Posters / Journal of Cystic Fibrosis 15 (2016) S51–S120
identification, manual and automated biochemical galleries, molecular method (PCR/sequencing) and MALDI-TOF VITEK MS (bioMerieux) ´ coupled with the SARAMIS database (AnagnosTec). Results: In 2011–2012, the use of the MALDI TOF MS technology increased progressively up to 80%, whereas the use of other technics decreased dramatically. Concerning the minor CF bacteria, the MALDI TOF appears to be convenient for their identification even if the small number of strains makes it difficult to perform the comparison. However, the MALDI TOF MS identified at least one strain of each species encountered in 2011–2012 except for the species Pseudomonas luteola identified only by biochemical gallery. Concerning the epidemiology of major CF pathogens, we observed a decrease of the rate of P. aeruginosa from 80.8% in 2008–2009 to 77.6% in 2011–2012, whereas the number of Burkholderia strains increased from 3.8 to 5.6%. Conclusion: Due to its rapidity, its simplicity and its efficiency as well as its low cost per analysis, the MALDI-TOF MS technology led to a decreased use of other methods, and a modification in the identification of P. aeruginosa and Burkholderia sp strains.
before patient’s death. Comprehensive comparative analysis of genomic sequences was performed. Results: While isolates MF16 and FFH2055 were genetically virtually identical, isolate 467 was revealed as their genomic derivative. 70% of total 1,225 point mutations detected in isolate 467 were AT→GC transitions, indicating deficiency in mismatch repair (MMR). Genes encoding MMR proteins contained missense mutations, localized in conserved domains of both MutS (628N→D) and MutL (317S→P). These mutations have not been reported among Argentinean B. contaminans isolates so far. Isolate 467 was found to have its mutation frequency ~100-fold higher than isolates FFH2055 and MF16. The distribution of mutations with respect to their localization (intragenic/intergenic) and protein effect (synonymous/nonsynonymous) corresponded to a random model. Hypermutability was preserved in isolates from 2 last years of chronic infection. Conclusion: Our results document low levels of purifying selection in CF sputum. Hypermutability thus enables rapid evolution that is not detrimental to B. contaminans during chronic infection.
90 Chronic infection with Achromobacter xylosoxidans in cystic fibrosis patients
92 Prevalence, colonization patterns and antibiotic susceptibility of Burkholderia cepacia complex isolates from a Spanish cystic fibrosis unit J. De Dios Caballero1 , L. Mart´ınez1 , M. Tato1 , M.I. Morosini1 , M. Cobo1 , ´ 1 . 1 Hospital Universitario Ramon y Cajal, R. del Campo1 , R. Canton Microbiolog´ıa, Madrid, Spain
V. D’Alessandro1 , M.F. Gil1 , M. Bettiol1 , B. Gatti1 , M.J. Palau1 , F. Renter´ıa1 , G. Diez1 . 1 Childrens Hospital ‘Sor Mar´ıa Ludovica’, La Plata, Argentina Objectives: To describe the occurrence of chronic infection with A. xylosoxidans and to investigate the antimicrobial sensitivity of isolates along a 6 year period. Methods: Retrospective microbiological analysis of respiratory samples obtained from CF patients between 2009–2014 was undertaken. Every patient showing A. xylosoxidans infection was classified as either “chronic” or “sporadic”. Chronic infection was defined as the persistence of three positive cultures within a six-month period. Sporadic infection when less than three cultures were positive in a year. In chronic patients we assessed age of acquisition of the infection, co-infection (defined as sputum culture positive for more than one microorganism) and antimicrobial susceptibility profile. Results: A total of 3120 sputum samples were obtained from 160 patients. 72 isolates of A. xylosoxidans were recovered from 16 patients. Among these, 4 (25%) were catalogued as chronically infected. The age of colonized were 2, 5 and 9 (two occurrences) years. All were co-infected, two with methicillin resistant S. aureus, one with P. aeruginosa and H. influenzae and other with B. cepacia complex. When comparing patient isolations, we found that the last isolate of A. xylosoxidans was more resistant than the initial one, showing resistance with ciprofloxacin, piperacillin-tazobactam and trimethoprim-sulfamethoxazole. Conclusion: We found in our cohort a 10% of prevalence of A.xylosoxidans and 2.5% of chronic infection. Although it seems to become more resistant with time, it remains to be determined whether or not is the same bacterial strain, (probably due to repeated use of antibiotics to treat respiratory infections). 91 Genomic evolution of Burkholderia contaminans ST872 during chronic CF infection J. Nunvar1 , R. Bloodworth2 , J. Degrossi3 , S. Cardona2 , P. Drevinek1 . 1 2nd Faculty of Medicine, Charles University in Prague, Department of Medical Microbiology, Prague, Czech Republic; 2 Faculty of Science, University of Manitoba, Department of Microbiology, Manitoba, Canada; 3 University of Buenos Aires, Buenos Aires, Argentina Objectives: To gain insight into long-term within-patient evolution of Burkholderia contaminans, a recently described B. cepacia complex species, we examined the nature and extent of genomic variability between longitudinal isolates. Methods: The genomic sequences of three B. contaminans isolates (multilocus sequence type 872) were determined. One sputum isolate (FFH2055) was obtained at the beginning of the chronic infection in a female Argentinean patient; other two (467 and MF16) were recovered from sputum and blood, respectively six years later, shortly
Objectives: To study the prevalence, antibiotic susceptibility and colonization patterns of Burkholderia cepacia complex (BCC) species isolated in our Hospital between 2010–2015. Methods: All Burkholderia spp. strains isolated from cystic fibrosis (CF)-patients were recovered for the study. Susceptibility testing was performed by automated microdilution (MicroScan). Clonality of the isolates was assessed by Pulse Field Gel Electrophoresis (PFGE) with SpeI. Identification of BCC species was performed by the sequencing of the recA gene. Results: A total of 313 CF-patients attended to our hospital in the study period, 55 strains of Burkholderia spp. being isolated in 14 of them (4.5% prevalence). B. contaminans (Bcon), B. vietnamiensis (Bv), B. cepacia (Bcep), B. stabilis (Bs) and B. multivorans (Bm) were seen in 36% (n = 5), 29% (n = 4), 14% (n = 2), 7% (n = 1), and 7% of the patients, respectively, being 1 patient (7%) colonized by B. gladioli (Bg). The most active antibiotics against BCC species were meropenem, minocycline and co-trimoxazole, being susceptible 76%, 65% and 59% of the isolates, respectively. PFGE patterns showed that each patient harbored his own clone but a possible case of Bcep cross-transmission was identified. Chronic colonization was found in 7 patients (50%) due to Bv (n = 2), Bs (n = 1), Bm (n = 1), Bcep (n = 1), Bcont (n = 1) and Bg (n = 1). Conclusion: BCC prevalence in our institution was higher than the one reported in other European countries but cross-transmission was not a frequent event. Bcon was the most prevalent species followed by Bv, and no B. cenocepacia was found. BCC chronic colonization was frequent but no species-specific association was seen. 93 The use of MALDI-TOF MS for the identification of non-fermenting Gram-negative bacilli isolated from cystic fibrosis patients L. O’Brien1 , G. Edwards1 , A. Hardy1 , M. Smith1 , H. Green2 , P.J. Barry2 , A.M. Jones2 , M. Cullen3 . 1 Public Health England, Microbiology, Manchester, United Kingdom; 2 Manchester Adult Cystic Fibrosis Centre, Manchester, United Kingdom; 3 University Hospital of South Manchester, Manchester, United Kingdom Objectives: Matrix-Assisted Laser Desorption/Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF MS) is emerging as a technology to rapidly identify bacterial pathogens in the medical microbiology laboratory. We aimed to audit the introduction of MALDI-TOF analysis for identification of non-fermenting Gram-negative bacilli (GNB) to microbiology diagnostics in a large adult CF centre. Methods: The Bruker microflex™ series MALDI Biotyper 3.1 instrument was used to perform MALDI-TOF MS in conjunction with Bruker flex