Wide Dispersion of ST175 Clone despite High Genetic Diversity of Carbapenem-Nonsusceptible Pseudomonas aeruginosa Clinical Strains in 16 Spanish Hospitals

García-Castillo, María; Campo, Rosa del; Morosini, María Isabel; Riera, Elena; Cabot, Gabriel; Willems, Rob J. L.; Mansfeld, Rosa van; Oliver, Antonio

doi:10.1128/jcm.00753-11

Cited by 78 publications

(66 citation statements)

References 35 publications

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“…MLST provides useful information about the population biology of international genetic lineages (clones and clonal complexes) producing infections worldwide (4,13). We have recently documented a high MLST-based genetic diversity among clinical carbapenem-nonsusceptible P. aeruginosa isolates in Spain (6). In this work, we detected widespread ST175 and ST646 clones.…”

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confidence: 49%

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Emergence of a mutL Mutation Causing Multilocus Sequence Typing–Pulsed-Field Gel Electrophoresis Discrepancy among Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient

et al. 2012

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A multilocus sequence type (MLST) shift (from ST242 to ST996) was detected in Pseudomonas aeruginosa isolates with a uniform pulsed-field gel electrophoresis (PFGE) pattern obtained from a chronically colonized patient. MLST mutational change involved the mutL gene with the consequent emergence of a hypermutable phenotype. This observation challenges the required neutrality of mutL as an appropriate marker in MLST and alerts researchers to the limitations of MLST-only-based population studies in chronic infections under constant antibiotic selective pressure. P ulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) methods are molecular typing tools used to study the genetic relationships among bacterial isolates (7). PFGE has been widely used to identify particular epidemic clones causing nosocomial outbreaks and to follow changes in the identities of isolates during chronic infections, such as in Pseudomonas aeruginosa cystic fibrosis (CF) lung infection (8,11). MLST provides useful information about the population biology of international genetic lineages (clones and clonal complexes) producing infections worldwide (4, 13). We have recently documented a high MLST-based genetic diversity among clinical carbapenem-nonsusceptible P. aeruginosa isolates in Spain (6). In this work, we detected widespread ST175 and ST646 clones. Despite these results, differences in their PFGE patterns were observed, probably due to local evolution (6).In the present work, we report a discrepancy between MLST and PFGE typing tools in a collection of 21 P. aeruginosa isolates obtained from a single CF patient attending our CF unit from 2003 to 2009. PFGE typing was performed as follows: from an overnight culture of 5 ml of LB broth (Difco, Detroit, MI) for each isolate, an aliquot of 1 ml was centrifuged and the pellet was mixed with 200 l of SE solution (75 mM NaCl, 25 mM EDTA, pH 7.5) in the same Eppendorf tube. Optical density was adjusted to between 0.6 and 2.5 using the Nanodrop system (Thermo Scientific, Wilmington, DE) at 590 nm. Subsequently, 200 l of 2% agarose was added to the Eppendorf tube, gently mixed, and deposited in appropriate plug molds. Overnight protein lysis was carried out at 56°C in 2 ml of lysis solution (0.5 M EDTA [pH 9], 10% Sarkosyl, and 25 mg/ml of proteinase K). Plugs were then washed twice in 2 ml of Tris-EDTA (TE) for 30 min. A third part of each plug was digested with 15 U of SpeI (Roche Diagnostics, Indianapolis, IN) at 37°C for at least 2 h. Electrophoresis was carried out in a 1.2% agarose gel in 0.5ϫ Tris-buffered EDTA (TBE) at 14°C with the following settings: 6 V/cm 2 , 5 to 40 s, and 22 h. For MLST, we followed the scheme developed by Keith Jolley at the University of Oxford (http://pubmlst.org/paeruginosa).The study of the sequential isolates obtained from the CF patient revealed an MLST shift during chronic infection. For seven consecutive years, 19 isolates grouped in ST242 (allele code 28,5,5,11,3,15,44). However, in the last 3 years two isolates clustered in ST996 (...

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“…In this work, we detected widespread ST175 and ST646 clones. Despite these results, differences in their PFGE patterns were observed, probably due to local evolution (6).…”

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confidence: 68%

Emergence of a mutL Mutation Causing Multilocus Sequence Typing–Pulsed-Field Gel Electrophoresis Discrepancy among Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient

et al. 2012

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“…olecular typing tools applied to Pseudomonas aeruginosa isolates recovered from cystic fibrosis (CF) patients have demonstrated not only local epidemiological differences (1)(2)(3)(4) but also the spread of particular multiresistant clones through different CF units (5)(6)(7)(8) and the in vivo evolution of single clones involving multilocus sequence type (MLST) shifts (9). Nevertheless, studies addressing differences in terms of population structure between nonpersistent and persistent isolates from CF patients have not yet been reported.…”

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confidence: 99%

“…Unlike other CF units, we could not demonstrate the spread of previously identified international CF epidemic clones. Nevertheless, one of the most prevalent STs was ST274, previously described in another Spanish CF unit, which is a multidrug-resistant clone circulating in Spain (1,18). Our CF unit follows the segregation measures internationally recommended (19), even though patient-to-patient transmission events were suspected in three patients.…”

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Population Structure and Antimicrobial Susceptibility of Both Nonpersistent and Persistent Pseudomonas aeruginosa Isolates Recovered from Cystic Fibrosis Patients

et al. 2013

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d Seventy-six Pseudomonas aeruginosa isolates recovered from chronically (n ‫؍‬ 18) and nonchronically (n ‫؍‬ 18) colonized cystic fibrosis (CF) patients (2002 to 2009) were grouped in separate polyclonal populations. International CF epidemic clones were not identified, but the high-risk clone ST274, also found circulating in Spanish hospitals, was present. Persistent isolates were more resistant to antibiotics than nonpersistent isolates.

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“…seudomonas aeruginosa has a nonclonal population structure with a few multidrug-resistant clusters, called "high-risk clones," that frequently produce acquired ␤-lactamases with an extended spectrum and are responsible for outbreaks in hospitals worldwide (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The quick implementation of infection control measures for relevant patients may tackle the spread of these epidemic clones, but current identification is long and complex since it is based on the analysis of nucleotidic sequences.…”

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Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Identifies Pseudomonas aeruginosa High-Risk Clones

et al. 2015

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We show here that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) accurately and quickly identified the five high-risk clones of Pseudomonas aeruginosa sequence type 111 (ST111), ST175, ST235, ST253, and ST395. The use of this screening technique by clinical microbiology laboratories may tackle the spread of high-risk clones by the quick implementation of hygiene control procedures for relevant patients. Pseudomonas aeruginosa has a nonclonal population structure with a few multidrug-resistant clusters, called "high-risk clones," that frequently produce acquired ␤-lactamases with an extended spectrum and are responsible for outbreaks in hospitals worldwide (1-13). The quick implementation of infection control measures for relevant patients may tackle the spread of these epidemic clones, but current identification is long and complex since it is based on the analysis of nucleotidic sequences. A quick and easy method for identifying high-risk clones of P. aeruginosa is then needed. Matrixassisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was recently integrated into the routine workflow of medical microbiology laboratories for microbial identification and has been further used for subspecies characterization (14). Here, we evaluated the ability of MALDI-TOF MS to identify high-risk clones of P. aeruginosa.Identification of peak biomarkers for five major high-risk clones of P. aeruginosa. In order to identify five major high-risk clones of P. aeruginosa (sequence type 111 [ST111], ST175, ST235, ST253, and ST395), we first defined recognition models with a training set of 46 isolates with known STs distributed homogeneously in the phylogenetic tree (Table 1 and Fig. 1). Frozen bacteria were streaked onto Mueller-Hinton agar (Bio-Rad) and incubated for 18 h at 37°C. As previously reported (15), each isolate was extracted with the ethanol-formic acid method recommended by Bruker Daltonik and analyzed with a Microflex LT mass spectrometer (Bruker Daltonik), which generated 24 raw spectra. We analyzed the spectra with the software ClinProTools 3.0 (Bruker Daltonik), which defined six models based on peak biomarkers (Table 1). The reliability and accuracy of each model were assessed through the recognition capabilities and the crossvalidation values. The models identified all the tested high-risk clones of P. aeruginosa with high recognition capabilities (Ͼ96%) and cross-validation values ranging from 72.5% to 100% (Table 1). Four models directly identified ST111, ST175, ST253, and ST395, while the identification of ST235 required a preliminary stage to identify cluster 1, which encompasses ST235 (Fig. 1).We then manually examined the spectra with ClinProTools 3.0 to assess the relevance of the peak biomarkers of each model (Table 1). Peaks were defined as biomarkers in a model because of their presence or absence or relative abundance between two tested classes. Figure 2 details only the peak biomarkers in which presence or absence was specif...

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Wide Dispersion of ST175 Clone despite High Genetic Diversity of Carbapenem-Nonsusceptible Pseudomonas aeruginosa Clinical Strains in 16 Spanish Hospitals

Cited by 78 publications

References 35 publications

Emergence of a mutL Mutation Causing Multilocus Sequence Typing–Pulsed-Field Gel Electrophoresis Discrepancy among Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient

Emergence of a mutL Mutation Causing Multilocus Sequence Typing–Pulsed-Field Gel Electrophoresis Discrepancy among Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient

Population Structure and Antimicrobial Susceptibility of Both Nonpersistent and Persistent Pseudomonas aeruginosa Isolates Recovered from Cystic Fibrosis Patients

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Identifies Pseudomonas aeruginosa High-Risk Clones

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