Programas de Vigilancia Microbiológica. Centro Nacional de Microbiología

Este libro recoge en pequeñas “cápsulas” de unas pocas páginas la información condensada de los Programas de Vigilancia Microbiológica del CNM en los últimos años. Una de las principales actividades del Centro Nacional de Microbiología (CNM) se fundamenta en dar soporte al Sistema Nacional de Salud en el control y prevención de las enfermedades infecciosas, incluyendo la detección precoz de infecciones emergentes o infrecuentes, de nuevas variantes problemáticas de agentes infecciosos más comunes, y la caracterización de brotes. Todo ello forma parte de la vigilancia microbiológica, que en el CNM se estructura en torno a los Programas de Vigilancia que abordan la mayoría de los principales microorganismos patógenos para el ser humano.Programa de Enfermedad Meningocócica Invasiva. Programa de Listeriosis. Programa de Infección Gonocócica. Sensibilidad Antimicrobiana, 1991-2020. Programa de Streptococcus pneumoniae. Programa de enfermedad invasiva por Streptococcus pyogenes. Programa de Haemophilus influenzae. Programa de Resistencia a Antibióticos. Programa de infecciones producidas por los estafilococos. Programa de infecciones entéricas bacterianas transmitidas por agua y alimentos. Programa de Legionelosis. Programa de infecciones causadas por especies toxigénicas del género Corynebacterium. Programa de resistencias en el complejo tuberculoso. Programa de micobacterias no tuberculosas. Programa de GRIPE. Programa de SARS-CoV-2. Programa de Enterovirus y Parálisis Flácida en menores de 15 años. Programa de Parotiditis. Programa de Sarampión y Rubeola. Enfermedades víricas transmitidas por vectores. Programa de Rabia. Programa de Variantes del Virus de la Hepatitis B de Impacto en Salud Pública y Estudio de Brotes de Hepatitis. Programa de Leishmaniasis Humana en el Area-9 de la Comunidad Autónoma de Madrid. Programa de la Enfermedad de Chagas. Programa de la Resistencia a los antifúngicos en España.N

Mutation Analysis in Regulator DNA-Binding Regions for Antimicrobial Efflux Pumps in 17,000 Pseudomonas aeruginosa Genomes

Mutations leading to upregulation of efflux pumps can produce multiple drug resistance in the pathogen Pseudomonas aeruginosa. Changes in their DNA binding regions, i.e., palindromic operators, can compromise pump depression and subsequently enhance resistance against several antibacterials and biocides. Here, we have identified (pseudo)palindromic repeats close to promoters of genes encoding 13 core drug-efflux pumps of P. aeruginosa. This framework was applied to detect mutations in these repeats in 17,292 genomes. Eighty-nine percent of isolates carried at least one mutation. Eight binary genetic properties potentially related to expression were calculated for mutations. These included palindromicity reduction, mutation type, positioning within the repeat and DNA-bending shift. High-risk ST298, ST308 and ST357 clones commonly carried four conserved mutations while ST175 and the cystic fibrosis-linked ST649 clones showed none. Remarkably, a T-to-C transition in the fourth position of the upstream repeat for mexEF-oprN was nearly exclusive of the high-risk ST111 clone. Other mutations were associated with high-risk sublineages using sample geotemporal metadata. Moreover, 1.5% of isolates carried five or more mutations suggesting they undergo an alternative program for regulation of their effluxome. Overall, P. aeruginosa shows a wide range of operator mutations with a potential effect on efflux pump expression and antibiotic resistance.This research was funded by Acción Estratégica en Salud from the ISCIII, grant MPY 509/19. This research was also supported by Personalized and Precision Medicine grant from the Instituto de Salud Carlos III (MePRAM Project, PMP22/00092), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU.S

Molecular studies of phages-Klebsiella pneumoniae in mucoid environment: innovative use of mucolytic agents prior to the administration of lytic phages

Mucins are important glycoproteins that form a protective layer throughout the gastrointestinal and respiratory tracts. There is scientific evidence of increase in phage-resistance in the presence of mucin for some bacterial pathogens. Manipulation in mucin composition may ultimately influence the effectiveness of phage therapy. In this work, two clinical strains of K. pneumoniae (K3574 and K3325), were exposed to the lytic bacteriophage vB_KpnS-VAC35 in the presence and absence of mucin on a long-term co-evolution assay, in an attempt to mimic in vitro the exposure to mucins that bacteria and their phages face in vivo. Enumerations of the bacterial and phage counts at regular time intervals were conducted, and extraction of the genomic DNA of co-evolved bacteria to the phage, the mucin and both was performed. We determined the frequency of phage-resistant mutants in the presence and absence of mucin and including a mucolytic agent (N-acetyl L-cysteine, NAC), and sequenced them using Nanopore. We phenotypically demonstrated that the presence of mucin induces the emergence of bacterial resistance against lytic phages, effectively decreased in the presence of NAC. In addition, the genomic analysis revealed some of the genes relevant to the development of phage resistance in long-term co-evolution, with a special focus on the mucoid environment. Genes involved in the metabolism of carbohydrates were mutated in the presence of mucin. In conclusion, the use of mucolytic agents prior to the administration of lytic phages could be an interesting therapeutic option when addressing K. pneumoniae infections in environments where mucin is overproduced.The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study has been funded by Instituto de Salud Carlos III (ISCIII) through the projects PI19/00878 and PI22/00323 and co-funded by the European Union, and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC). (SEIMC, http://www.seimc.org/). This research was also supported by CIBERINFEC (CIBER21/13/00095) and by Personalized and precision medicine grant from the Instituto de Salud Carlos III (MePRAM Project, PMP22/00092). MT was financially supported by the Miguel Servet Research Programme (SERGAS and ISCIII). OP, LF-G, and ML were financially supported by the grants IN606A-2020/035, IN606B-2021/013, and IN606C-2022/002, respectively (GAIN, Xunta de Galicia). IB was financially supported by the pFIS program (ISCIII, FI20/00302). Finally, to thank to PIRASOA laboratory which is the reference laboratory for molecular typing of nosocomial pathogens and detection of mechanisms of resistance to antimicrobials of health interest in Andalusia, Virgen Macarena Hospital, Seville, to send us the clinical isolates. Thanks to Alvaro Pascual and Luis Martínez-Martínez from Virgen Macarena Hospital, Seville and Reina Sofia Hospital, Cordoba.S

Vigilancia molecular de Klebsiella pneumoniae, Enterobacter cloacae complex y Escherichia coli productores de carbapene¬masas en España. Informe anual RedLabRA 2022

Incluye PDF y Epub del documentoEn el seno del Plan Nacional frente a la Resistencia a los Antibióticos (PRAN) y en el marco de la vigilancia de las IRAS y las resistencias en la Red Nacional de Vigilancia Epidemiológica (RENAVE), se reconoció la necesidad de implementar una red nacional de laboratorios para la vigilancia de microorganismos resistentes, con capacidad para abordar la caracterización molecular de los patógenos responsables de los principales problemas de resistencia a antimicrobianos. Un grupo de trabajo multidisciplinar del PRAN, formado por más de 50 profesionales, elaboró el documento “Red de laboratorios para la vigilancia de los microorganismos resistentes” (1) que se aprobó por la Comisión de Salud Pública y el Consejo Interterritorial en noviembre de 2018. Las recomendaciones plasmadas en este documento abogan por la integración de la secuenciación genómica en la vigilancia de los patógenos multirresistentes, alineándose con el Marco Estratégico que ha desarrollado el ECDC sobre este tema. El documento recoge como objetivos específicos de la Red: - Obtener un diagnóstico microbiológico completo y de calidad en todos los casos de infección y/o colonización por microorganismos resistentes objeto de vigilancia. - Asegurar que la información microbiológica se incluye en la notificación de casos de acuerdo a los protocolos de la Red Nacional de Vigilancia Epidemiológica. - Estandarizar los procedimientos de detección y caracterización de los mecanismos de resistencia. - Establecer los mecanismos de intercambio de información entre los laboratorios de la red según las prioridades que se establezcan.N

Informe anual RedLabRA 2021

Incluye PDF y Epub del documentoEn el seno del Plan Nacional frente a la Resistencia a los Antibióticos (PRAN) y en el marco de la vigilancia de las IRAS y las resistencias en la Red Nacional de Vigilancia Epidemiológica (RENAVE), se reconoció la necesidad de implementar una red nacional de laboratorios para la vigilancia de microorganismos resistentes, con capacidad para abordar la caracterización molecular de los patógenos responsables de los principales problemas de resistencia a antimicrobianos. Un grupo de trabajo multidisciplinar del PRAN, formado por más de 50 profesionales, elaboró el documento “Red de laboratorios para la vigilancia de los microorganismos resistentes” (1) que se aprobó por la Comisión de Salud Pública y el Consejo Interterritorial en noviembre de 2018. Las recomendaciones plasmadas en este documento abogan por la integración de la secuenciación genómica en la vigilancia de los patógenos multirresistentes, alineándose con el Marco Estratégico que ha desarrollado el European Centre for Disease Prevention and Control (ECDC) sobre este tema.N

Proteomic Study of the Interactions between Phages and the Bacterial Host Klebsiella pneumoniae

Phages and bacteria have acquired resistance mechanisms for protection. In this context, the aims of the present study were to analyze the proteins isolated from 21 novel lytic phages of Klebsiella pneumoniae in search of defense mechanisms against bacteria and also to determine the infective capacity of the phages. A proteomic study was also conducted to investigate the defense mechanisms of two clinical isolates of K. pneumoniae infected by phages. For this purpose, the 21 lytic phages were sequenced and de novo assembled. The host range was determined in a collection of 47 clinical isolates of K. pneumoniae, revealing the variable infective capacity of the phages. Genome sequencing showed that all of the phages were lytic phages belonging to the order Caudovirales. Phage sequence analysis revealed that the proteins were organized in functional modules within the genome. Although most of the proteins have unknown functions, multiple proteins were associated with defense mechanisms against bacteria, including the restriction-modification system, the toxin-antitoxin system, evasion of DNA degradation, blocking of host restriction and modification, the orphan CRISPR-Cas system, and the anti-CRISPR system. Proteomic study of the phage-host interactions (i.e., between isolates K3574 and K3320, which have intact CRISPR-Cas systems, and phages vB_KpnS-VAC35 and vB_KpnM-VAC36, respectively) revealed the presence of several defense mechanisms against phage infection (prophage, defense/virulence/resistance, oxidative stress and plasmid proteins) in the bacteria, and of the Acr candidate (anti-CRISPR protein) in the phages. IMPORTANCE Researchers, including microbiologists and infectious disease specialists, require more knowledge about the interactions between phages and their bacterial hosts and about their defense mechanisms. In this study, we analyzed the molecular mechanisms of viral and bacterial defense in phages infecting clinical isolates of K. pneumoniae. Viral defense mechanisms included restriction-modification system evasion, the toxin-antitoxin (TA) system, DNA degradation evasion, blocking of host restriction and modification, and resistance to the abortive infection system, anti-CRISPR and CRISPR-Cas systems. Regarding bacterial defense mechanisms, proteomic analysis revealed expression of proteins involved in the prophage (FtsH protease modulator), plasmid (cupin phosphomannose isomerase protein), defense/virulence/resistance (porins, efflux pumps, lipopolysaccharide, pilus elements, quorum network proteins, TA systems, and methyltransferases), oxidative stress mechanisms, and Acr candidates (anti-CRISPR protein). The findings reveal some important molecular mechanisms involved in the phage-host bacterial interactions; however, further study in this field is required to improve the efficacy of phage therapy.This study was funded by grant PI19/00878 and PI22/00323 awarded to M.T. within the State Plan for R1D1I 2013-2016 (National Plan for Scientific Research, Technological Development, and Innovation 2008-2011) and cofinanced by the ISCIII-Deputy General Directorate for Evaluation and Promotion of Research/European Regional Development Fund “A Way of Making Europe” and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI; RD16/0016/0006 and RD16/0016/0008), CIBERINFEC (CIBER21/13/00012, CB21/13/00049, CIBER21/13/00084, and CIBER21/13/00095), and Personalized Medicine Project (MePRAM; PMP/00092) and also by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC; http://www.seimc.org/). M.T. was financially supported by the Miguel Servet Research Program (SERGAS and ISCIII). I.B. was financially supported by pFIS program (ISCIII, FI20/00302). O.P., L.F.-G., and M.L. were financially supported by grants IN606A-2020/035, IN606B-2021/013, and IN606C-2022/002, respectively (GAIN; Xunta de Galicia). The authors acknowledge CESGA (www.cesga.es) in Santiago de Compostela, Spain, for providing access to computing facilities and the RIAIDT-USC analytical facilities. Finally, We thank researchers from the Spanish Network of Bacteriophages and Transducer Elements (FAGOMA) for contributing the lytic phages. I.B., L.B., O.P., and L.F.-G. developed the experiments, analyzed the results, and wrote the original manuscript. M.L., C.O.C. and A.B.P. helped to prepare the visual presentation of the results. F.F.C., Á.P., L.M.-M., and J.O.-I. rewrote the manuscript. M.T. financed and directed the experiments and supervised the writing of the originalmanuscript. We declare that there are no conflicts of interest.S

Fecal carriage of extended-spectrum beta-lactamase-producing Enterobacterales in healthy Spanish schoolchildren

Background: Extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) are a serious threat among emerging antibiotic resistant bacteria. Particularly, the number of cases of ESBL-E infections reported in children has been increasing in recent years, and approved antibiotic treatments for this age group are limited. However, information regarding the prevalence of colonization in European children, risk factors associated with colonization, and the characteristics of the colonizing strains is scarce. The aims of this study were to determine the prevalence of ESBL-E colonization in fecal samples of apparently healthy schoolchildren, to identify lifestyle routines associated with colonization, and to characterize clonal relationships and mechanisms of resistance in ESBL-E isolates. Methods: A cohort of 887 healthy children (3-13 years old) from seven primary and secondary schools in the Madrid metropolitan area was recruited between April-June 2018, and sociodemographic information and daily habits were collected. Fecal samples were screened for ESBL-E carriage in selective medium. ESBL-E isolates were further characterized by assessing molecular epidemiology (PFGE and MLST), ESBL gene carriage, and antibiotic resistance profile. This information was analyzed in conjunction with the metadata of the participants in order to identify external factors associated with ESBL-E carriage. Results: Twenty four ESBL-E, all but one Escherichia coli, were detected in 23 children (prevalence: 2.6%; 95% CI: 1.6-3.6%). Of these, seven contained the blaCTX-M-14 allele, five the blaCTX-M-15, five the blaSHV-12, three the blaCTX-M-27, three the blaCTX-M-32, and one the blaCTX-M-9. Significant clonal diversity was observed among the isolates that grouped into 22 distinct clusters (at <85% similarity of PFGE profile). ESBL-producing E. coli isolates belonged to 12 different STs, with ST10 (25%) and ST131 (17%) being the most frequent. Apart from ß-lactams, resistance to trimethoprim/sulfamethoxazole (46%), ciprofloxacin (33%), levofloxacin (33%), tobramycin (21%), and gentamicin (8%) were the most frequently detected. Conclusion: The prevalence of ESBL-E in the studied cohort of children was lower than the average colonization rate previously detected in Europe for both children and adults. E. coli was the main ESBL-producing species detected and CTX-M were the most frequently identified ESBLs. High ST diversity suggests polyclonal dissemination. Compared to other STs, ST131 isolates were associated with resistance to various antimicrobials.ML-S was supported by the Sara Borrell Program of the Instituto de Salud Carlos III (ISCIII) (CD17CIII/00017). ZM was supported by the Río Hortega Program of the ISCIII. AÁ was supported by the Garantía Juvenil Program of the Comunidad Autónoma de Madrid. SS was supported by the Miguel Servet program of ISCIII (CPII18CIII/00005). This study was funded by the ISCIII, Ministry of Economy and Competitiveness (Spain), under projects PI16CIII/00024, PI18CIII/00030, MPY380/18, and MPY516/19.S

Pseudomonas aeruginosa antibiotic susceptibility profiles, genomic epidemiology and resistance mechanisms: a nation-wide five-year time lapse analysis

Background: Pseudomonas aeruginosa healthcare-associated infections are one of the top antimicrobial resistance threats world-wide. In order to analyze the current trends, we performed a Spanish nation-wide high-resolution analysis of the susceptibility profiles, the genomic epidemiology and the resistome of P. aeruginosa over a five-year time lapse. Methods: A total of 3.180 nonduplicated P. aeruginosa clinical isolates from two Spanish nation-wide surveys performed in October 2017 and 2022 were analyzed. MICs of 13 antipseudomonals were determined by ISO-EUCAST. Multidrug resistance (MDR)/extensively drug resistance (XDR)/difficult to treat resistance (DTR)/pandrug resistance (PDR) profiles were defined following established criteria. All XDR/DTR isolates were subjected to whole genome sequencing (WGS). Findings: A decrease in resistance to all tested antibiotics, including older and newer antimicrobials, was observed in 2022 vs 2017. Likewise, a major reduction of XDR (15.2% vs 5.9%) and DTR (4.2 vs 2.1%) profiles was evidenced, and even more patent among ICU isolates [XDR (26.0% vs 6.0%) and DTR (8.9% vs 2.6%)] (p < 0.001). The prevalence of Extended-spectrum β-lactamase/carbapenemase production was slightly lower in 2022 (2.1%. vs 3.1%, p = 0.064). However, there was a significant increase in the proportion of carbapenemase production among carbapenem-resistant strains (29.4% vs 18.1%, p = 0.0246). While ST175 was still the most frequent clone among XDR, a slight reduction in its prevalence was noted (35.9% vs 45.5%, p = 0.106) as opposed to ST235 which increased significantly (24.3% vs 12.3%, p = 0.0062). Interpretation: While the generalized decrease in P. aeruginosa resistance, linked to a major reduction in the prevalence of XDR strains, is encouraging, the negative counterpart is the increase in the proportion of XDR strains producing carbapenemases, associated to the significant advance of the concerning world-wide disseminated hypervirulent high-risk clone ST235. Continued high-resolution surveillance, integrating phenotypic and genomic data, is necessary for understanding resistance trends and analyzing the impact of national plans on antimicrobial resistance.This work was supported by MSD and by the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea —NextGenerationEU through grants PI21/00017 and Personalized and precision medicine grant (MePRAM Project, PMP22/00092).S

Phenotypic and molecular characterization of IMP-producing Enterobacterales in Spain: Predominance of IMP-8 in Klebsiella pneumoniae and IMP-22 in Enterobacter roggenkampii

Objectives: Little is known about IMP-producing Enterobacterales (IMP-Ent) in Europe. We analyzed at genomic and phenotypic level IMP-Ent isolates circulating in Spain in a 9-year period. Materials and methods: IMP-Ent isolates submitted to our reference laboratory were included. Antibiotic susceptibility was performed using microdilution method (EUCAST), and IMP-carbapenemase activity was measured with carbapenemase inhibitors, the β-CARBA method, the modified Hodge test (MHT), and the modified carbapenemase inhibition method (mCIM). All isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: Fifty IMP-Ent isolates, collected from 19 hospitals in 13 Spanish provinces, were detected: Klebsiella pneumoniae (IMP-Kpn) (24; 48%), Enterobacter roggenkampii (13; 26%), Enterobacter hormaechei (8, 16%), Klebsiella oxytoca (two; 4%), Enterobacter asburiae (one, 2%), Serratia marcescens (one; 2%) and Escherichia coli (one; 2%). All isolates were positive by the MHT and β-CARBA tests; 48 (96%) were mCIM positive; 12 (24%) and 26 (52%) displayed positive inhibition with dipicolinic (meropenem) and EDTA (ertapenem), respectively. Five IMP-carbapenemase types were identified: IMP-8 (22; 44%), IMP-22 (17; 34%), IMP-13 (7; 14%), IMP-28 (two; 4%), and IMP-15 (two; 4%), predominating IMP-8 in K. pneumoniae and IMP-22 in E. roggenkampii. IMP-28 was exclusively identified in K. oxytoca and IMP-15 in E. hormaechei. Predominant STs were ST405 (29.2%), ST15 (25%) and ST464 (20.8%) in IMP-Kpn; ST96 (100%) in E. roggenkampii and ST182 (62.5%) in E. hormachei. Colistin and amikacin were the most active non-carbapenem antibiotics against IMP-Ent. Conclusion: IMP-Ent isolates remain infrequent in Spain, although in recent years have been circulating causing nosocomial outbreaks, being IMP-8-producing K. pneumoniae and IMP-22-producing E. roggenkampii the most frequently detected in this study. Inhibition with EDTA or dipicolinic acid presented false negative results in some IMP-producing strains. Active microbiological and molecular surveillance is essential for a better comprehension and control of IMP-Ent dissemination.This research was supported by grants from the Instituto de Salud Carlos III (numbers PI18CIII/00030 and PI21CIII/00039). This research was also supported by CIBER-Consorcio Centro de Investigación Biomédica en Red (CB21/13/00095 and CB21/13/000968), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea – NextGenerationEU. This work was supported by Plan Nacional de I + D + i 2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16CIII/0004/0002 and REIPI RD16/0016/0007) and co-financed by the European Development Regional Fund (EDRF), “A way to achieve Europe,” Operative program Intelligent Growth, 2014–2020.S