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1. Comparison of susceptibility testing methods for determining the activity of colistin against Gram-negative bacilli of clinical origin.

Author

Haeili M, Kafshdouz M, Pishnian Z, Feizabadi MM, and Martinez-Martinez L

Subjects
Drug Resistance, Bacterial, Humans, Anti-Bacterial Agents pharmacology, Colistin pharmacology, Gram-Negative Bacteria drug effects, Gram-Negative Bacterial Infections microbiology, Microbial Sensitivity Tests methods
Abstract

Purpose: Despite being in clinical use for decades, colistin susceptibility testing remains challenging because of its inherent cationic properties. We aimed to compare the performance characteristics of different methods for testing susceptibility to colistin in a series of clinical isolates of Gram-negative bacilli., Methodology: One hundred and nine clinical isolates of Klebsiella pneumoniae (n=34), Escherichia coli (n=20), Acinetobacter baumannii (n=17) and Pseudomonas aeruginosa (n=38) were studied for colistin susceptibility using broth microdilution (BMID), broth macrodilution (BMAD), agar dilution (AD) as well as disc-diffusion (DD) utilizing two different commercial disc sources., Results: By using BMID as reference method, 88 and 21 isolates were found to be colistin susceptible and resistant, respectively. Overall, acceptable essential agreement (EA) and categorical agreement (CA) were observed between BMAD and reference method (100 %). Whereas the AD method revealed the lowest rate of EA (61.7, 11.7, 5.0 and 5.2 % for K. pneumoniae, A. baumannii, E. coli and P. aeruginosa, respectively), it showed acceptable or near acceptable CA for K. pneumoniae (100 %), E. coli (100 %) and A. baumannii (88.2 %) isolates but not for P. aeruginosa (13.1 %). DD failed to detect resistance in colistin-resistant (colR) P. aeruginosa (n=5, very major errors of 100 %) but successfully identified all high-level colistin-resistant A. baumannii and K. pneumoniae isolates., Conclusion: We found BMAD to be very reliable for colistin MIC determination. Methods AD and DD should not be used for colistin susceptibility testing in P. aeruginosa isolates as these are associated with false-resistant and -susceptible results, respectively.

Published
2019
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2. Challenges to accurate susceptibility testing and interpretation of quinolone resistance in Enterobacteriaceae: results of a Spanish multicentre study.

Author

Rodriguez-Martinez JM, Machuca J, Calvo J, Diaz-de-Alba P, Rodríguez-Mirones C, Gimeno C, Martinez-Martinez L, and Pascual Á

Subjects
Diagnostic Errors, Humans, Microbial Sensitivity Tests methods, Reproducibility of Results, Spain, Enterobacteriaceae drug effects, Laboratory Proficiency Testing, Microbial Sensitivity Tests standards, Quinolones pharmacology
Abstract

Objectives: The objective of this study was to evaluate the proficiency of Spanish laboratories with respect to accurate susceptibility testing and the detection and interpretation of quinolone resistance phenotypes in Enterobacteriaceae., Methods: Thirteen strains of Enterobacteriaceae were sent to 62 participating centres throughout Spain; strains harboured GyrA/ParC modifications, reduced permeability and/or plasmid-mediated quinolone resistance genes. The centres were requested to evaluate nalidixic acid and five quinolones, provide raw/interpreted clinical categories and to detect/infer resistance mechanisms. Consensus results from reference centres were used to assign minor, major and very major errors (mEs, MEs and VMEs, respectively)., Results: Susceptibility testing in the participating centres was frequently performed using the MicroScan WalkAway, Vitek 2 and Wider systems (48%, 30% and 8%, respectively). CLSI/EUCAST breakpoints were used in 71%/29% of the determinations. The percentage of VMEs for all quinolones was well below 2%. Only ofloxacin and moxifloxacin showed higher values for raw VMEs (6.6%), which decreased to 0% and 2.9%, respectively, in the interpreted VMEs. These errors were particularly associated with the CC-03 strain [qnrS2 + aac(6')-Ib-cr]. For MEs, percentages were always <10%, except in the case of ofloxacin and nalidixic acid. There was a significantly higher percentage of all types of errors for strains whose MICs were at the border of clinical breakpoints., Conclusions: The use of different breakpoints and methods, the complexity of mutation-driven and transferable resistance mechanisms and the absence of specific tests for detecting low-level resistance lead to high variability and represent a challenge to accuracy in susceptibility testing, particularly in strains with MICs on the border of clinical breakpoints., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Published
2015
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3. Use of positive blood cultures for direct identification and susceptibility testing with the vitek 2 system.

Author

de Cueto M, Ceballos E, Martinez-Martinez L, Perea EJ, and Pascual A

Subjects
Anti-Bacterial Agents pharmacology, Bacteria drug effects, Blood microbiology, Gram-Positive Bacteria drug effects, Gram-Positive Bacteria isolation & purification, Humans, Bacteria isolation & purification, Microbial Sensitivity Tests methods
Abstract

In order to further decrease the time lapse between initial inoculation of blood culture media and the reporting of results of identification and antimicrobial susceptibility tests for microorganisms causing bacteremia, we performed a prospective study in which specially processed fluid from positive blood culture bottles from Bactec 9240 (Becton Dickinson, Cockeysville, Md.) containing aerobic media were directly inoculated into Vitek 2 system cards (bio-Mérieux, France). Organism identification and susceptibility results were compared with those obtained from cards inoculated with a standardized bacterial suspension obtained following subculture to agar; 100 consecutive positive monomicrobic blood cultures, consisting of 50 gram-negative rods and 50 gram-positive cocci, were included in the study. For gram-negative organisms, 31 of the 50 (62%) showed complete agreement with the standard method for species identification, while none of the 50 gram-positive cocci were correctly identified by the direct method. For gram-negative rods, there were 50% categorical agreements between the direct and standard methods for all drugs tested. The very major error rate was 2.4%, and the major error rate was 0.6%. The overall error rate for gram-negatives was 6.6%. Complete agreement in clinical categories of all antimicrobial agents evaluated was obtained for 19 of 50 (38%) gram-positive cocci evaluated; the overall error rate was 8.4%, with 2.8% minor errors, 2.4% major errors, and 3.2% very major errors. These findings suggest that the Vitek 2 cards inoculated directly from positive Bactec 9240 bottles do not provide acceptable bacterial identification or susceptibility testing in comparison with corresponding cards tested by a standard method.

Published
2004
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4. Molecular characterization of multidrug resistant Enterobacterales strains isolated from liver and kidney transplant recipients in Spain

Author

Fernandez-Martinez, Marta, Gonzalez-Rico, Claudia, Gozalo-Marguello, Monica, Marco, Francesc, Gracia-Ahufinger, Irene, Aranzamendi, Maitane, Sanchez-Diaz, Ana M., Vicente-Rangel, Teresa, Chaves, Fernando, Calvo Montes, Jorge, Martinez-Martinez, Luis, Carmen Farinas, Maria, ENTHERE Study Grp, Spanish Soc Infect Dis Clinical Mi, Spanish Network Res Infect Dis REI, Universidad de Cantabria, [Fernandez-Martinez, Marta] Hosp Univ Marques de Valdecilla, Serv Microbiol, Santander, Spain, [Gozalo-Marguello, Monica] Hosp Univ Marques de Valdecilla, Serv Microbiol, Santander, Spain, [Calvo Montes, Jorge] Hosp Univ Marques de Valdecilla, Serv Microbiol, Santander, Spain, [Fernandez-Martinez, Marta] Inst Invest Valdecilla IDIVAL, Santander, Spain, [Gonzalez-Rico, Claudia] Inst Invest Valdecilla IDIVAL, Santander, Spain, [Gozalo-Marguello, Monica] Inst Invest Valdecilla IDIVAL, Santander, Spain, [Calvo Montes, Jorge] Inst Invest Valdecilla IDIVAL, Santander, Spain, [Carmen Farinas, Maria] Inst Invest Valdecilla IDIVAL, Santander, Spain, [Gonzalez-Rico, Claudia] Hosp Univ Marques Valdecilla, Serv Enfermedades Infecciosas, Santander, Spain, [Carmen Farinas, Maria] Hosp Univ Marques Valdecilla, Serv Enfermedades Infecciosas, Santander, Spain, [Marco, Francesc] Univ Barcelona, Hosp Clin ISGlobal, Ctr Diagnost Biomed, Serv Microbiol, Barcelona, Spain, [Gracia-Ahufinger, Irene] Hosp Univ Reina Sofia, Unidad Microbiol, Cordoba, Spain, [Martinez-Martinez, Luis] Hosp Univ Reina Sofia, Unidad Microbiol, Cordoba, Spain, [Gracia-Ahufinger, Irene] Inst Maimonides Invest Biomed Cordoba IMIBIC, Cordoba, Spain, [Martinez-Martinez, Luis] Inst Maimonides Invest Biomed Cordoba IMIBIC, Cordoba, Spain, [Aranzamendi, Maitane] Hosp Univ Cruces, Serv Microbiol, Baracaldo, Vizcaya, Spain, [Aranzamendi, Maitane] Inst Invest Sanitaria Biocruces, Vizcaya, Spain, [Sanchez-Diaz, Ana M.] Hosp Univ Ramon y Cajal, Serv Microbiol, Madrid, Spain, [Vicente-Rangel, Teresa] Hosp Gen Univ Gregorio Maranon, Serv Microbiol Clin & Enfermedades Infecciosas, Madrid, Spain, [Chaves, Fernando] Hosp Univ 12 Octubre, Serv Microbiol, Madrid, Spain, [Martinez-Martinez, Luis] Univ Cordoba, Hosp Univ Reina Sofia, IMIBIC, Dept Microbiol,Unidad Microbiol, Cordoba, Spain, Plan Nacional de I+D+i and Instituto de Salud Carlos III, Spanish Network for Research in Infectious Diseases, European Development Regional Fund 'A way to achieve Europe', Operative Program Intelligent Growth, Subdireccion General de Redes y Centros de Investigacion Cooperativa, Ministerio de Ciencia, Innovacion y Universidades, ENTHERE Study Group, the Group for Study of Infection in Transplantation of the Spanish Society of Infectious Diseases and Clinical Microbiology (GESITRA-SEIMC) and the Spanish Network for Research in Infectious Diseases (REIPI), [Fernández-Martínez,M, Gozalo-Margüello,M, Calvo Montes,J] Servicio de Microbiología, Hospital Universitario Marqués de Valdecilla, Santander, Spain. [Fernández-Martínez,M, González-Rico,C, Calvo Montes,J, Fariñas,MC] Instituto de Investigación Valdecilla-IDIVAL, Santander, Spain. [González-Rico,C, Fariñas,MC] Servicio de Enfermedades Infecciosas, Hospital Universitario Marqués de Valdecilla, Santander, Spain. [Marco,F] Servicio de Microbiología, Centro Diagnóstico Biomédico, Hospital Clínic. ISGlobal, Universidad de Barcelona, Barcelona, Spain. [Gracia-Ahufinger,I, Martínez-Martínez,L] Unidad de Microbiología, Hospital Universitario Reina Sofía, Córdoba, Spain. [Gracia-Ahufinger,I, Martínez-Martínez,L] Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain. [Aranzamendi,M] Servicio de Microbiología, Hospital Universitario de Cruces, Baracaldo, Vizcaya, Spain. [Aranzamendi,M] Instituto de Investigación Sanitaria Biocruces, Vizcaya, Spain. [Sánchez-Díaz,AM] Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Madrid, Spain. [Vicente-Rangel,T] Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain. [Chaves,F] Servicio de Microbiología, Hospital Universitario 12 de Octubre, Madrid, Spain. [Martínez-Martínez,L] Departamento of Microbiología, Unidad de Microbiología, Hospital Universitario Reina Sofía, IMIBIC, Universidad de Córdoba, Córdoba, Spain., and Te study was co-fnanced by the Euro pean Development Regional Fund 'A way to achieve Europe' and the Operative Program Intelligent Growth 2014‐2020.

Subjects
0301 basic medicine, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Surgical Procedures, Operative::Transplantation::Organ Transplantation::Pancreas Transplantation [Medical Subject Headings], Klebsiella pneumoniae, Epidemiology, España, Riñón, medicine.disease_cause, Kidney, Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans [Medical Subject Headings], Lactamase-producing enterobacteriaceae, Resistencia a múltiples medicamentos, Phenomena and Processes::Microbiological Phenomena::Drug Resistance, Microbial::Drug Resistance, Bacterial::Drug Resistance, Multiple, Bacterial [Medical Subject Headings], 0302 clinical medicine, Enterobacterales, Drug Resistance, Multiple, Bacterial, Prevalence, Colonization, Multidrug-resistant, 030212 general & internal medicine, Prospective Studies, Prospective cohort study, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Epidemiologic Methods::Data Collection::Vital Statistics::Morbidity::Prevalence [Medical Subject Headings], Organisms::Bacteria::Gram-Negative Bacteria::Gram-Negative Facultatively Anaerobic Rods::Enterobacteriaceae [Medical Subject Headings], High-risk clones, Multidisciplinary, Surveillance, biology, Antimicrobials, Bacterial Infections, Hospitals, Citrobacter freundii, Organisms::Bacteria::Gram-Negative Bacteria::Gram-Negative Facultatively Anaerobic Rods::Enterobacteriaceae::Escherichia::Escherichia coli [Medical Subject Headings], Anti-Bacterial Agents, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Diagnosis::Diagnostic Techniques and Procedures::Clinical Laboratory Techniques::Microbiological Techniques::Microbial Sensitivity Tests [Medical Subject Headings], Analytical, Diagnostic and Therapeutic Techniques and Equipment::Surgical Procedures, Operative::Transplantation::Organ Transplantation::Kidney Transplantation [Medical Subject Headings], Medicine, Organisms::Bacteria::Gram-Negative Bacteria::Gram-Negative Facultatively Anaerobic Rods::Enterobacteriaceae::Klebsiella::Klebsiella pneumoniae [Medical Subject Headings], Pancreas Transplantation, Infection, Chemicals and Drugs::Organic Chemicals::Amides::Lactams::beta-Lactams::Carbapenems [Medical Subject Headings], Chemicals and Drugs::Chemical Actions and Uses::Pharmacologic Actions::Therapeutic Uses::Anti-Infective Agents::Anti-Bacterial Agents [Medical Subject Headings], Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Epidemiologic Methods::Epidemiologic Study Characteristics as Topic::Epidemiologic Studies::Cohort Studies::Longitudinal Studies::Prospective Studies [Medical Subject Headings], Analytical, Diagnostic and Therapeutic Techniques and Equipment::Surgical Procedures, Operative::Transplantation::Organ Transplantation::Liver Transplantation [Medical Subject Headings], Science, 030106 microbiology, Microbial Sensitivity Tests, Infections, Microbiology, Article, Diseases::Bacterial Infections and Mycoses::Bacterial Infections [Medical Subject Headings], 03 medical and health sciences, Enterobacteriaceae, Enterobacter cloacae, medicine, Escherichia coli, Infección, Humans, Clinical microbiology, Geographical Locations::Geographic Locations::Europe::Spain [Medical Subject Headings], Transplantation, business.industry, biology.organism_classification, Kidney Transplantation, Transplant Recipients, Liver Transplantation, Multiple drug resistance, Organisms::Bacteria::Gram-Negative Bacteria::Gram-Negative Facultatively Anaerobic Rods::Enterobacteriaceae::Citrobacter::Citrobacter freundii [Medical Subject Headings], Organisms::Bacteria::Gram-Negative Bacteria::Gram-Negative Facultatively Anaerobic Rods::Enterobacteriaceae::Enterobacter::Enterobacter cloacae [Medical Subject Headings], Carbapenems, Spain, Trasplante, business
Abstract

The objective of this study was to analyse the mechanisms of resistance to carbapenems and other extended-spectrum-?-lactams and to determine the genetic relatedness of multidrug-resistant Enterobacterales (MDR-E) causing colonization or infection in solid-organ transplantation (SOT) recipients. Prospective cohort study in kidney (n= 142), liver (n= 98) or kidney/pancreas (n= 7) transplant recipients between 2014 and 2018 in seven Spanish hospitals. We included 531 MDR-E isolates from rectal swabs obtained before transplantation and weekly for 4?6 weeks after the procedure and 10 MDR-E from clinical samples related to an infection. Overall, 46.2% Escherichia coli, 35.3% Klebsiella pneumoniae, 6.5% Enterobacter cloacae, 6.3% Citrobacter freundii and 5.7% other species were isolated. The number of patients with MDR-E colonization post-transplantation (176; 71.3%) was 2.5-fold the number of patients colonized pre-transplantation (71; 28.7%). Extended spectrum ?-lactamases (ESBLs) and carbapenemases were detected in 78.0% and 21.1% of MDR-E isolates respectively. In nine of the 247 (3.6%) transplant patients, the microorganism causing an infection was the same strain previously cultured from surveillance rectal swabs. In our study we have observed a low rate of MDR-E infection in colonized patients 4?6 weeks post-transplantation. E. coli producing blaCTX-M-G1 and K. pneumoniae harbouring blaOXA-48 alone or with blaCTX-M-G1 were the most prevalent MDR-E colonization strains in SOT recipients. Acknowledgements The authors thank Mª Jesús Lecea and Laura Álvarez for technical assistance. Tis research was supported by ‘Plan Nacional de I+D+i and Instituto de Salud Carlos III (Fondo de Investigaciones Sanitarias 13/01191), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, and the Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0007, RD16/0016/0010, RD16/0016/0012, RD16/0016/0011, RD16/0016/0008, RD16/0016/0002). Te study was co-fnanced by the European Development Regional Fund “A way to achieve Europe” and the Operative Program Intelligent Growth 2014‐2020.

Published
2021

5. Hidden dissemination of carbapenem-susceptible OXA-48-producing Proteus mirabilis.

Author

Pedraza, Rosa, Kieffer, Nicolas, Guzmán-Puche, Julia, Artacho, María José, Pitart, Cristina, Hernández-García, Marta, Vila, Jordi, Cantón, Rafael, and Martinez-Martinez, Luis

Subjects
PROTEUS (Bacteria), HYDROLASES, MENTAL health surveys, RESEARCH funding, CARBAPENEMS, MICROBIAL sensitivity tests, ANTIBIOTICS, PHARMACODYNAMICS
Abstract

Objectives: To detect a potential hidden dissemination of the blaOXA-48 gene among Proteus mirabilis isolates obtained from a single centre.Methods: P. mirabilis from diverse clinical samples presenting an ESBL phenotype or obtained from blood cultured from 2017 to 2019 were evaluated. Bacterial identification was performed using MALDI-TOF MS. MICs were determined using International Organization for Standardization (ISO) standard microdilution and interpreted following EUCAST guidelines. WGS was performed using both short- and long-read technologies and assemblies were done using Unicycler. Resistomes were assessed using the ResFinder database. SNPs were detected using the PATRIC bioinformatics platform. Cloning experiments were performed using the pCRII-TOPO cloning kit.Results: Thirty-one out of 108 (28.7%) isolates were positive for blaOXA-48 and blaCTX-M-15. Twenty-nine out of 31 of the isolates were susceptible to temocillin, piperacillin/tazobactam, ertapenem and meropenem, whereas only 2/31 showed a resistance phenotype against these antibiotics. Both blaOXA-48 and blaCTX-M-15 genes were detected within the same chromosomally integrated new transposon in all isolates. The resistant isolates displayed a single mutation located in the putative promoter upstream of blaOXA-48. Cloning experiments confirmed that the mutation was responsible for the resistance phenotype.Conclusions: The presence of a chromosomal copy of blaOXA-48 did not confer resistance to carbapenems, but a single mutation in the promoter could lead to an increase in resistance. This study shows a hidden circulation of OXA-48-positive, but carbapenem- and piperacillin/tazobactam-susceptible, P. mirabilis isolates that can become resistant to β-lactams after a single mutation. [ABSTRACT FROM AUTHOR]

Published
2022
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6. Interplay among Resistance Profiles, High-Risk Clones, and Virulence in the Caenorhabditis elegans Pseudomonas aeruginosa Infection Model

Author

Carla López-Causapé, Antonio Oliver, Rosa del Campo, Xavier Mulet, Antonio Doménech-Sánchez, Gabriel Cabot, Rafael Cantón, Alfonso Navas, Irina Sánchez-Diener, Carmen Peña, Laura Zamorano, Luis Martínez-Martínez, Susana C. Arcos, [Sanchez-Diener, Irina] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Serv Microbiol, Palma De Mallorca, Majorca, Spain, [Zamorano, Laura] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Serv Microbiol, Palma De Mallorca, Majorca, Spain, [Lopez-Causape, Carla] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Serv Microbiol, Palma De Mallorca, Majorca, Spain, [Cabot, Gabriel] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Serv Microbiol, Palma De Mallorca, Majorca, Spain, [Mulet, Xavier] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Serv Microbiol, Palma De Mallorca, Majorca, Spain, [Oliver, Antonio] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Serv Microbiol, Palma De Mallorca, Majorca, Spain, [Sanchez-Diener, Irina] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Unidad Invest, Palma De Mallorca, Majorca, Spain, [Zamorano, Laura] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Unidad Invest, Palma De Mallorca, Majorca, Spain, [Lopez-Causape, Carla] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Unidad Invest, Palma De Mallorca, Majorca, Spain, [Cabot, Gabriel] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Unidad Invest, Palma De Mallorca, Majorca, Spain, [Mulet, Xavier] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Unidad Invest, Palma De Mallorca, Majorca, Spain, [Oliver, Antonio] Hosp Son Espases, Inst Invest Sanitaria Palma IdISPa, Unidad Invest, Palma De Mallorca, Majorca, Spain, [Pena, Carmen] Hosp Virgen Lirios, Serv Med Interna, Alcoy, Spain, [del Campo, Rosa] Hosp Univ Ramon & Cajal, Serv Microbiol, Madrid, Spain, [Canton, Rafael] Hosp Univ Ramon & Cajal, Serv Microbiol, Madrid, Spain, [Martinez-Martinez, Luis] Hosp Univ Ramon & Cajal, Serv Microbiol, Madrid, Spain, [Domenech-Sanchez, Antonio] Inst Ramon & Cajal Invest Sanitaria IRYCIS, Madrid, Spain, [Martinez-Martinez, Luis] Univ Islas Baleares, Area Microbiol, Saniconsult Iber SL, Palma De Mallorca, Majorca, Spain, [Martinez-Martinez, Luis] Univ Islas Baleares, IUNICS, Palma De Mallorca, Majorca, Spain, [Martinez-Martinez, Luis] Hosp Univ Reina Sofia, Unidad Gest Clin Microbiol, Cordoba, Spain, [Martinez-Martinez, Luis] Univ Cordoba, Dept Microbiol, Cordoba, Spain, [Martinez-Martinez, Luis] Inst Maimonides Invest Biomed Cordoba IMIBIC, Cordoba, Spain, [Arcos, Susana C.] Univ Autonoma, Consejo Super Invest, Ctr Nacl Biotecnol, Museo Nacl Ciencias Nat, Madrid, Spain, [Navas, Alfonso] Univ Autonoma, Consejo Super Invest, Ctr Nacl Biotecnol, Museo Nacl Ciencias Nat, Madrid, Spain, Planes Nacionales de I + D + i, Instituto de Salud Carlos III, Subdireccion General de Redes y Centros de Investigacion Cooperativa, Ministerio de Economia y Competitividad, Spanish Network for Research in Infectious Diseases, European Development Regional Fund, P-FIS fellowship from Instituto de Salud Carlos III, Ministerio de Economoia y Competitividad, operative program Intelligent Growth, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), and European Commission

Subjects
0301 basic medicine, Clone (cell biology), Bacteremia, Pathogenesis, medicine.disease_cause, Type three secretion system, In-vitro, Drug Resistance, Multiple, Bacterial, Genotype, Pseudomonas aeruginosa, Caenorhabditis elegans, Virulence, Multidrug resistant, Extensively drug resistant, High-risk clones, Fitness, Type III Secretion Systems, Pharmacology (medical), Blood-stream infections, Caenorhabditis elegans, Cross Infection, extensively drug resistant, Virulence, Drug discovery, Host, Anti-Bacterial Agents, Infectious Diseases, high-risk clones, Pseudomonas aeruginosa, 030106 microbiology, Multidrug-resistance, Microbial Sensitivity Tests, Biology, Microbiology, Epidemiology and Surveillance, 03 medical and health sciences, Antibiotic resistance, Bacterial Proteins, medicine, Cystic-fibrosis, Animals, Humans, Pathogenicity, Pseudomonas Infections, Pharmacology, multidrug resistant, biology.organism_classification, Clone Cells, Multiple drug resistance, virulence, Disease Models, Animal, Antibiotic-resistance
Abstract

The increasing prevalence of nosocomial infections produced by multidrugresistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa is frequently linked to widespread international strains designated high-risk clones. In this work, we attempted to decipher the interplay between resistance profiles, high-risk clones, and virulence, testing a large (n 140) collection of well-characterized P. aeruginosa isolates from different sources (bloodstream infections, nosocomial outbreaks, cystic fibrosis, and the environment) in a Caenorhabditis elegans infection model. Consistent with previous data, we documented a clear inverse correlation between antimicrobial resistance and virulence in the C. elegans model. Indeed, the lowest virulence was linked to XDR profiles, which were typically linked to defined high-risk clones. However, virulence varied broadly depending on the involved highrisk clone; it was high for sequence type 111 (ST111) and ST235 but very low for ST175. The highest virulence of ST235 could be attributed to its exoU type III secretion system (TTSS) genotype, which was found to be linked with higher virulence in our C. elegans model. Other markers, such as motility or pigment production, were not essential for virulence in the C. elegans model but seemed to be related with the higher values of the statistical normalized data. In contrast to ST235, the ST175 high-risk clone, which is widespread in Spain and France, seems to be associated with a particularly low virulence in the C. elegans model. Moreover, the previously described G154R AmpR mutation, prevalent in ST175, was found to contribute to the reduced virulence, although it was not the only factor involved. Altogether, our results provide a major step forward for understanding the interplay between P. aeruginosa resistance profiles, high-risk clones, and virulence., This work was supported by the Planes Nacionales de IDi 2008-2011/2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015/0006, RD12/0015/004, RD16/0016/ 0004, RD16/0016/008, and RD16/0016/0011) and grants PI15/00088 and PI12/00734. This work was cofinanced by grants from the European Development Regional Fund “A way to achieve Europe” and operative program Intelligent Growth (2014-2020). I.S.-D. is the recipient of a P-FIS fellowship from Instituto de Salud Carlos III, Ministerio de Economía y Competitividad.

Published
2017

7. Genomics And Susceptibility Profiles Of Extensively Drug-resistant Pseudomonas Aeruginosa Isolates From Spain

Author

Ester del Barrio-Tofiño, Luisa Sorlí, José Antonio Martínez, Concepción Segura, Fe Tubau, Antonio Oliver, Luis Martínez-Martínez, Natividad Benito, Alba Rivera, Gabriel Cabot, Elena Resino-Foz, Juan Pablo Horcajada, Raquel Durá-Navarro, Laura Zamorano, María Milagro Montero, Cristina Labayru-Echverria, Silvia Gómez-Zorrilla, Marta Fernández-Martínez, Ignacio Ayestarán, Esther Viedma, Carlos Dueñas, Izaskun Alejo-Cancho, Carla López-Causapé, Claudia González-Rico, Nuria Tormo, [del Barrio-Tofino, Ester] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Dept Microbiol, Intens Care Unit, Palma De Mallorca, Spain, [Lopez-Causape, Carla] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Dept Microbiol, Intens Care Unit, Palma De Mallorca, Spain, [Cabot, Gabriel] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Dept Microbiol, Intens Care Unit, Palma De Mallorca, Spain, [Ayestaran, Ignacio] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Dept Microbiol, Intens Care Unit, Palma De Mallorca, Spain, [Zamorano, Laura] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Dept Microbiol, Intens Care Unit, Palma De Mallorca, Spain, [Oliver, Antonio] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Dept Microbiol, Intens Care Unit, Palma De Mallorca, Spain, [del Barrio-Tofino, Ester] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Unidad Invest, Palma De Mallorca, Spain, [Lopez-Causape, Carla] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Unidad Invest, Palma De Mallorca, Spain, [Cabot, Gabriel] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Unidad Invest, Palma De Mallorca, Spain, [Ayestaran, Ignacio] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Unidad Invest, Palma De Mallorca, Spain, [Zamorano, Laura] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Unidad Invest, Palma De Mallorca, Spain, [Oliver, Antonio] Hosp Univ Son Espases, Inst Invest Illes Balears IdISBa, Unidad Invest, Palma De Mallorca, Spain, [Rivera, Alba] Hosp Santa Creu & Sant Pau, Dept Microbiol & Infect Dis, Barcelona, Spain, [Benito, Natividad] Hosp Santa Creu & Sant Pau, Dept Microbiol & Infect Dis, Barcelona, Spain, [Segura, Concepcion] Hosp del Mar, Lab Referencia Catalunya, Infect Pathol & Antimicrobial Res Grp IPAR, Inst Hosp del Mar Invest Med IMIM, Barcelona, Spain, [Milagro Montero, Maria] Hosp del Mar, Lab Referencia Catalunya, Infect Pathol & Antimicrobial Res Grp IPAR, Inst Hosp del Mar Invest Med IMIM, Barcelona, Spain, [Sorli, Luisa] Hosp del Mar, Lab Referencia Catalunya, Infect Pathol & Antimicrobial Res Grp IPAR, Inst Hosp del Mar Invest Med IMIM, Barcelona, Spain, [Pablo Horcajada, Juan] Hosp del Mar, Lab Referencia Catalunya, Infect Pathol & Antimicrobial Res Grp IPAR, Inst Hosp del Mar Invest Med IMIM, Barcelona, Spain, [Segura, Concepcion] Hosp del Mar, Dept Infect Dis, Infect Pathol & Antimicrobial Res Grp IPAR, Inst Hosp del Mar Invest Med IMIM, Barcelona, Spain, [Milagro Montero, Maria] Hosp del Mar, Dept Infect Dis, Infect Pathol & Antimicrobial Res Grp IPAR, Inst Hosp del Mar Invest Med IMIM, Barcelona, Spain, [Sorli, Luisa] Hosp del Mar, Dept Infect Dis, Infect Pathol & Antimicrobial Res Grp IPAR, Inst Hosp del Mar Invest Med IMIM, Barcelona, Spain, [Pablo Horcajada, Juan] Hosp del Mar, Dept Infect Dis, Infect Pathol & Antimicrobial Res Grp IPAR, Inst Hosp del Mar Invest Med IMIM, Barcelona, Spain, [Tubau, Fe] Hosp Univ Bellvitge, Dept Microbiol & Infect Dis, Barcelona, Spain, [Gomez-Zorrilla, Silvia] Hosp Univ Bellvitge, Dept Microbiol & Infect Dis, Barcelona, Spain, [Tormo, Nuria] Consorcio Hosp Gen Univ Valencia, Dept Microbiol & Infect Dis, Valencia, Spain, [Dura-Navarro, Raquel] Consorcio Hosp Gen Univ Valencia, Dept Microbiol & Infect Dis, Valencia, Spain, [Viedma, Esther] Hosp Univ 12 Octubre, Dept Microbiol & Infect Dis, Madrid, Spain, [Resino-Foz, Elena] Hosp Univ 12 Octubre, Dept Microbiol & Infect Dis, Madrid, Spain, [Fernandez-Martinez, Marta] Hosp Univ Marques de Valdecilla, Inst Invest Valdecilla IDIVAL, Dept Microbiol & Infect Dis, Santander, Spain, [Gonzalez-Rico, Claudia] Hosp Univ Marques de Valdecilla, Inst Invest Valdecilla IDIVAL, Dept Microbiol & Infect Dis, Santander, Spain, [Alejo-Cancho, Izaskun] Hosp Univ Clin, Dept Microbiol & Infect Dis, Barcelona, Spain, [Antonio Martinez, Jose] Hosp Univ Clin, Dept Microbiol & Infect Dis, Barcelona, Spain, [Labayru-Echverria, Cristina] Hosp Univ Burgos, Dept Microbiol & Infect Dis, Burgos, Spain, [Duenas, Carlos] Hosp Univ Burgos, Dept Microbiol & Infect Dis, Burgos, Spain, [Martinez-Martinez, Luis] Univ Cordoba, Hosp Univ Reina Sofia, Dept Microbiol, Unit Microbiol, Cordoba, Spain, [Martinez-Martinez, Luis] Inst Maimonides Invest Biomed Cordoba IMIBIC, Cordoba, Spain, Plan Nacional de I+D+i, Instituto de Salud Carlos III, Subdireccion General de Redes y Centros de Investigacion Cooperativa, Ministerio de Economia, Industria y Competitividad, Spanish Network for Research in Infectious Diseases, and European Development Regional Fund (ERDF)

Subjects
0301 basic medicine, Imipenem, Ceftazidime, Penicillanic Acid, Aztreonam, Drug resistance, medicine.disease_cause, Antimicrobial resistance, chemistry.chemical_compound, polycyclic compounds, Pharmacology (medical), Aminoglycoside resistance, Molecular Epidemiology, extensively drug resistant, Beta-lactam resistance, Ceftolozane-tazobactam, Broth microdilution, 3. Good health, Anti-Bacterial Agents, Infectious Diseases, high-risk clones, Amikacin, whole-genome sequencing, Molecular epidemiology, Pseudomonas aeruginosa, Erratum, Convergent evolution, Mutations, medicine.drug, Fluoroquinolones, Field gel-electrophoresis, Tazobactam, Carbapenem resistance, Cxa-101 fr264205, Epidemiologia molecular, 030106 microbiology, Microbial Sensitivity Tests, Biology, beta-Lactam Resistance, beta-Lactamases, Microbiology, 03 medical and health sciences, Polymyxin resistance, Bacterial Proteins, Pseudomonas, Drug Resistance, Bacterial, medicine, Humans, Pseudomonas Infections, Polymyxins, Pharmacology, biochemical phenomena, metabolism, and nutrition, bacterial infections and mycoses, Cephalosporins, Aminoglycosides, chemistry, Spain
Abstract

This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) Pseudomonas aeruginosa clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (>95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis–multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% ( n = 101) of the XDR isolates, distantly followed by ST244 ( n = 16), ST253 ( n = 12), ST235 ( n = 8), and ST111 ( n = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital ( n = 44) were fully sequenced on an Illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the β-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of ampC mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in β-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance.

Published
2017

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