Juan de Dios Caballero

Shortcomings of the Commercial MALDI-TOF MS Database and Use of MLSA as an Arbiter in the Identification of Nocardia Species

Nocardia species are difficult to identify, a consequence of the ever increasing number of species known and their homogeneous genetic characteristics. 16S rRNA analysis has been the gold standard for identifying these organisms, but proteomic techniques such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) and housekeeping gene analysis, have also been explored. One hundred high (n = 25), intermediate (n = 20), and low (n = 55) prevalence (for Spain) Nocardia strains belonging to 30 species were identified via 16S rRNA and MALDI-TOF MS analysis. The manufacturer-provided database MALDI Biotyper library v4.0 (5.627 entries, Bruker Daltonik) was employed. In the high prevalence group (Nocardia farcinica, N. abscessus, N. cyriacigeorgica and N. nova), the 16S rRNA and MALDI-TOF MS methods provided the same identification for 76% of the strains examined. For the intermediate prevalence group (N. brasiliensis, N. carnea, N. otitidiscaviarum and N. transvalensis complex), this figure fell to 45%. In the low-prevalence group (22 species), these two methods were concordant only in six strains at the species level. Tetra-gene multi-locus sequencing analysis (MLSA) involving the concatemer gyrB-16S rRNA-hsp65-secA1 was used to arbitrate between discrepant identifications (n = 67). Overall, the MLSA confirmed the results provided at species level by 16S rRNA analysis in 34.3% of discrepancies, and those provided by MALDI-TOF MS in 13.4%. MALDI-TOF MS could be a strong candidate for the identification of Nocardia species, but only if its reference spectrum database improves, especially with respect to unusual, recently described species and species included in the described Nocardia complexes.

First Report of Macrolide Resistance in a Mycoplasma pneumoniae Isolate Causing Community-Acquired Pneumonia in Spain

Mycoplasma pneumoniae is a common cause of community acquired pneumonia (CAP) ([1][1]). Macrolides are first-choice agents, but resistance is possible by point mutations in domain V of the 23S rRNA gene ([1][1], [2][2]). Asiatic countries have the highest proportion of macrolide resistance, but in

Emergence of cfr-Mediated Linezolid Resistance in a Methicillin-Resistant Staphylococcus aureus Epidemic Clone Isolated from Patients with Cystic Fibrosis

ABSTRACT Resistance to linezolid (LZD) in methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients with cystic fibrosis (CF) is due mainly to ribosomal mutations. We report on four CF patients with LZD-resistant MRSA bronchopulmonary infections by strains carrying the cfr gene. Strains from one patient also harbored the G2576U mutation (23S rRNA) and the G139R substitution (L3 protein). All strains belonged to the epidemic clone ST125 MRSA IVc. Our results support the monitoring of LZD resistance emergence in CF and non-CF MRSA isolates.

Antibiotic resistance and population structure of cystic fibrosis Pseudomonas aeruginosa isolates from a Spanish multi-centre study

The first Spanish multi-centre study on the microbiology of cystic fibrosis (CF) was conducted from 2013 to 2014. The study involved 24 CF units from 17 hospitals, and recruited 341 patients. The aim of this study was to characterise Pseudomonas aeruginosa isolates, 79 of which were recovered from 75 (22%) patients. The study determined the population structure, antibiotic susceptibility profile and genetic background of the strains. Fifty-five percent of the isolates were multi-drug-resistant, and 16% were extensively-drug-resistant. Defective mutS and mutL genes were observed in mutator isolates (15.2%). Considerable genetic diversity was observed by pulsed-field gel electrophoresis (70 patterns) and multi-locus sequence typing (72 sequence types). International epidemic clones were not detected. Fifty-one new and 14 previously described array tube (AT) genotypes were detected by AT technology. This study found a genetically unrelated and highly diverse CF P. aeruginosa population in Spain, not represented by the epidemic clones widely distributed across Europe, with multiple combinations of virulence factors and high antimicrobial resistance rates (except for colistin).

Bronchopulmonary infection–colonization patterns in Spanish cystic fibrosis patients: Results from a national multicenter study

BACKGROUND Clinical and demographical knowledge on Spanish cystic fibrosis (CF) patients is incomplete as no national registry exists. CF-microbiology has not been studied at national level. The results of the first Spanish multicenter study on CF microbiology are presented. METHODS 24 CF-Units for adult (n=12) and pediatric (n=12) patients from 17 hospitals provided sputa and clinical data from 15 consecutive patients. Cultures and susceptibility testing were performed. Colonization impact on pulmonary function was assessed. RESULTS 341 patients [mean (SD) age 21 (11) years, 180≥18years, mean (SD) FEV1=68 (25)%] were included. Pseudomonas aeruginosa was reported as chronic, intermittent or absent in 46%, 22% and 32% of patients, respectively. The annual prevalence was 62%. Positive P. aeruginosa and methicillin-resistant Staphylococcus aureus cultures were significantly associated with lower FEV1 (p<0.001 and p=0.003, respectively). CONCLUSIONS The representative subset of the Spanish CF-population which has been clinically, demographically and microbiologically characterized will serve as a reference for future CF studies in Spain.

Microbiological diagnostic procedures for respiratory cystic fibrosis samples in Spain: towards standard of care practices

BackgroundThe microbiological procedures for cystic fibrosis (CF) samples of 17 participating Spanish centers were examined to verify their compliance with current international and national guidelines and to implement the best standards of care for microbiology practices. A 47-item questionnaire covering different CF microbiology aspects was sent to participant laboratories. Telephone interviews were performed when necessary. Data about samples processing for bacteria, mycobacteria and fungi were collected.ResultsGene sequencing (71%), MALDI-TOF (59%) or both (94%) were available for most laboratories. Susceptibility testing was performed by automated microdilution systems (94%) and manual diffusion methods (59%). However, a low use of selective media for Staphylococcus aureus (59%) and Burkholderia cepacia complex (71%), and of epidemiological typing methods (41%) was reported.ConclusionsMost Spanish laboratories are in agreement with consensus guidelines for the processing of CF respiratory samples, but need to improve in the use of specific selective media and typing methods for epidemiologic studies.

Individual Patterns of Complexity in Cystic Fibrosis Lung Microbiota, Including Predator Bacteria, over a 1-Year Period

ABSTRACT Cystic fibrosis (CF) lung microbiota composition has recently been redefined by the application of next-generation sequencing (NGS) tools, identifying, among others, previously undescribed anaerobic and uncultivable bacteria. In the present study, we monitored the fluctuations of this ecosystem in 15 CF patients during a 1-year follow-up period, describing for the first time, as far as we know, the presence of predator bacteria in the CF lung microbiome. In addition, a new computational model was developed to ascertain the hypothetical ecological repercussions of a prey-predator interaction in CF lung microbial communities. Fifteen adult CF patients, stratified according to their pulmonary function into mild (n = 5), moderate (n = 9), and severe (n = 1) disease, were recruited at the CF unit of the Ramón y Cajal University Hospital (Madrid, Spain). Each patient contributed three or four induced sputum samples during a 1-year follow-up period. Lung microbiota composition was determined by both cultivation and NGS techniques and was compared with the patients’ clinical variables. Results revealed a particular microbiota composition for each patient that was maintained during the study period, although some fluctuations were detected without any clinical correlation. For the first time, Bdellovibrio and Vampirovibrio predator bacteria were shown in CF lung microbiota and reduced-genome bacterial parasites of the phylum Parcubacteria were also consistently detected. The newly designed computational model allows us to hypothesize that inoculation of predators into the pulmonary microbiome might contribute to the control of chronic colonization by CF pathogens in early colonization stages. IMPORTANCE The application of NGS to sequential samples of CF patients demonstrated the complexity of the organisms present in the lung (156 species) and the constancy of basic individual colonization patterns, although some differences between samples from the same patient were observed, probably related to sampling bias. Bdellovibrio and Vampirovibrio predator bacteria were found for the first time by NGS as part of the CF lung microbiota, although their ecological significance needs to be clarified. The newly designed computational model allows us to hypothesize that inoculation of predators into the lung microbiome can eradicate CF pathogens in early stages of the process. Our data strongly suggest that lower respiratory microbiome fluctuations are not necessarily related to the patient’s clinical status. IMPORTANCE The application of NGS to sequential samples of CF patients demonstrated the complexity of the organisms present in the lung (156 species) and the constancy of basic individual colonization patterns, although some differences between samples from the same patient were observed, probably related to sampling bias. Bdellovibrio and Vampirovibrio predator bacteria were found for the first time by NGS as part of the CF lung microbiota, although their ecological significance needs to be clarified. The newly designed computational model allows us to hypothesize that inoculation of predators into the lung microbiome can eradicate CF pathogens in early stages of the process. Our data strongly suggest that lower respiratory microbiome fluctuations are not necessarily related to the patient’s clinical status.

Antimicrobial susceptibility of non-fermenting Gram-negative pathogens isolated from cystic fibrosis patients

Non-fermenting Gram-negative bacteria (NFGNB) are increasingly cultured in respiratory samples from cystic fibrosis (CF) patients. This study determined the antimicrobial susceptibility of clinical CF respiratory isolates from distinct geographical regions. A total of 286 isolates (106 Stenotrophomonas maltophilia, 100 Burkholderia spp., 59 Achromobacter spp., 12 Pandoraea spp., 9 Ralstonia spp.) from the Netherlands, Northern Ireland, Spain, USA and Australia were tested. MIC50/90 values and susceptibility categorisation were determined. Trimethoprim/sulfamethoxazole (SXT) was the most active compound for all micro-organisms (MIC50, 0.12-4 mg/L; MIC90, 1-16 mg/L). For S. maltophilia, 47% and 62% of isolates were susceptible to SXT according to CLSI and EUCAST breakpoints, respectively. Ceftazidime presented lower susceptibility (35%; MIC50, 32 mg/L; MIC90, 256 mg/L). MIC90 values for tobramycin and colistin were >128 mg/L and >16 mg/L, respectively. Regarding Burkholderia, 72%, 56% and 44% were susceptible to SXT, ceftazidime and meropenem, respectively. For both ceftazidime and meropenem, MIC50 and MIC90 values were within the intermediate or resistant category. The most active antibiotics for Achromobacter spp. were SXT (MIC50, 0.5 mg/L; MIC90, 8 mg/L) and imipenem (MIC50, 2 mg/L; MIC90, 8 mg/L). SXT, imipenem and ciprofloxacin were active against 12 Pandoraea spp. (MIC50, 0.12-4 mg/L; MIC90, 1-8 mg/L). Ciprofloxacin (MIC50, 4 mg/L) and SXT (MIC50, 1 mg/L) were the only active antibiotics for Ralstonia spp. There were no statistically significant differences in susceptibility rates between countries. NFGNB other than Pseudomonas aeruginosa are potential pathogens in CF. SXT was demonstrated to be the most active compound against these isolates.

98 Antibiotic prescription patterns for the treatment of Spanish cystic fibrosis patients: Results from a national multicentre survey study

Objectives To describe antibiotic (AB) prescription to Spanish cystic fibrosis (CF) patients over a 1-yr period, administration route and frequency of combined regimes. Oral azithromycin (AZT), dornase-alpha (Da) and inhaled hypertonic saline (IHS) use were recorded. Methods 24 CF Units (12 adult/12 pediatric) from 17 Spanish hospitals participated in a multicentre study (2013). Centres attend ∼75% of the Spanish CF population. Clinicians were asked to record prescribed treatments over a 1-yr period to the first 15 consecutive patients attending routinely to their units. Results 341 patients were included (12% of the Spanish CF population): Females 53%; mean age 21±11 yr; ≥18 yr 53%; mean %FEV 1 68±25; median exacerbations/yr 2 [0–12]; hospitalised patients 29%. 338 patients (99%) were receiving AB (Table) with 160 (47%) oral + inhaled, 10 (3%) oral + i.v., 4 (1%) inhaled+i.v. and 88 (26%) oral + inhaled + i.v. More than one inhaled antibiotic was administered to 67 patients (20%). Patients with inhaled colistin, tobramycin and aztreonam were 186 (68%), 90 (33%) and 22 (8%), respectively. Inhaled i.v. formulations were administered to 37 patients. AZT, IHS, Da and probiotics were used in 198 (58%), 204 (60%), 28 (8%) and 73 (23%) patients, respectively. Table . Number (%) of patients by type of therapy Number (%) of patients Oral (N = 302, 89%) Inhaled (N = 273, 80%) i.v. (N = 105, 31%) Betalactams 133 (44) 44 (14) 99 (94) Aminoglycosides − 103 (38) 68 (65) Quinolones 207 (69) − 7 (7) Colistin − 186 (68) 8 (8) Tetracyclines 19 (6) − 1 (1) Glycopeptides − 9 (3) 11 (11) Linezolid 22 (7) − 3 (3) Co-trimoxazole 109 (36) − 2 (2) Antifungals 23 (8) 1 (0.4) 1 (1) Conclusion Antibiotic management of CF patients is challenging since the proportion of older patients with more advanced disease is high.

WS19.2 Staphylococcus aureus population from cystic fibrosis patients in Spain: Results from a point-prevalence multicentre study

Objectives To characterize Staphylococcus aureus isolates colonising the lung of Spanish cystic fibrosis (CF) patients. Methods A prospective, multicentre study was performed (March-Nov. 2013) covering 341 CF-patients (180 adults, 161 children) from 17 hospitals. Respiratory samples (one per patient) and clinical and microbiological data were sent to our centre. S. aureus isolates were identified (MALDI-TOF MS) and antibiotic susceptibility (MicroScan) determined. Genetic relationships between the isolates were assessed by PFGE- Sma I, spa -typing and MLST, including a concatenate analysis of the alleles. SCC mec and agr type were characterized in methicillin-resistant S. aureus (MRSA). Results A total of 206 S. aureus isolates (60%) were obtained in 97 adults and 109 children. Prevalence in the different CF-Units varied between 13.3–86.6%. Pseudomonas aeruginosa co-colonisation was observed in 36 patients. Twenty-eight MRSA (13.7%) were detected in 11 Units, corresponding to SCCmec-IVc (22/28), SCCmec-IVa (3/28), and SCCmec-V (3/28). Eighty-seven isolates displaying unrelated PFGE were further analysed, showing a high genetic diversity by spa (the most frequent were CC002, CC012) and MLST (CC5, CC8, CC30, and CC45) typing. Three isolates grouped into the ST398, linked to MRSA carriage in pigs. Glycopeptide resistance was absent, whereas a cfr -mediated-linezolid resistant MRSA isolate was detected. Conclusion Overall prevalence of S. aureus in the Spanish CF-population was 60%, being 13.7% of the isolates MRSA. Genetic relationship between isolates was scarce although some widespread nosocomial and community clones were detected, including ST398 from animal origin.