Social Behavior of Antibiotic Resistant Mutants Within Pseudomonas aeruginosa Biofilm Communities

The complex spatial structure and the heterogeneity within biofilms lead to the emergence of specific social behaviors. However, the impact of resistant mutants within bacterial communities is still mostly unknown. Thus, we determined whether antibiotic resistant mutants display selfish or altruistic behaviors in mixed Pseudomonas aeruginosa biofilms exposed to antibiotics. ECFP-tagged P. aeruginosa strain PAO1 and its EYFP-tagged derivatives hyperproducing the β-lactamase AmpC or the efflux pump MexAB-OprM were used to develop single or mixed biofilms. Mature biofilms were challenged with different concentrations of β-lactams to monitor biofilm structural dynamics, using confocal laser scanning microscopy (CLSM), and population dynamics, through enumeration of viable cells. While exposure of single wild-type PAO1 biofilms to β-lactams lead to a major reduction in bacterial load, it had little effect on biofilms formed by the resistant mutants. However, the most reveling finding was that bacterial load of wild-type PAO1 was significantly increased when growing in mixed biofilms compared to single biofilms. In agreement with CFU enumeration data, CLSM images revealed the amplification of the resistant mutants and their protection of susceptible populations. These findings show that mutants expressing diverse resistance mechanisms, including β-lactamases, but also, as evidenced for the first time, efflux pumps, protect the whole biofilm community, preserving susceptible populations from the effect of antibiotics. Thus, these results are a step forward to understanding antibiotic resistance dynamics in biofilms, as well as the population biology of bacterial pathogens in chronic infections, where the coexistence of susceptible and resistant variants is a hallmark

The imperative for quality control programs in Monkeypox virus DNA testing by PCR: CIBERINFEC quality control

Monkeypox; PCR; Quality controlVerola del mico; PCR; Control de qualitatViruela del mono; PCR; Control de calidadTo evaluate molecular assays for Mpox diagnosis available in various clinical microbiology services in Spain through a quality control (QC) approach. A total of 14 centers from across Spain participated in the study. The Reference Laboratory dispatched eight serum samples and eight nucleic acid extracts to each participating center. Some samples were spiked with Mpox or Vaccinia virus to mimic positive samples for Mpox or other orthopox viruses. Participating centers provided information on the results obtained, as well as the laboratory methods used. Among the 14 participating centers seven different commercial assays were employed, with the most commonly used kit being LightMix Modular Orthopox/Monkeypox (Mpox) Virus (Roche®). Of the 12 centers conducting Mpox determinations, concordance ranged from 62.5% (n = 1) to 100% (n = 11) for eluates and from 75.0% (n = 1) to 100% (n = 10) for serum. Among the 10 centers performing Orthopoxvirus determinations, a 100% concordance was observed for eluates, while for serum, concordance ranged from 87.5% (n = 6) to 100% (n = 4). Repeatedly, 6 different centers reported a false negative in serum samples for Orthopoxvirus diagnosis, particularly in a sample with borderline Ct = 39. Conversely, one center, using the TaqMan™ Mpox Virus Microbe Detection Assay (Thermo Fisher), reported false positives in Mpox diagnosis for samples spiked with vaccinia virus due to cross-reactions. We observed a positive correlation of various diagnostic assays for Mpox used by the participating centers with the reference values. Our results highlight the significance of standardization, validation, and ongoing QC in the microbiological diagnosis of infectious diseases, which might be particularly relevant for emerging viruses.This research was supported by CIBER (Strategic Action for Monkeypox)—Consorcio Centro de Investigación Biomédica en Red—(CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU. A. d. S. is supported by ‘Instituto de Salud Carlos III’ (grant number JR22/00055)

The imperative for quality control programs in Monkeypox virus DNA testing by PCR: CIBERINFEC quality control

© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.To evaluate molecular assays for Mpox diagnosis available in various clinical microbiology services in Spain through a quality control (QC) approach. A total of 14 centers from across Spain participated in the study. The Reference Laboratory dispatched eight serum samples and eight nucleic acid extracts to each participating center. Some samples were spiked with Mpox or Vaccinia virus to mimic positive samples for Mpox or other orthopox viruses. Participating centers provided information on the results obtained, as well as the laboratory methods used. Among the 14 participating centers seven different commercial assays were employed, with the most commonly used kit being LightMix Modular Orthopox/Monkeypox (Mpox) Virus (Roche®). Of the 12 centers conducting Mpox determinations, concordance ranged from 62.5% (n = 1) to 100% (n = 11) for eluates and from 75.0% (n = 1) to 100% (n = 10) for serum. Among the 10 centers performing Orthopoxvirus determinations, a 100% concordance was observed for eluates, while for serum, concordance ranged from 87.5% (n = 6) to 100% (n = 4). Repeatedly, 6 different centers reported a false negative in serum samples for Orthopoxvirus diagnosis, particularly in a sample with borderline Ct = 39. Conversely, one center, using the TaqMan™ Mpox Virus Microbe Detection Assay (Thermo Fisher), reported false positives in Mpox diagnosis for samples spiked with vaccinia virus due to cross-reactions. We observed a positive correlation of various diagnostic assays for Mpox used by the participating centers with the reference values. Our results highlight the significance of standardization, validation, and ongoing QC in the microbiological diagnosis of infectious diseases, which might be particularly relevant for emerging viruses.This research was supported by CIBER (Strategic Action for Monkeypox)—Consorcio Centro de Investigación Biomédica en Red—(CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU. A. d. S. is supported by ‘Instituto de Salud Carlos III’ (grant number JR22/00055).Peer reviewe

Novel treatment strategies for chronic respiratory infections based on the analysis of antimicrobial resistance dynamics in Pseudomonas aeruginosa biofilms

[eng] Pseudomonas aeruginosa is the major cause of chronic respiratory infections (CRI) and the main driver of morbidity and mortality in cystic fibrosis (CF) patients. The establishment of P. aeruginosa CRI requires a complex adaptive process that includes the selection of an important number of mutations required for long-term persistence and the transition from the planktonic to biofilm mode of growth, a hallmark of chronic infections. The complex spatial structure of the biofilm and the antibiotic selective pressure lead to evolve of heterogeneous communities conformed by different subpopulations that coexist and interact establishing diverse social behaviors. Moreover, the prolonged antibiotic treatments required in CRI unavoidably entail an increase of resistance. Traditional therapeutic strategies to control P. aeruginosa infection in CF patients based on the use of a single nebulized antibiotic seem to be ineffective over time. First, this work determined whether antibiotic resistant mutants display selfish or altruistic behaviors in mixed P. aeruginosa biofilms exposed to antibiotics. ECFP-tagged strain PAO1 and its EYFP-tagged derivatives hyperproducing the β-lactamase AmpC or the efflux pump MexAB-OprM were used to develop single or mixed biofilms. Mature biofilms were challenged with different concentrations of β-lactams to monitor biofilm structural dynamics, using confocal laser scanning microscopy (CLSM), and population dynamics, through enumeration of viable cells. While exposure of single wild-type PAO1 biofilms to β-lactams lead to a major reduction in bacterial load, it had little effect on biofilms formed by the resistant mutants. However, the most revelling finding was that bacterial load of wild-type PAO1 was significantly increased when growing in mixed biofilms compared to single biofilms. In agreement with CFU enumeration data, CLSM images revealed the amplification of the resistant mutants and their protection of susceptible populations. These findings show that mutants expressing diverse resistance mechanisms, including β-lactamases, but also, as evidenced for the first time, efflux pumps, protect the whole biofilm community, preserving susceptible populations from the effect of antibiotics. Finally, this work also evaluated the therapeutic efficacy and dynamics of antibiotic resistance in P. aeruginosa biofilms under sequential therapy with inhaled aztreonam (ATM) and tobramycin (TOB). Biofilms of laboratory and clinical strains were developed using the flow cell system. Mature biofilms were challenged with different concentrations of ATM and TOB and their alternations at different time point. The number of viable cells and resistant mutants were determined, and biofilm structural dynamics were monitored by CLSM. TOB monotherapy produced an intense decrease of CFUs that was not always correlated with a reduction of biomass and/or bactericidal effect on biofilms, particularly for the CF strains. ATM monotherapy bactericidal effect was lower, but effects on biofilm biomass and/or structure, including intense filamentation, were documented. The alternation of TOB and ATM led to an enhancement of the antibiofilm activity against laboratory and CF strains compared to individual regimens, potentiating the bactericidal effect and/or the reduction of biomass. These results support the clinical evaluation of sequential regimens with inhaled antibiotics in CF, as opposed to current maintenance treatments with just one antibiotic in monotherapy. On the whole, this work provides new insights into the complexity of resistance development in biofilms, highlighting the significance of resistant mutants within heterogenous populations, and also represent a step forward for the development of new strategies for combating P. aeruginosa CRI.[spa] Pseudomonas aeruginosa es la principal causa de infecciones respiratorias crónicas (IRC) y el principal responsable de la elevada morbilidad y mortalidad de los pacientes con fibrosis quística (FQ). La instauración de la IRC requiere un complejo proceso adaptativo que incluye la selección de un número importante de mutaciones, necesarias para la persistencia a largo plazo y conlleva la transición desde el modo de crecimiento planctónico al de biopelícula, un sello distintivo de las infecciones crónicas. La compleja estructura espacial de las biopelículas junto con la presión selectiva de antibióticos, da lugar a la evolución de comunidades heterogéneas conformadas por diferentes subpoblaciones que coexisten e interactúan estableciendo diversos comportamientos sociales. Además, lo prolongados tratamientos antibióticos empleados en la IRC conllevan, inevitablemente, un aumento de la resistencia. Las estrategias terapéuticas tradicionales para controlar la infección por P. aeruginosa en pacientes con FQ están basadas en el uso de un único antibiótico nebulizado y parecen ser ineficaces con el paso del tiempo. En primer lugar, este trabajo determinó si los mutantes resistentes de P. aeruginosa mostraban comportamientos egoístas o altruistas al ser estudiado en biopelículas mixtas durante el tratamiento antibióticos. Se emplearon para el estudio de biopelículas simples o mixtas la cepa PAO1 marcada con ECFP y derivados marcados con EYFP que hiperproducían la β-lactamasa AmpC o la bomba de expulsión MexAB-OprM. Las biopelículas maduras fueron expuestas a diferentes concentraciones de β-lactámicos para monitorear la dinámica estructural, utilizando microscopía de barrido láser confocal (CLSM) y la dinámica de poblacional, a través del recuento de células viables. Si bien el tratamiento con β-lactámicos de biopelículas formadas por PAO1 condujo a una reducción importante de la carga bacteriana, tuvo poco efecto sobre las biopelículas formadas por los mutantes resistentes. Sin embargo, el hallazgo más revelador fue que la carga bacteriana de PAO1 aumentó significativamente cuando se crecía en biopelículas mixtas en comparación con las biopelículas individuales. De acuerdo con los recuentos de células viables, las imágenes CLSM mostraron la amplificación de los mutantes resistentes y la protección sobre las poblaciones sensibles. Estos hallazgos demuestran por primera vez que los mutantes que expresan diversos mecanismos de resistencia, tanto hiperproducción de β-lactamasa como hiperexpresión de bombas de flujo, protegen a toda la comunidad, preservando a las poblaciones susceptibles del efecto de los antibióticos. Por último, este trabajo también evaluó la eficacia terapéutica y la dinámica de la resistencia a los antibióticos en biopelículas expuestas a tratamiento secuencial con aztreonam (ATM) y tobramicina (TOB) simulando la terapia inhalada. Para su estudio se desarrollaron biopelículas de cepas clínicas y de laboratorio utilizando el sistema de celda de flujo. Las biopelículas maduras fueron tratadas con diferentes concentraciones de ATM y TOB y sus alternancias a diferentes tiempos. Se determinó tanto el número de células viables como de mutantes resistentes y se controló la dinámica estructural de las biopelículas mediante CLSM. La monoterapia con TOB produjo una intensa disminución de las células viables, que no siempre se correlacionó con una reducción de la biomasa y/o el efecto bactericida sobre las biopelículas, particularmente para las cepas de FQ. El efecto bactericida de la monoterapia con ATM fue menor, pero tuvo efecto sobre la biomasa y/o la estructura de las biopelículas, incluyendo un inteso efecto de filamentación. La alternancia de TOB y ATM supuso una mejora de la actividad antibiopelícula tanto frente a las cepas clínicas como de laboratorio, en comparación con los regímenes individuales, potenciando el efecto bactericida y/o la reducción de la biomasa. Estos resultados apoyan la evaluación clínica de regímenes secuenciales con antibióticos inhalados en la FQ, a diferencia de los tratamientos de mantenimiento actuales que emplean un solo antibiótico en monoterapia. En general, este trabajo proporciona nuevos conocimientos sobre la complejidad del desarrollo de resistencia en las biopelículas, destacando la importancia de los mutantes resistentes dentro de las poblaciones heterogéneas, y también representa un paso adelante para el desarrollo de nuevas estrategias que puedan combatir la IRC por P. aeruginosa.[cat] Pseudomonas aeruginosa és la principal causa d`infeccions respiratòries (IRC) i el principal responsable de l´elevada morbiditat i mortalitat dels pacients amb fibrosi quística (FQ). La instauració de la IRC requereix un complex procés adaptatiu que inclou la selecció d´un número important de mutacions, necessàries per la persistència a llarg termini i comporta la transició des de el mode de creixement planctònic al de biopel·lícula, un segell distintiu de les infeccions cròniques. La complexa estructura espacial de les biopel·lícules junt amb la pressió selectiva d´antibiòtics donen lloc a l´evolució de comunitats heterogènies conformades per diferents subpoblacions que coexisteixen e interactuen establint diversos comportament socials. A més, els perllongats tractaments antibiòtics empleats en la IRC comporten, inevitablement, un augment de la resistència. Les estratègies terapèutiques tradicionals per controlar la infecció per P. aeruginosa amb pacients amb FQ estan basades en l´ús d´un únic antibiòtic nebulitzat i semblen ser eficaços amb el pas del temps. En primer lloc, aquest treball va determinar si els mutants resistents de P. aeruginosa mostraven comportaments egoistes o altruistes en ser estudiats amb biopel·lícules mixtes durant el tractament antibiòtic. Es van utilitzar per l´estudi de biopel·lícules simples o mixtes la soca PAO1 marcada amb ECFP i derivats marcats amb EYFP que híper produïen b-lactamasa AmpC o la bomba d´expulsió MexAB-OprM. Les biopel·lícules madures van ser exposades a diferents concentracions de b-lactàmics per monitoritzar la dinàmica estructural, utilitzant microscopía làser confocal (CLSM) i la dinàmica poblacional, a través del recompte de cèl·lules viables. Si bé el tractament amb b-lactàmics de biopel·lícules formades amb PAO1 condueix a una reducció important de la càrrega bacteriana, va tenir poc efecte sobre les biopel·lícules formades per els mutants resistents. No obstant, el descobriment més revelador , va ser que la càrrega bacteriana de PAO1 va augmentar significativament quan creixia amb biopel·lícules mixtes, amb comparació amb biopel·lícules individuals. D´acord amb els recomptes de cèl·lules viables , les imatges CLSM van mostrar l´amplificació dels mutants resistents i la protecció sobre les poblacions susceptibles. Aquets descobriments demostren per primera vegada que els mutants que expressen diversos mecanismes de resistència, tant de híper producció de b-lactamases com híper expressió de bombes de flux, protegeixen a tota la comunitat, preservant a les poblacions susceptibles de l´efecte dels antibiòtics. Per últim, aquest treball també va avaluar l’eficàcia terapèutica i la dinàmica de la resistència els antibiòtics amb biopel·lícules exposades a tractament seqüencial amb aztreonam (ATM) i tobramicina (TOB) simulant la teràpia inhalada. Per el seu estudi es van desenvolupar biopel·licules de soques clínics i de laboratori utilitzant el sistema del cel·la de flux. Les biopel·lícules madures van ser tractades amb diferents concentracions de ATM i TOB i les seves alternances a diferents temps. Es va determinar tant el número de cèl·lules viables com de mutants resistents i es va controlar la dinàmica estructural de les biopel·lícules mitjançant CLSM. La monoteràpia amb TOB va produir una intensa disminució de les cèl·lules viables, que no sempre es va correlacionar amb una reducció de la biomassa i/o l’efecte bactericida sobre les biopel·lícules, particularment per les soques de FQ. L’efecte bactericida de la monoteràpia amb ATM va ser menor, però va tenir efecte sobre la biomassa i/o l’estructura de les biopel·lícules, afegint un intens efecte de filamentació. L’alternança de TOB i ATM va suposar una millora de l’activitat antibiopel·lícula tant davant les soques clínics com del laboratori, amb comparació amb el règims individuals, potenciant l’efecte bactericida i/o la reducció de la biomassa. Aquests resultats secunden l’avaluació clínica de règims seqüencials amb antibiòtics inhalats en la FQ, a diferencia dels tractaments de manteniment actuals que utilitzen un sol antibiòtic en monoteràpia. En general aquest treball proporciona nous coneixements sobre la complexitat del desenvolupament de resistència en les biopel·lícules, destacant la importància dels mutants resistents dins de les poblacions heterogènies, i també representen un pas endavant per el desenvolupament de noves estratègies que puguin combatre la IRC per P.aeruginosa

Clonal dissemination, emergence of mutator lineages and antibiotic resistance evolution in Pseudomonas aeruginosa cystic fibrosis chronic lung infection.

Chronic respiratory infection by Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis (CF). We investigated the interplay between three key microbiological aspects of these infections: the occurrence of transmissible and persistent strains, the emergence of variants with enhanced mutation rates (mutators) and the evolution of antibiotic resistance. For this purpose, 10 sequential isolates, covering up to an 8-year period, from each of 10 CF patients were studied. As anticipated, resistance significantly accumulated overtime, and occurred more frequently among mutator variants detected in 6 of the patients. Nevertheless, highest resistance was documented for the nonmutator CF epidemic strain LES-1 (ST-146) detected for the first time in Spain. A correlation between resistance profiles and resistance mechanisms evaluated [efflux pump (mexB, mexD, mexF, and mexY) and ampC overexpression and OprD production] was not always obvious and hypersusceptibility to certain antibiotics (such as aztreonam or meropenem) was frequently observed. The analysis of whole genome macrorestriction fragments through Pulsed-Field Gel Electrophoresis (PFGE) revealed that a single genotype (clone FQSE-A) produced persistent infections in 4 of the patients. Multilocus Sequence typing (MLST) identified clone FQSE-A as the CF epidemic clone ST-274, but striking discrepancies between PFGE and MLST profiles were evidenced. While PFGE macrorestriction patterns remained stable, a new sequence type (ST-1089) was detected in two of the patients, differing from ST-274 by only two point mutations in two of the genes, each leading to a nonpreviously described allele. Moreover, detailed genetic analyses revealed that the new ST-1089 is a mutS deficient mutator lineage that evolved from the epidemic strain ST-274, acquired specific resistance mechanisms, and underwent further interpatient spread. Thus, presented results provide the first evidence of interpatient dissemination of mutator lineages and denote their potential for unexpected short-term sequence type evolution, illustrating the complexity of P. aeruginosa population biology in CF

Sequential Treatment of Biofilms with Aztreonam and Tobramycin Is a Novel Strategy for Combating Pseudomonas aeruginosa Chronic Respiratory Infections

31495 El Camino Real, San Juan Capistrano CA 92675exterior, upper courtyard, with poo

A paper biosensor for overcoming matrix effects interfering with the detection of sputum pyocyanin with competitive immunoassays

Detecting sputum pyocyanin (PYO) with a competitive immunoassay is a promising approach for diagnosing Pseudomonas aeruginosa respiratory infections. However, it is not possible to perform a negative control to evaluate matrix-effects in competitive immunoassays, and the highly complex sputum matrix often interferes with target detection. Here, we show that these issues are alleviated by performing competitive immunoassays with a paper biosensor. The biosensing platform consists of a paper reservoir, which contains antibody-coated gold nanoparticles, and a substrate containing a competing recognition element, which is a piece of paper modified with an albumin-antigen conjugate. Detection of PYO with a limit of detection of 4.7·10-3 µM and a dynamic range between 4.7·10-1 µM and 47.6 µM is accomplished by adding the sample to the substrate with the competing element and pressing the reservoir against it for 5 min. When tested with patient samples, the biosensor was able to qualitatively differentiate spiked from non-spiked samples, whereas ELISA did not show a clear cut-off between them. Furthermore, the relative standard deviation was lower when determining sputum with the paper-based biosensor. These features, along with a mild liquefaction step that circumvents the use of harsh chemicals or instruments, make our biosensor a good candidate for diagnosing Pseudomonas infections at the bedside through the detection of sputum PYO.R.R acknowledges funding from the following grants: PDC2021-121819-I00/AEI /https://doi.org/10.13039/501100011033 from Agencia Estatal de Investigación and the EU Recovery and Resilience Facility-NextGenerationEU; AP_2021_006/Acciones Puntuales de Investigación from Govern de les Illes Balears (CAIB); DTS21/00025 from Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union. M-P.M acknowledges funding from the following grants: RTI2018-096278-B-C21 from Spanish Ministry of Science and Innovation; TV32018-201825-30-31 from Fundació Marató de TV3. C.A. acknowledge INTRES: Invertir, investigar, innovar Junior fellowshipfrom IdISBa/Impost turisme sostenible/Agència d’Estratègia Turística de les Illes Balears/Govern de les Illes Balears. A.C. contract was funded by the abovementioned grant PDC2021-121819-I00. We thank A. Busquets (Scientific and Technical Services, Balearic Islands University) for technical assistance with TEM analyses. TEM equipment was co-funded by FEDER funds and the Balearic Islands Government (Direcció Generanl d’Innovació i Recerca). We thank IdISBa the financial support to cover the open access publication fee (Liberi program).Peer reviewe

Prevalence of the fimbrial operon mrkABCD, mrkA expression, biofilm formation and effect of biocides on biofilm formation in carbapenemase-producing Klebsiella pneumoniae isolates belonging or not belonging to high-risk clones

[Background] The role of mrkA adhesin expression, biofilm production, biofilm viability and biocides in the biofilm of carbapenemase-producing Klebsiella pneumoniae isolates was investigated.[Methods] Seventeen isolates representing different sequence types and carbapenemases were investigated. mrkA expression was determined by real-time reverse transcription polymerase chain reaction. Biofilm production (25°C and 37°C, with and without humidity) was determined by the crystal violet assay. The effect of isopropanol, povidone-iodine, sodium hypochlorite, chlorhexidine digluconate, benzalkonium chloride, ethanol and triclosan on biofilm was determined. The effect of povidone-iodine on biofilm biomass and thickness was also determined by confocal laser scanning microscopy.[Results] mrkA expression ranged from 28.2 to 1.3 [high or intermediate level; 64% of high-risk (HR) clones] and from 21.5 to 1.3 (50% of non-HR clones). At 25°C, biofilm formation was observed in 41% of isolates (absence of humidity) and 35% of isolates (presence of humidity), whereas at 37°C, biofilm formation was observed in 76% of isolates with and without humidity. At 25°C, biofilm producers were more frequently observed in HR clones (45% with humidity and 55% without humidity) than non-HR clones (17% with and without humidity). Biofilm viability from day 21 was higher at 25°C than 37°C. The greatest decrease in biofilm formation was observed with povidone-iodine (29% decrease), which also decreased biofilm thickness.[Conclusions] Biofilm formation in carbapenemase-producing K. pneumoniae is related to mrkA expression. Biofilm formation is affected by temperature (37°C>25°C), whereas humidity has little effect. Biofilm viability is affected by temperature (25°C>37°C). At 25°C, HR clones are more frequently biofilm producers than non-HR clones. Povidone-iodine can decrease biofilm production and biofilm thickness.This work was supported by the Plan Nacional de I+D+i 2013–2016 and the 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; three groups in collaboration RD16/0016/0001, RD16/CIII/0004/0002 and RD16/0016/0004), co-financed by European Development Regional Fund ‘A way to achieve Europe’, Operative program Intelligent Growth 2014–2020. This work was funded, in part, by a grant (PI15/01172) from the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad.Peer reviewe

Transferable AmpCs in Klebsiella pneumoniae : interplay with peptidoglycan recycling, mechanisms of hyperproduction, and virulence implications

Chromosomal and transferable AmpC β-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used Klebsiella pneumoniae (KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when blaDHA-1 was expressed in an ampG-defective mutant, Galleria mellonella killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained in vitro, showing slight or insignificant virulence attenuations together with high-level resistance to β-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene mpl was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most β-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable β-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed