Dynamics of the MRSA Population in a Chilean Hospital: a Phylogenomic Analysis (2000-2016)

La diseminación mundial de Staphylococcus aureus resistente a meticilina (SARM) está asociada a la aparición y el establecimiento de clones en zonas geográficas específicas. El clon chileno-cordobés (ChC) (ST5-SCCmecI) ha sido el clon de SARM predominante en Chile desde su primera descripción en 1998, a pesar del informe de otros clones de SARM emergentes en los últimos años. Aquí, caracterizamos la historia evolutiva de MRSA desde 2000 hasta 2016 en un centro de salud terciario chileno utilizando análisis filogenómicos. Secuenciamos 469 aislamientos de SARM recogidos entre 2000 y 2016. Evaluamos las tendencias temporales de los clones circulantes y realizamos una reconstrucción filogenómica para caracterizar la dinámica clonal. Encontramos un aumento significativo en la diversidad y riqueza de tipos de secuencia (STs; Spearman r = 0,8748, P , 0,0001) con un índice de diversidad de Shannon que aumentó de 0,221 en el año 2000 a 1,33 en 2016, y una diversidad efectiva (número de Hill; q = 2) que aumentó de 1,12 a 2,71. El análisis de la tendencia temporal reveló que en el periodo de 2000 a 2003 la mayoría de los aislados (94,2%; n = 98) pertenecían al clon ChC. Sin embargo, desde entonces, la frecuencia del clon ChC ha disminuido con el tiempo, representando el 52% de la colección en el período de 2013 a 2016. Este descenso estuvo acompañado por el aumento de dos linajes emergentes de SARM, ST105-SCCmecII y ST72-SCCmecVI. En conclusión, el clon ChC sigue siendo el linaje MRSA más frecuente, pero este linaje está siendo reemplazado gradualmente por varios clones emergentes, el más importante de los cuales es el clon ST105-SCCmecII. Hasta donde sabemos, éste es el mayor estudio de la dinámica clonal del SARM realizado en Sudamérica. © 2023 Martínez et al.The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in recent years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary health care center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000 and 2016. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r = 0.8748, P , 0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016, and an effective diversity (Hill number; q = 2) increasing from 1.12 to 2.71. The temporal trend analysis revealed that in the period 2000 to 2003 most of the isolates (94.2%; n = 98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013 to 2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage, but this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. To the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. © 2023 Martínez et al

Dynamics of the Mrsa Population in a Chilean Hospital: a Phylogenomic analysis (2000-2016)

The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in recent years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary health care center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000 and 2016. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r = 0.8748, P \u3c 0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016, and an effective diversity (Hill number; q = 2) increasing from 1.12 to 2.71. The temporal trend analysis revealed that in the period 2000 to 2003 most of the isolates (94.2%; n = 98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013 to 2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage, but this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. to the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health pathogen that disseminates through the emergence of successful dominant clones in specific geographic regions. Knowledge of the dissemination and molecular epidemiology of MRSA in Latin America is scarce and is largely based on small studies or more limited typing techniques that lack the resolution to represent an accurate description of the genomic landscape. We used whole-genome sequencing to study 469 MRSA isolates collected between 2000 and 2016 in Chile providing the largest and most detailed study of clonal dynamics of MRSA in South America to date. We found a significant increase in the diversity of MRSA clones circulating over the 17-year study period. Additionally, we describe the emergence of two novel clones (ST105-SCCmecII and ST72-SCCmecVI), which have been gradually increasing in frequency over time. Our results drastically improve our understanding of the dissemination and update our knowledge about MRSA in Latin America

Antibiotic Consumption During the Coronavirus Disease 2019 Pandemic and Emergence of Carbapenemase-Producing Klebsiella pneumoniae Lineages Among Inpatients in a Chilean Hospital: A Time-Series Study and Phylogenomic Analysis.

BACKGROUND: The impact of coronavirus disease 2019 (COVID-19) on antimicrobial use (AU) and resistance has not been well evaluated in South America. These data are critical to inform national policies and clinical care. METHODS: At a tertiary hospital in Santiago, Chile, between 2018 and 2022, subdivided into pre- (3/2018-2/2020) and post-COVID-19 onset (3/2020-2/2022), we evaluated intravenous AU and frequency of carbapenem-resistant Enterobacterales (CRE). We grouped monthly AU (defined daily doses [DDD]/1000 patient-days) into broad-spectrum β-lactams, carbapenems, and colistin and used interrupted time-series analysis to compare AU during pre- and post-pandemic onset. We studied the frequency of carbapenemase-producing (CP) CRE and performed whole-genome sequencing analyses of all carbapenem-resistant (CR) Klebsiella pneumoniae (CRKpn) isolates collected during the study period. RESULTS: Compared with pre-pandemic, AU (DDD/1000 patient-days) significantly increased after the pandemic onset, from 78.1 to 142.5 (P < .001), 50.9 to 110.1 (P < .001), and 4.1 to 13.3 (P < .001) for broad-spectrum β-lactams, carbapenems, and colistin, respectively. The frequency of CP-CRE increased from 12.8% pre-COVID-19 to 51.9% after pandemic onset (P < .001). The most frequent CRE species in both periods was CRKpn (79.5% and 76.5%, respectively). The expansion of CP-CRE harboring blaNDM was particularly noticeable, increasing from 40% (n = 4/10) before to 73.6% (n = 39/53) after pandemic onset (P < .001). Our phylogenomic analyses revealed the emergence of two distinct genomic lineages of CP-CRKpn: ST45, harboring blaNDM, and ST1161, which carried blaKPC. CONCLUSIONS: AU and the frequency of CP-CRE increased after COVID-19 onset. The increase in CP-CRKpn was driven by the emergence of novel genomic lineages. Our observations highlight the need to strengthen infection prevention and control and antimicrobial stewardship efforts

Influencia de la deficiencia de Il-10 sobre el fenotipo mastocitario, la microbiota y la respuesta inmune intestinal en un modelo murino

Los mastocitos (MC) pueden participar en la respuesta a microorganismos mediante diversos receptores de reconocimiento de patrones (PRRs). Luego de su activación a través de estos receptores, los MC pueden orquestar una respuesta mediante la secreción de mediadores inmunológicos como las citocinas. Entre éstas, la interleucina 10 (IL-10) es una citocina importante por sus características inmunomoduladoras, así como también, por su capacidad de regular la expresión de proteasas en MC. Adicionalmente, gracias a la existencia de modelos murinos modificados genéticamente, como los ratones IL-10 deficientes (IL-10-/-), que desarrollan colitis de forma espontánea, es posible investigar el rol potencial de la IL-10 en la respuesta de MC frente a la activación con antígenos de diversos microorganismos. Por otra parte, el uso de este modelo animal permite investigar la influencia de esta citocina sobre la composición de la microbiota intestinal. Este trabajo ha explorado el rol funcional de la IL-10 en mastocitos diferenciados in vitro, así como los efectos de la deficiencia de IL-10 sobre la composición de la microbiota y la expresión de factores relacionados con la respuesta inmune, antes (6 semanas) y al inicio (20 semanas) de la colitis. Para este propósito, se ha caracterizado el efecto de la deficiencia de IL-10 sobre el fenotipo mastocitario y tras su activación vía PRRs. Adicionalmente, se evaluó el efecto que produce la carencia de IL-10 sobre la composición de la microbiota, la expresión de TLRs y citocinas proinflamatorias, así como la producción de IgA luminal, en las mismas etapas y tras el tratamiento con antibióticos. Los resultados obtenidos indicaron que la deficiencia de IL-10 produjo distintos efectos dependiendo del fenotipo mastocitario, de la edad y del tipo de ligando PRR. Así, en ausencia de IL-10, MC de tipo mucosa (MLMC) mostraron una menor expresión de TLR4 y NOD2 a las 6 semanas y TLR7 a las 20 semanas. Además, ambos fenotipos de MC (mucosa y conectivo), mostraron una menor secreción de IL-6 y TNFα tras la activación de TLR2 La activación de TLR4 y TLR7 en MLMC generó una menor secreción de IL-6 a las 6 semanas, mientras MLMC secretaron menos TNFα a las 20 semanas. Finalmente, tras la estimulación de NOD2 no se observó secreción de citocinas en ninguno de los fenotipos mastocitarios. Por otra parte, se observó que en animales IL-10-/- existen factores que potencialmente favorecerían el desarrollo de colitis. Así, los ratones IL-10-/- a las 6 semanas mostraron representantes del filo Verrucomicrobia y una menor abundancia relativa de los taxa Rikenellaceae y Lachnospiraceae. Mientras que a las 20 semanas en los ratones IL-10-/- se observaron microorganismos del filo TM7, una menor expresión de IL-1β, IL-6, TLR6, -7 y -8, y un incremento de TNFα e IgA. Adicionalmente, el uso de antibióticos antes del inicio de la colitis indujo una disminución en la diversidad y una reestructuración de la microbiota, junto con una disminución en la expresión de TLRs, citocinas y menor producción de IgA luminal. En resumen, estos hallazgos proveen nuevas perspectivas sobre la función de los MC y la IL-10 en la interacción microorganismo-huésped. Muestran cómo la ausencia de IL-10 puede afectar la composición de la microbiota y la expresión de factores asociados a la respuesta inmune. Y sugieren que la modificación temprana de la microbiota mediante la utilización de antibióticos en individuos genéticamente susceptibles podría alterar la progresión de la colitis.Mast cells (MC) can participate in the response to microorganisms by various pattern recognition receptors (PRRs). After their activation through these receptors, MC can orchestrate a response by secreting immunological mediators such as cytokines. Among these, interleukin 10 (IL-10) is an important cytokine due to its immunomodulatory characteristics, as well as its ability to regulate the expression of MC proteases. Additionally, thanks to the existence of genetically modified murine models, such as IL-10 deficient (IL-10-/-) mice that develop colitis spontaneously, it is possible to investigate the potential role of IL-10 in MC response to activation with antigens of different microorganisms. On the other hand, the use of this animal model allows investigating the influence of this cytokine on the composition of the intestinal microbiota. This work has explored the functional role of IL-10 in differentiated MC in vitro, as well as the effects of IL-10 deficiency on the composition of the microbiota and the expression of factors related to the immune response, before (6 weeks) and at the onset (20 weeks) of colitis. For this purpose, the effect of IL-10 deficiency has been characterized on MC of different phenotype and after its activation via PRRs. Additionally, the effect produced by the lack of IL-10 on the microbiota composition, the expression of TLRs and proinflammatory cytokines, as well as the production of luminal IgA, in the same stages and after antibiotics treatment was evaluated. The results obtained indicated that the IL-10 deficiency produced different effects depending on the MC phenotype, age and type of PRR ligand. Thus, in the absence of IL-10, mucosal-like MC (MLMC) showed lower expression of TLR4 and NOD2 at week 6 and TLR7 at week 20. In addition, both MC phenotypes (mucosa and connective), showed a lower secretion of IL-6 and TNFα after TLR2 activation. The TLR4 and TLR7 activation in MLMC generated a lower secretion of IL-6 at week 6, while MLMC secreted less TNFα at week 20. Finally, after NOD2 stimulation, no cytokine secretion was observed in any of the MC phenotypes. On the other hand, it was observed that in IL-10-/- animals there are factors that potentially favor the development of colitis. Thus, IL-10-/- mice at week 6 showed representatives of Verrucomicrobia phylum and a lower relative abundance of Rikenellaceae and Lachnospiraceae taxa. Meanwhile at week 20 in IL-10-/- mice, microorganisms of the phylum TM7 were observed, as well as, a lower expression of IL-1β, IL-6, TLR6, -7 and -8, and an increase of TNFα and IgA. Additionally, the use of antibiotics before the development of colitis induced a decrease in diversity and a restructuring of the microbiota, together with a decrease in TLRs and cytokines expression, and a lower production of luminal IgA. In summary, these findings provide new insights on the role of MC and IL-10 in the host-microorganism interaction. They show how the IL-10 deficiency can affect the microbiota composition and the expression of factors associated with the immune response. And they suggest that early modification of the microbiota through the use of antibiotics in genetically susceptible individuals could alter the colitis progression

Applications in the search for genomic selection signatures in fish

© 2014 Lopez_dinamarca, Neira and Yáñez. Selection signatures are genomic regions harboring DNA sequences functionally involved in the genetic variation of traits subject to selection. Selection signatures have been intensively studied in recent years because of their relevance to evolutionary biology and their potential association with genes that control phenotypes of interest in wild and domestic populations. Selection signature research in fish has been confined to a smaller scale, due in part to the relatively recent domestication of fish species and limited genomic resources such as molecular markers, genetic mapping, DNA sequences, and reference genomes. However, recent genomic technology advances are paving the way for more studies that may contribute to the knowledge of genomic regions underlying phenotypes of biological and productive interest in fish

IL-10 Modulates the Expression and Activation of Pattern Recognition Receptors in Mast Cells

Mast cells (MCs) are involved in several immune-related responses, including those in bacterial infections, autoimmune diseases, inflammatory bowel diseases, and cancer, among others. MCs identify microorganisms by pattern recognition receptors (PRRs), activating a secretory response. Interleukin (IL)-10 has been described as an important modulator of MC responses; however, its role in PRR-mediated activation of MC is not fully understood. We analyzed the activation of TLR2, TLR4, TLR7 and Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mucosal-like MCs (MLMCs) and peritoneum-derived cultured MCs (PCMCs) from IL-10−/− and wild-type (WT) mice. IL-10−/− mice showed a reduced expression of TLR4 and NOD2 at week 6 and TLR7 at week 20 in MLMC. In MLMC and PCMC, TLR2 activation induced a reduced secretion of IL-6 and TNFα in IL-10−/− MCs. TLR4- and TLR7-mediated secretion of IL-6 and TNFα was not detected in PCMCs. Finally, no cytokine release was induced by NOD2 ligand, and responses to TLR2 and TLR4 were lower in MCs at 20 weeks. These findings indicate that PRR activation in MCs depends on the phenotype, ligand, age, and IL-10

Influencia de la deficiencia de Il-10 sobre el fenotipo mastocitario, la microbiota y la respuesta inmune intestinal en un modelo murino /

Los mastocitos (MC) pueden participar en la respuesta a microorganismos mediante diversos receptores de reconocimiento de patrones (PRRs). Luego de su activación a través de estos receptores, los MC pueden orquestar una respuesta mediante la secreción de mediadores inmunológicos como las citocinas. Entre éstas, la interleucina 10 (IL-10) es una citocina importante por sus características inmunomoduladoras, así como también, por su capacidad de regular la expresión de proteasas en MC. Adicionalmente, gracias a la existencia de modelos murinos modificados genéticamente, como los ratones IL-10 deficientes (IL-10-/-), que desarrollan colitis de forma espontánea, es posible investigar el rol potencial de la IL-10 en la respuesta de MC frente a la activación con antígenos de diversos microorganismos. Por otra parte, el uso de este modelo animal permite investigar la influencia de esta citocina sobre la composición de la microbiota intestinal. Este trabajo ha explorado el rol funcional de la IL-10 en mastocitos diferenciados in vitro, así como los efectos de la deficiencia de IL-10 sobre la composición de la microbiota y la expresión de factores relacionados con la respuesta inmune, antes (6 semanas) y al inicio (20 semanas) de la colitis. Para este propósito, se ha caracterizado el efecto de la deficiencia de IL-10 sobre el fenotipo mastocitario y tras su activación vía PRRs. Adicionalmente, se evaluó el efecto que produce la carencia de IL-10 sobre la composición de la microbiota, la expresión de TLRs y citocinas proinflamatorias, así como la producción de IgA luminal, en las mismas etapas y tras el tratamiento con antibióticos. Los resultados obtenidos indicaron que la deficiencia de IL-10 produjo distintos efectos dependiendo del fenotipo mastocitario, de la edad y del tipo de ligando PRR. Así, en ausencia de IL-10, MC de tipo mucosa (MLMC) mostraron una menor expresión de TLR4 y NOD2 a las 6 semanas y TLR7 a las 20 semanas. Además, ambos fenotipos de MC (mucosa y conectivo), mostraron una menor secreción de IL-6 y TNFα tras la activación de TLR2 La activación de TLR4 y TLR7 en MLMC generó una menor secreción de IL-6 a las 6 semanas, mientras MLMC secretaron menos TNFα a las 20 semanas. Finalmente, tras la estimulación de NOD2 no se observó secreción de citocinas en ninguno de los fenotipos mastocitarios. Por otra parte, se observó que en animales IL-10-/- existen factores que potencialmente favorecerían el desarrollo de colitis. Así, los ratones IL-10-/- a las 6 semanas mostraron representantes del filo Verrucomicrobia y una menor abundancia relativa de los taxa Rikenellaceae y Lachnospiraceae. Mientras que a las 20 semanas en los ratones IL-10-/- se observaron microorganismos del filo TM7, una menor expresión de IL-1β, IL-6, TLR6, -7 y -8, y un incremento de TNFα e IgA. Adicionalmente, el uso de antibióticos antes del inicio de la colitis indujo una disminución en la diversidad y una reestructuración de la microbiota, junto con una disminución en la expresión de TLRs, citocinas y menor producción de IgA luminal. En resumen, estos hallazgos proveen nuevas perspectivas sobre la función de los MC y la IL-10 en la interacción microorganismo-huésped. Muestran cómo la ausencia de IL-10 puede afectar la composición de la microbiota y la expresión de factores asociados a la respuesta inmune. Y sugieren que la modificación temprana de la microbiota mediante la utilización de antibióticos en individuos genéticamente susceptibles podría alterar la progresión de la colitis.Mast cells (MC) can participate in the response to microorganisms by various pattern recognition receptors (PRRs). After their activation through these receptors, MC can orchestrate a response by secreting immunological mediators such as cytokines. Among these, interleukin 10 (IL-10) is an important cytokine due to its immunomodulatory characteristics, as well as its ability to regulate the expression of MC proteases. Additionally, thanks to the existence of genetically modified murine models, such as IL-10 deficient (IL-10-/-) mice that develop colitis spontaneously, it is possible to investigate the potential role of IL-10 in MC response to activation with antigens of different microorganisms. On the other hand, the use of this animal model allows investigating the influence of this cytokine on the composition of the intestinal microbiota. This work has explored the functional role of IL-10 in differentiated MC in vitro, as well as the effects of IL-10 deficiency on the composition of the microbiota and the expression of factors related to the immune response, before (6 weeks) and at the onset (20 weeks) of colitis. For this purpose, the effect of IL-10 deficiency has been characterized on MC of different phenotype and after its activation via PRRs. Additionally, the effect produced by the lack of IL-10 on the microbiota composition, the expression of TLRs and proinflammatory cytokines, as well as the production of luminal IgA, in the same stages and after antibiotics treatment was evaluated. The results obtained indicated that the IL-10 deficiency produced different effects depending on the MC phenotype, age and type of PRR ligand. Thus, in the absence of IL-10, mucosal-like MC (MLMC) showed lower expression of TLR4 and NOD2 at week 6 and TLR7 at week 20. In addition, both MC phenotypes (mucosa and connective), showed a lower secretion of IL-6 and TNFα after TLR2 activation. The TLR4 and TLR7 activation in MLMC generated a lower secretion of IL-6 at week 6, while MLMC secreted less TNFα at week 20. Finally, after NOD2 stimulation, no cytokine secretion was observed in any of the MC phenotypes. On the other hand, it was observed that in IL-10-/- animals there are factors that potentially favor the development of colitis. Thus, IL-10-/- mice at week 6 showed representatives of Verrucomicrobia phylum and a lower relative abundance of Rikenellaceae and Lachnospiraceae taxa. Meanwhile at week 20 in IL-10-/- mice, microorganisms of the phylum TM7 were observed, as well as, a lower expression of IL-1β, IL-6, TLR6, -7 and -8, and an increase of TNFα and IgA. Additionally, the use of antibiotics before the development of colitis induced a decrease in diversity and a restructuring of the microbiota, together with a decrease in TLRs and cytokines expression, and a lower production of luminal IgA. In summary, these findings provide new insights on the role of MC and IL-10 in the host-microorganism interaction. They show how the IL-10 deficiency can affect the microbiota composition and the expression of factors associated with the immune response. And they suggest that early modification of the microbiota through the use of antibiotics in genetically susceptible individuals could alter the colitis progression

Vaccination with DNA Encoding Truncated Enterohemorrhagic Escherichia coli (EHEC) Factor for Adherence-1 Gene (efa-1′) Confers Protective Immunity to Mice Infected with E. coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is the predominant causative agent of hemorrhagic colitis in humans and is the cause of haemolytic uraemic syndrome and other illnesses. Cattle have been implicated as the main reservoir of this organism. Here, we evaluated the immunogenicity and protective efficacy of a DNA vaccine encoding conserved sequences of truncated EHEC factor for adherence-1 (efa-1′ ) in a mouse model. Intranasal administration of plasmid DNA carrying the efa-1′ gene (pVAXefa-1′ ) into C57BL/6 mice elicited both humoral and cellular immune responses. In animals immunized with pVAXefa-1′ , EHEC-secreted protein-specific IgM and IgG antibodies were detected in sera at day 45. Anti-EHEC-secreted protein sIgA was also detected in nasal and bronchoalveolar lavages. In addition, antigen-specific T-cell-proliferation, IL-10, and IFN-γ were observed upon re-stimulation with either heat-killed bacteria or EHEC-secreted proteins. Vaccinated animals were also protected against challenge with E. coli O157:H7 strain EDL933. These results suggest that DNA vaccine encoding efa-1′ have therapeutic potential in interventions against EHEC infections. This approach could lead to a new strategy in the production of vaccines that prevent infections in cattle

Table1_Role of the multi-drug efflux systems on the baseline susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam in clinical isolates of non-carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa.XLSX

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the pathogens that urgently needs new drugs and new alternatives for its control. The primary strategy to combat this bacterium is combining treatments of beta-lactam with a beta-lactamase inhibitor. The most used combinations against P. aeruginosa are ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T). Although mechanisms leading to CZA and C/T resistance have already been described, among which are the resistance-nodulation-division (RND) efflux pumps, the role that these extrusion systems may play in CZA, and C/T baseline susceptibility of clinical isolates remains unknown. For this purpose, 161 isolates of non-carbapenemase-producing (Non-CP) CRPA were selected, and susceptibility tests to CZA and C/T were performed in the presence and absence of the RND efflux pumps inhibitor, Phenylalanine-arginine β-naphthylamide (PAβN). In the absence of PAβN, C/T showed markedly higher activity against Non-CP-CRPA isolates than observed for CZA. These results were even more evident in isolates classified as extremely-drug resistant (XDR) or with difficult-to-treat resistance (DTR), where CZA decreased its activity up to 55.2% and 20.0%, respectively, whereas C/T did it up to 82.8% (XDR), and 73.3% (DTR). The presence of PAβN showed an increase in both CZA (37.6%) and C/T (44.6%) activity, and 25.5% of Non-CP-CRPA isolates increased their susceptibility to these two combined antibiotics. However, statistical analysis showed that only the C/T susceptibility of Non-CP-CRPA isolates was significantly increased. Although the contribution of RND activity to CZA and C/T baseline susceptibility was generally low (two-fold decrease of minimal inhibitory concentrations [MIC]), a more evident contribution was observed in a non-minor proportion of the Non-CP-CRPA isolates affected by PAβN [CZA: 25.4% (15/59); C/T: 30% (21/70)]. These isolates presented significantly higher MIC values for C/T. Therefore, we conclude that RND efflux pumps are participating in the phenomenon of baseline susceptibility to CZA and, even more, to C/T. However, the genomic diversity of clinical isolates is so great that deeper analyzes are necessary to determine which elements are directly involved in this phenomenon.</p

Table2_Role of the multi-drug efflux systems on the baseline susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam in clinical isolates of non-carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa.XLSX

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the pathogens that urgently needs new drugs and new alternatives for its control. The primary strategy to combat this bacterium is combining treatments of beta-lactam with a beta-lactamase inhibitor. The most used combinations against P. aeruginosa are ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T). Although mechanisms leading to CZA and C/T resistance have already been described, among which are the resistance-nodulation-division (RND) efflux pumps, the role that these extrusion systems may play in CZA, and C/T baseline susceptibility of clinical isolates remains unknown. For this purpose, 161 isolates of non-carbapenemase-producing (Non-CP) CRPA were selected, and susceptibility tests to CZA and C/T were performed in the presence and absence of the RND efflux pumps inhibitor, Phenylalanine-arginine β-naphthylamide (PAβN). In the absence of PAβN, C/T showed markedly higher activity against Non-CP-CRPA isolates than observed for CZA. These results were even more evident in isolates classified as extremely-drug resistant (XDR) or with difficult-to-treat resistance (DTR), where CZA decreased its activity up to 55.2% and 20.0%, respectively, whereas C/T did it up to 82.8% (XDR), and 73.3% (DTR). The presence of PAβN showed an increase in both CZA (37.6%) and C/T (44.6%) activity, and 25.5% of Non-CP-CRPA isolates increased their susceptibility to these two combined antibiotics. However, statistical analysis showed that only the C/T susceptibility of Non-CP-CRPA isolates was significantly increased. Although the contribution of RND activity to CZA and C/T baseline susceptibility was generally low (two-fold decrease of minimal inhibitory concentrations [MIC]), a more evident contribution was observed in a non-minor proportion of the Non-CP-CRPA isolates affected by PAβN [CZA: 25.4% (15/59); C/T: 30% (21/70)]. These isolates presented significantly higher MIC values for C/T. Therefore, we conclude that RND efflux pumps are participating in the phenomenon of baseline susceptibility to CZA and, even more, to C/T. However, the genomic diversity of clinical isolates is so great that deeper analyzes are necessary to determine which elements are directly involved in this phenomenon.</p