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1.
Proc Natl Acad Sci U S A ; 119(38): e2203593119, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36095213

RESUMO

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae, modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. An analysis of large K. pneumoniae genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine-to-thymine transition at position 25 (25c > t) in ompK36. We show that the 25c > t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates K. pneumoniae in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c > t transition tips the balance toward treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c > t transition mediates an intramolecular messenger RNA (mRNA) interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.


Assuntos
Antibacterianos , Proteínas de Bactérias , Carbapenêmicos , Klebsiella pneumoniae , Porinas , Resistência beta-Lactâmica , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Carbapenêmicos/uso terapêutico , Modelos Animais de Doenças , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Camundongos , Testes de Sensibilidade Microbiana , Mutação , Filogenia , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/microbiologia , Porinas/classificação , Porinas/genética , RNA Mensageiro/metabolismo , Resistência beta-Lactâmica/genética
2.
PLoS Pathog ; 18(7): e1010334, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35816554

RESUMO

Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by KP sequence types ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. We show that in the absence of antibiotics KP mutants harbouring these L3 insertions exhibit both an in vitro and in vivo competitive disadvantage relative to the isogenic parental strain expressing wild type OmpK36. We propose that this explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ácido Aspártico , Proteínas de Bactérias/metabolismo , Células Clonais , Humanos , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Testes de Sensibilidade Microbiana , Porinas/genética , Porinas/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo
3.
J Antimicrob Chemother ; 78(7): 1599-1605, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-37161536

RESUMO

OBJECTIVES: This study investigated fosfomycin susceptibility and mechanisms of resistance in a collection of 99 Staphylococcus aureus isolated from cases of hospital-acquired pneumonia, previously collected from a multicentre survey carried out in Italy. METHODS: Fosfomycin susceptibility was tested by reference agar dilution. Bioinformatic and gene expression analysis, mutant selection experiments and WGS were executed to characterize fosfomycin resistance mechanisms. RESULTS: Fosfomycin resistance rates were 0% (0 of 35) among MSSA and 22% (14 of 64) among MRSA, with no evidence of clonal expansion. Resistance mechanisms were putatively identified in 8 of the 14 resistant strains, including: (i) chromosomal mutations causing loss of function of the UhpT transporter; (ii) overexpression of the gene encoding the Tet38 efflux pump; and (iii) overexpression of a fosB gene encoding a fosfomycin-inactivating enzyme, which was found to be resident in the chromosome of several S. aureus lineages but not always associated with fosfomycin resistance. The latter mechanism, which had not been previously described and was confirmed by results of in vitro mutant selection experiments, was associated in two cases with transposition of an IS1182 element upstream of the chromosomal fosB gene, apparently providing an additional promoter. CONCLUSIONS: This study showed that some S. aureus clonal lineages carry a resident chromosomal fosB gene and can evolve to fosfomycin resistance by overexpression of this gene.


Assuntos
Fosfomicina , Staphylococcus aureus Resistente à Meticilina , Fosfomicina/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus , Regulação para Cima , Testes de Sensibilidade Microbiana , Cromossomos , Expressão Gênica , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico
4.
J Antimicrob Chemother ; 78(7): 1672-1676, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-37209112

RESUMO

BACKGROUND: Cefiderocol is a catechol-substituted cephalosporin with potent in vitro activity against carbapenem-resistant (CR) Gram-negative bacteria (GNB). Cefiderocol susceptibility testing is complex because iron concentrations need to be taken into consideration. Here, we assessed the clinical performance of Bruker's UMIC® Cefiderocol and corresponding iron-depleted CAMHB to determine MIC by broth microdilution (BMD) for clinically relevant GNB. METHODS: MICs of cefiderocol for 283 GN clinical isolates were determined by BMD using iron-depleted CAMHB. Frozen panels were used as a reference. The concentration range of cefiderocol was 0.03-32 mg/L. The isolates, with different degrees of susceptibility to cefiderocol, included Enterobacterales (n = 180), Pseudomonas aeruginosa (n = 49), Acinetobacter baumannii (n = 44) and Stenotrophomonas maltophilia (n = 10). RESULTS: The rates of categorical agreement (CA), essential agreement (EA) and bias were calculated to evaluate the performance of the UMIC® Cefiderocol, as compared with the reference method. Overall, the UMIC® Cefiderocol showed 90.8% EA (95% CI: 86.9%-93.7%) with a bias of -14.5% and a CA of 90.1% (95% CI: 86.1%-93.1%). For Enterobacterales, the UMIC® Cefiderocol showed 91.7% EA (95% CI: 86.7%-94.9%) with a bias of -25.0% and a CA of 87.8% (95% CI: 82.2%-91.8%). For non-fermenters, the UMIC® Cefiderocol showed 89.3% EA (95% CI: 81.9%-93.9%) (not significantly different from 90.0%, Student t-test) with a bias of -3.9% and a CA of 94.2% (95% CI: 87.7%-97.3%). CONCLUSIONS: UMIC® Cefiderocol is a valid method for the determination of cefiderocol MICs even if higher than expected discrepancies were observed with NDM-producing Enterobacterales, which presented in most cases MIC values close to the breakpoint.


Assuntos
Antibacterianos , Cefalosporinas , Humanos , Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Bactérias Gram-Negativas , Ferro , Pseudomonas aeruginosa , Testes de Sensibilidade Microbiana , Cefiderocol
5.
J Antimicrob Chemother ; 78(11): 2752-2761, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37807834

RESUMO

BACKGROUND: Cefiderocol is a novel siderophore cephalosporin with promising activity against most carbapenem-resistant Gram-negative bacteria (CRGNB). However, extensive postmarketing experiences are lacking. This study aimed to analyse the early experience on cefiderocol postmarketing use at three tertiary care hospitals in Italy. METHODS: We retrospectively included patients with infections caused by CRGNB treated with cefiderocol at three Italian tertiary care hospitals from 1 March 2021 to 30 June 2022. A multivariate Cox model was used to identify predictors of 30 day mortality. A propensity score (PS) analysis with inverse probability weighting (IPW) was also performed to compare the treatment effect of cefiderocol monotherapy (CM) versus combination regimens (CCRs). RESULTS: The cohort included 142 patients (72% male, median age 67 years, with 89 cases of Acinetobacter baumannii infection, 22 cases of Klebsiella pneumoniae, 27 cases of Pseudomonas aeruginosa and 4 of other pathogens). The 30 day all-cause mortality was 37% (52/142). We found no association between bacterial species and mortality. In multivariate analysis, a Charlson Comorbidity Index >3 was an independent predictor of mortality (HR 5.02, 95% CI 2.37-10.66, P < 0.001). In contrast, polymicrobial infection (HR 0.41, 95% CI 0.21-0.82, P < 0.05) was associated with lower mortality. There was no significant difference in mortality between patients receiving CM (n = 70) and those receiving a CCR (n = 72) (33% versus 40%, respectively), even when adjusted for IPW-PS (HR 1.11, 95% CI 0.63-1.96, P = 0.71). CONCLUSIONS: Real-life data confirm that cefiderocol is a promising option against carbapenem-resistant Gram-negative infections, even as monotherapy.


Assuntos
Infecções por Acinetobacter , Infecções por Bactérias Gram-Negativas , Humanos , Masculino , Idoso , Feminino , Antibacterianos/uso terapêutico , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Carbapenêmicos/uso terapêutico , Estudos Retrospectivos , Cefalosporinas/uso terapêutico , Cefalosporinas/farmacologia , Bactérias Gram-Negativas , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia , Cefiderocol
6.
Proc Natl Acad Sci U S A ; 117(40): 25043-25054, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32968015

RESUMO

Molecular and genomic surveillance systems for bacterial pathogens currently rely on tracking clonally evolving lineages. By contrast, plasmids are usually excluded or analyzed with low-resolution techniques, despite being the primary vectors of antibiotic resistance genes across many key pathogens. Here, we used a combination of long- and short-read sequence data of Klebsiella pneumoniae isolates (n = 1,717) from a European survey to perform an integrated, continent-wide study of chromosomal and plasmid diversity. This revealed three contrasting modes of dissemination used by carbapenemase genes, which confer resistance to last-line carbapenems. First, blaOXA-48-like genes have spread primarily via the single epidemic pOXA-48-like plasmid, which emerged recently in clinical settings and spread rapidly to numerous lineages. Second, blaVIM and blaNDM genes have spread via transient associations of many diverse plasmids with numerous lineages. Third, blaKPC genes have transmitted predominantly by stable association with one successful clonal lineage (ST258/512) yet have been mobilized among diverse plasmids within this lineage. We show that these plasmids, which include pKpQIL-like and IncX3 plasmids, have a long association (and are coevolving) with the lineage, although frequent recombination and rearrangement events between them have led to a complex array of mosaic plasmids carrying blaKPC Taken altogether, these results reveal the diverse trajectories of antibiotic resistance genes in clinical settings, summarized as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage. Our study provides a framework for the much needed incorporation of plasmid data into genomic surveillance systems, an essential step toward a more comprehensive understanding of resistance spread.


Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Klebsiella/genética , Klebsiella pneumoniae/genética , beta-Lactamases/genética , Antibacterianos/uso terapêutico , Carbapenêmicos/uso terapêutico , Linhagem da Célula/genética , Cromossomos Bacterianos/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Genoma Bacteriano/genética , Humanos , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/patogenicidade , Plasmídeos/genética , Análise de Sequência de DNA/métodos
7.
J Antimicrob Chemother ; 77(8): 2199-2208, 2022 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-35512342

RESUMO

OBJECTIVES: To investigate the in vitro activity of fosfomycin, colistin and combinations thereof against planktonic and biofilm cultures of Gram-negative pathogens, mostly showing MDR phenotypes, at concentrations achievable via inhalation of aerosolized drugs. METHODS: Activity against planktonic cultures was tested by the chequerboard assay with 130 strains, including 52 Pseudomonas aeruginosa, 47 Klebsiella pneumoniae, 19 Escherichia coli, 7 Stenotrophomonas maltophilia and 5 Acinetobacter baumannii. Activity against biofilm cultures was tested by biofilm chequerboard and quantitative antibiofilm assays with a subset of 20 strains. In addition, 10 of these strains were tested in mutant prevention concentration (MPC) assays. RESULTS: Against planktonic cultures, synergism between fosfomycin and colistin was detected with a minority (10%) of strains (eight K. pneumoniae and five P. aeruginosa), while antagonism was never observed. Synergism between fosfomycin and colistin against biofilms was observed with the majority of tested strains (16/20 in biofilm chequerboard assays, and 18/20 in the quantitative antibiofilm assays), including representatives of each species and regardless of their resistance genotype or phenotype. Furthermore, combination of fosfomycin and colistin was found to significantly reduce the MPC of individual drugs. CONCLUSIONS: Fosfomycin and colistin in combination, at concentrations achievable via inhalation of nebulized drugs, showed notable synergy against MDR Gram-negative pathogens grown in biofilm, and were able to reduce the emergence of fosfomycin- and colistin-resistant subpopulations.


Assuntos
Colistina , Fosfomicina , Antibacterianos/farmacologia , Biofilmes , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla , Sinergismo Farmacológico , Fosfomicina/farmacologia , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Plâncton
8.
Euro Surveill ; 27(43)2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36305334

RESUMO

A nosocomial outbreak by cefiderocol (FDC)-resistant NDM-1-producing Klebsiella pneumoniae (NDM-Kp) occurred in a large tertiary care hospital from August 2021-June 2022 in Florence, Italy, an area where NDM-Kp strains have become endemic. Retrospective analysis of NDM-Kp from cases observed in January 2021-June 2022 revealed that 21/52 were FDC-resistant. The outbreak was mostly sustained by clonal expansion of a mutant with inactivated cirA siderophore receptor gene, which exhibited high-level resistance to FDC (MIC ≥ 32 mg/L) and spread independently of FDC exposure.


Assuntos
Infecção Hospitalar , Infecções por Klebsiella , Humanos , Klebsiella pneumoniae/genética , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/epidemiologia , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/epidemiologia , Estudos Retrospectivos , Proteínas de Bactérias/genética , beta-Lactamases/genética , Surtos de Doenças , Antibacterianos , Testes de Sensibilidade Microbiana , Cefiderocol
9.
J Antimicrob Chemother ; 76(2): 355-361, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33188415

RESUMO

BACKGROUND: Previous studies showed that the epidemic of carbapenem-resistant Klebsiella pneumoniae (CR-KP) observed in Italy since 2010 was sustained mostly by strains of clonal group (CG) 258 producing KPC-type carbapenemases. In the framework of the National Antibiotic-Resistance Surveillance (AR-ISS), a countrywide survey was conducted in 2016 to explore the evolution of the phenotypic and genotypic characteristics of CR-KP isolates. METHODS: From March to July 2016, hospital laboratories participating in AR-ISS were requested to provide consecutive, non-duplicated CR-KP (meropenem and/or imipenem MIC >1 mg/L) from invasive infections. Antibiotic susceptibility was determined according to EUCAST recommendations. A WGS approach was adopted to characterize the isolates by investigating phylogeny, resistome and virulome. RESULTS: Twenty-four laboratories provided 157 CR-KP isolates, of which 156 were confirmed as K. pneumoniae sensu stricto by WGS and found to carry at least one carbapenemase-encoding gene, corresponding in most cases (96.1%) to blaKPC. MLST- and SNP-based phylogeny revealed that 87.8% of the isolates clustered in four major lineages: CG258 (47.4%), with ST512 as the most common clone, CG307 (19.9%), ST101 (15.4%) and ST395 (5.1%). A close association was identified between lineages and antibiotic resistance phenotypes and genotypes, virulence traits and capsular types. Colistin resistance, mainly associated with mgrB mutations, was common in all major lineages except ST395. CONCLUSIONS: This WGS-based survey showed that, although CG258 remained the most common CR-KP lineage in Italy, a polyclonal population has emerged with the spread of the new high-risk lineages CG307, ST101 and ST395, while KPC remained the most common carbapenemase.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Humanos , Itália/epidemiologia , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , beta-Lactamases/genética
10.
Infect Immun ; 88(8)2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32513853

RESUMO

MCR-1 is a plasmid-encoded phosphoethanolamine transferase able to modify the lipid A structure. It confers resistance to colistin and was isolated from human, animal, and environmental strains of Enterobacteriaceae, raising serious global health concerns. In this paper, we used recombinant mcr-1-expressing Escherichia coli to study the impact of MCR-1 products on E. coli-induced activation of inflammatory pathways in activated THP-1 cells, which was used as a model of human macrophages. We found that infection with recombinant mcr-1-expressing E. coli significantly modulated p38-MAPK and Jun N-terminal protein kinase (JNK) activation and pNF-κB nuclear translocation as well as the expression of genes for the relevant proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and IL-1ß compared with mcr-1-negative strains. Caspase-1 activity and IL-1ß secretion were significantly less activated by mcr-1-positive E. coli strains than the mcr-1-negative parental strain. Similar results were obtained with clinical isolates of mcr-1-positive E. coli, suggesting that, in addition to colistin resistance, the expression of mcr-1 allows the escape of early host innate defenses and may promote bacterial survival.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação da Expressão Gênica/imunologia , MAP Quinase Quinase 4/genética , NF-kappa B/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Caspase 1/genética , Caspase 1/imunologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/microbiologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Citoplasma/microbiologia , Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Inflamação , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , MAP Quinase Quinase 4/imunologia , Viabilidade Microbiana , NF-kappa B/imunologia , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Transdução de Sinais , Células THP-1 , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-31964792

RESUMO

This study reports on the characterization of two ceftazidime-avibactam (CZA)-resistant KPC-producing Klebsiella pneumoniae strains (KP-14159 and KP-8788) sequentially isolated from infections occurred in a patient never treated with CZA. Whole-genome sequencing characterization using a combined short- and long-read sequencing approach showed that both isolates belonged to the same ST258 strain, had altered outer membrane porins (a truncated OmpK35 and an Asp137Thr138 duplication in the L3 loop of OmpK36), and carried novel pKpQIL plasmid derivatives (pIT-14159 and pIT-8788, respectively) harboring two copies of the Tn4401a KPC-3-encoding transposon. Plasmid pIT-8788 was a cointegrate of pIT-14159 with a ColE replicon (that was also present in KP-14159) apparently evolved in vivo during infection. pIT-8788 was maintained at a higher copy number than pIT-14159 and, upon transfer to Escherichia coli DH10B, was able to increase the CZA MIC by 32-fold. The present findings provide novel insights about the mechanisms of acquired resistance to CZA, underscoring the role that the evolution of broadly disseminated pKpQIL plasmid derivatives may have in increasing the blaKPC gene copy number and KPC-3 expression in bacterial hosts. Although not self-transferable, similar elements, with multiple copies of Tn4401 and maintained at a high copy number, could mediate transferable CZA resistance upon mobilization.


Assuntos
Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Proteínas de Bactérias/genética , Elementos de DNA Transponíveis/genética , Combinação de Medicamentos , Escherichia coli/genética , Dosagem de Genes/genética , Genoma Bacteriano/genética , Humanos , Transplante de Rim/efeitos adversos , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Porinas/genética , Sequenciamento Completo do Genoma , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/genética
12.
Artigo em Inglês | MEDLINE | ID: mdl-33106265

RESUMO

This study reports on the characterization of a Klebsiella pneumoniae clinical isolate showing high-level resistance to ceftazidime-avibactam associated with the production of KPC-53, a KPC-3 variant exhibiting a Leu167Glu168 duplication in the Ω-loop and a loss of carbapenemase activity. Whole-genome sequencing (WGS) revealed the presence of two copies of blaKPC-53, located on a pKpQIL-like plasmid and on a plasmid prophage of the Siphoviridae family, respectively. The present findings provide new insights into the mechanisms of resistance to ceftazidime-avibactam.


Assuntos
Infecções por Klebsiella , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Compostos Azabicíclicos/farmacologia , Proteínas de Bactérias/genética , Ceftazidima/farmacologia , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana , beta-Lactamases/genética
13.
J Antimicrob Chemother ; 75(4): 979-983, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31958125

RESUMO

OBJECTIVES: To assess the in vitro antibacterial activity of ceftazidime/avibactam against a recent Italian collection of carbapenem-resistant Enterobacterales (CRE) isolated from urine specimens. METHODS: Consecutive Gram-negative isolates from urine specimens, collected from inpatients in five Italian hospitals during the period October 2016 to February 2017, were screened for CRE phenotype using chromogenic selective medium and identified using MALDI-TOF MS. Antimicrobial susceptibility testing was performed by reference broth microdilution (BMD) and, for ceftazidime/avibactam, also by Etest® CZA. Results were interpreted according to the EUCAST breakpoints. All confirmed CRE were subjected to real-time PCR targeting blaKPC-type, blaVIM-type, blaNDM-type and blaOXA-48-type carbapenemase genes. Non-MBL-producing isolates resistant to ceftazidime/avibactam were subjected to WGS and their resistome and clonality were analysed. RESULTS: Overall, 318 non-replicate presumptive CRE were collected following screening of 9405 isolates of Enterobacterales (3.4%) on chromogenic selective medium. Molecular analysis revealed that 216 isolates were positive for a carbapenemase gene (of which 92.1%, 2.8%, 1.4% and 1.4% were positive for blaKPC-type, blaOXA-48-type, blaNDM-type and blaVIM-type, respectively). Against the confirmed carbapenemase-producing Enterobacterales (CPE), ceftazidime/avibactam was the most active compound, followed by colistin (susceptibility rates 91.6% and 69.4%, respectively). Compared with BMD, Etest® for ceftazidime/avibactam yielded consistent results (100% category agreement). All class B ß-lactamase producers were resistant to ceftazidime/avibactam, while OXA-48 and KPC producers were susceptible, with the exception of seven KPC-producing isolates (4.2%). The latter exhibited an MIC of 16 to >32 mg/L, belonged to ST512, produced KPC-3 and showed alterations in the OmpK35 and Ompk36 porins. CONCLUSIONS: Ceftazidime/avibactam showed potent in vitro activity against a recent Italian collection of CPE from urine.


Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos , Ceftazidima , Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Carbapenêmicos , Ceftazidima/farmacologia , Combinação de Medicamentos , Itália , Testes de Sensibilidade Microbiana , beta-Lactamases/genética
14.
Mol Biol Rep ; 47(10): 8301-8304, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32920756

RESUMO

The interest on the role of angiogenesis in the pathogenesis and progression of human interstitial lung diseases is growing, with conventional sprouting (SA) and non-sprouting intussusceptive angiogenesis (IA) being differently represented in specific pulmonary injury patterns. The role of viruses as key regulators of angiogenesis is known for several years. A significantly enhanced amount of new vessel growth, through a mechanism of IA, has been reported in lungs of patients who died from Covid-19; among the angiogenesis-related genes, fibroblast growth factor 2 (FGF2) was found to be upregulated. These findings are intriguing. FGF2 plays a role in some viral infections: the upregulation is involved in the MERS-CoV-induced strong apoptotic response crucial for its highly lytic replication cycle in lung cells, whereas FGF2 is protective against the acute lung injury induced by H1N1 influenza virus, improving the lung wet-to-dry weight ratio. FGF2 plays a role also in regulating IA, acting on pericytes (crucial for the formation of intraluminal pillars), and endothelium, and FGF2-induced angiogenesis may be promoted by inflammation and hypoxia. IA is a faster and probably more efficient process than SA, able to modulate vascular remodeling through pruning of redundant or inefficient blood vessels. We can speculate that IA might have the function of restoring a functional vascular plexus consequently to extensive endothelialitis and alveolar capillary micro-thrombosis observed in Covid-19. Anti-Vascular endothelial growth factor (anti-VEGF) strategies are currently investigated for treatment of severe and critically ill Covid-19 patients, but also FGF2, and its expression and/or signaling, might represent a promising target.


Assuntos
Infecções por Coronavirus/patologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neovascularização Patológica/virologia , Pneumonia Viral/patologia , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19 , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/etiologia , Sistemas de Liberação de Medicamentos , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Humanos , Intussuscepção/virologia , Neovascularização Patológica/genética , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/etiologia
16.
J Antimicrob Chemother ; 74(12): 3453-3461, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31652323

RESUMO

OBJECTIVES: To determine the prevalence of Staphylococcus aureus from hospital-acquired pneumonia (HAP) in Italy and the susceptibility to ceftobiprole and comparators of MSSA and MRSA isolates. A secondary objective was to characterize the clonality and acquired resistance and virulence genes of MRSA. METHODS: Consecutive non-replicate isolates from HAP were collected from 13 laboratories distributed across Italy, from January to May 2016. Antimicrobial susceptibility testing was performed by broth microdilution, and results were interpreted according to the EUCAST breakpoints. All MRSA isolates were subjected to WGS using an Illumina platform. Clonality and resistance and virulence gene content were investigated with bioinformatics tools. RESULTS: Among 333 isolates from HAP, S. aureus was the third most common pathogen (18.6%). The proportion of MRSA was 40.3%. Susceptibility to ceftobiprole was 100% for MSSA and 95.5% for MRSA. Lower susceptibility rates of 78.4% and 94.6% in MSSA and 36.4% and 12.1% in MRSA isolates were observed for erythromycin and levofloxacin, respectively. The MRSA from HAP mostly belonged to clonal complex (CC) 22 (47.0%), CC5 (25.8%) and CC8 (15.2%), with a minority of other lineages (ST1, ST6, ST7, ST30, ST152 and ST398). Acquired resistance and virulence genes in most cases exhibited a clonal distribution. The three ceftobiprole-resistant isolates exhibited an MIC of 4 mg/L and belonged to ST228-MRSA-I of CC5. CONCLUSIONS: S. aureus is an important cause of HAP in Italy. Ceftobiprole exhibited good in vitro activity against S. aureus isolated from HAP, including MRSA. A trend to replacement of ST228 with ST22 was noticed compared with previous studies.


Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Infecção Hospitalar/epidemiologia , Pneumonia Bacteriana/epidemiologia , Infecções Estafilocócicas/epidemiologia , Antibacterianos/uso terapêutico , Técnicas de Tipagem Bacteriana , Cefalosporinas/uso terapêutico , Infecção Hospitalar/microbiologia , DNA Bacteriano/genética , Humanos , Itália/epidemiologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Prevalência , Vigilância em Saúde Pública , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Virulência/genética , Sequenciamento Completo do Genoma
17.
Artigo em Inglês | MEDLINE | ID: mdl-29941651

RESUMO

TEM-184, a novel TEM-derived extended-spectrum ß-lactamase (ESBL), was isolated from an Escherichia coli ST354 clinical strain. Compared to TEM-1, TEM-184 contains the mutations Q6K, E104K, I127V, R164S, and M182T. Kinetic analysis of this enzyme revealed extended-spectrum activity against aztreonam in particular. TEM-184 was also susceptible to inhibitors, including clavulanic acid, tazobactam, and avibactam.


Assuntos
Antibacterianos/farmacologia , Aztreonam/farmacologia , beta-Lactamases/genética , Sequência de Aminoácidos , Compostos Azabicíclicos/farmacologia , Ácido Clavulânico/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Humanos , Cinética , Testes de Sensibilidade Microbiana/métodos , Alinhamento de Sequência , Tazobactam/farmacologia , Inibidores de beta-Lactamases/farmacologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-29661874

RESUMO

Carbapenem-resistant Klebsiella pneumoniae causes important health care-associated infections worldwide. An outbreak of sequence type 11 (ST11) OXA-48-producing K. pneumoniae (OXA-48-Kp) isolates occurred in Tzaneio Hospital in 2012 and was contained until 2014, when OXA-48-Kp reemerged. The present study involved 19 bloodstream infection (BSI) OXA-48-Kp isolates recovered from 19 intensive care unit (ICU) patients hospitalized between August 2014 and July 2016. MICs were determined by broth microdilution. Beta-lactamase genes were detected by PCR. All isolates were typed by pulsed-field gel electrophoresis/multilocus sequence typing (PFGE/MLST), and 10 representative isolates were typed by next-generation sequencing (NGS). Of the 19 study patients, 9 had previous hospitalizations, and 10 carried OXA-48-Kp prior to BSI isolation; median time from ICU admission to BSI was 29 days. Four OXA-48-Kp isolates belonged to PFGE profile A (ST147) and were pandrug resistant (PDR), while 15 isolates exhibited PFGE profile B (ST101) and were extensively drug resistant. Genes detected via NGS resistome analysis accounted for most of the resistance phenotypes, except for tigecycline and fosfomycin. Insertional inactivation of mgrB (distinct per clone) conferred colistin resistance in all 19 isolates. NGS single nucleotide polymorphism (SNP) analysis validated the clonal relatedness of the ST147 and ST101 strains and revealed the possible presence of two index ST147 strains and the microevolution of ST101 strains. Distinct, but highly related, IncL OXA-48-encoding plasmid lineages were identified; plasmids of the ST147 strains were identical with the plasmid of ST11 OXA-48-Kp which caused the 2012 outbreak. In conclusion, biclonal circulation of OXA-48-Kp and, alarmingly, emergence of a PDR clone are reported. These observations, along with the challenging phenotypic detection of OXA-48 producers and the high reported transmissibility of blaOXA-48, necessitate intensive efforts to prevent their further spread.


Assuntos
Antibacterianos/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Eletroforese em Gel de Campo Pulsado , Feminino , Humanos , Unidades de Terapia Intensiva , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Polimorfismo de Nucleotídeo Único/genética , beta-Lactamases/genética
19.
J Antimicrob Chemother ; 73(3): 664-671, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29216350

RESUMO

Objectives: Pseudomonas aeruginosa is a major cause of severe healthcare-associated infections and often shows MDR phenotypes. Ceftolozane/tazobactam is a new cephalosporin/ß-lactamase inhibitor combination with potent activity against P. aeruginosa. This survey was carried out to evaluate the susceptibility of P. aeruginosa, circulating in Italy, to ceftolozane/tazobactam and comparators and to investigate the molecular epidemiology of carbapenemase-producing strains. Methods: Consecutive non-replicate P. aeruginosa clinical isolates (935) from bloodstream infections and lower respiratory tract infections were collected from 20 centres distributed across Italy from September 2013 to November 2014. Antimicrobial susceptibility testing was performed by broth microdilution and results were interpreted according to the EUCAST breakpoints. Isolates resistant to ceftolozane/tazobactam were investigated for carbapenemase genes by PCR, and for carbapenemase activity by spectrophotometric assay. WGS using an Illumina platform was performed on carbapenemase-producing isolates. Results: Ceftolozane/tazobactam was the most active molecule, retaining activity against 90.9% of P. aeruginosa isolates, followed by amikacin (88.0% susceptibility) and colistin (84.7% susceptibility). Overall, 48 isolates (5.1%) were positive for carbapenemase genes, including blaVIM (n = 32), blaIMP (n = 12) and blaGES-5 (n = 4), while the remaining ceftolozane/tazobactam-resistant isolates tested negative for carbapenemase production. Carbapenemase producers belonged to 10 different STs, with ST175 (n = 12) and ST621 (n = 11) being the most common lineages. Genome analysis revealed different trajectories of spread for the different carbapenemase genes. Conclusions: Ceftolozane/tazobactam exhibited potent in vitro activity against P. aeruginosa causing invasive infections in Italy. Carbapenemase production was the most common mechanism of resistance to ceftolozane/tazobactam.


Assuntos
Proteínas de Bactérias/biossíntese , Cefalosporinas/farmacologia , Infecção Hospitalar/epidemiologia , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Tazobactam/farmacologia , beta-Lactamases/biossíntese , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bacteriemia/epidemiologia , Proteínas de Bactérias/genética , Cefalosporinas/uso terapêutico , Infecção Hospitalar/microbiologia , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Monitoramento Epidemiológico , Humanos , Itália/epidemiologia , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/enzimologia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/microbiologia , Tazobactam/uso terapêutico , Sequenciamento Completo do Genoma , beta-Lactamases/genética
20.
Euro Surveill ; 23(45)2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30424831

RESUMO

BackgroundThe mcr-1 gene is a transferable resistance determinant against colistin, a last-resort antimicrobial for infections caused by multi-resistant Gram-negatives.AimTo study carriage of antibiotic-resistant bacteria in healthy school children as part of a helminth control and antimicrobial resistance survey in the Bolivian Chaco region.MethodsFrom September to October 2016 we collected faecal samples from healthy children in eight rural villages. Samples were screened for mcr-1- and mcr-2 genes. Antimicrobial susceptibility testing was performed, and a subset of 18 isolates representative of individuals from different villages was analysed by whole genome sequencing (WGS).ResultsWe included 337 children (mean age: 9.2 years, range: 7-11; 53% females). The proportion of mcr-1 carriers was high (38.3%) and present in all villages; only four children had previous antibiotic exposure. One or more mcr-1-positive isolates were recovered from 129 positive samples, yielding a total of 173 isolates (171 Escherichia coli, 1 Citrobacter europaeus, 1 Enterobacter hormaechei). No mcr-2 was detected. Co-resistance to other antimicrobials varied in mcr-positive E. coli. All 171 isolates were susceptible to carbapenems and tigecycline; 41 (24.0%) were extended-spectrum ß-lactamase producers and most of them (37/41) carried blaCTX-M-type genes. WGS revealed heterogeneity of clonal lineages and mcr-genetic supports.ConclusionThis high prevalence of mcr-1-like carriage, in absence of professional exposure, is unexpected. Its extent at the national level should be investigated with priority. Possible causes should be studied; they may include unrestricted use of colistin in veterinary medicine and animal breeding, and importation of mcr-1-positive bacteria via food and animals.


Assuntos
Portador Sadio/epidemiologia , Farmacorresistência Bacteriana/genética , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Escherichia coli/genética , Escherichia coli/isolamento & purificação , População Rural , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Bolívia/epidemiologia , Carbapenêmicos/farmacologia , Criança , Farmacorresistência Bacteriana Múltipla , Enterobacteriaceae/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli , Feminino , Microbioma Gastrointestinal , Humanos , Masculino , Prevalência
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