RESUMO
Multidrug-resistant (MDR) Pseudomonas aeruginosa infections are a major clinical challenge. Many isolates are carbapenem resistant, which severely limits treatment options; thus, novel therapeutic combinations, such as imipenem-relebactam (IMI/REL), ceftazidime-avibactam (CAZ/AVI), ceftolozane-tazobactam (TOL/TAZO), and meropenem-vaborbactam (MEM/VAB) were developed. Here, we studied two extensively drug-resistant (XDR) P. aeruginosa isolates, collected in the United States and Mexico, that demonstrated resistance to IMI/REL. Whole-genome sequencing (WGS) showed that both isolates contained acquired GES ß-lactamases, intrinsic PDC and OXA ß-lactamases, and disruptions in the genes encoding the OprD porin, thereby inhibiting uptake of carbapenems. In one isolate (ST17), the entire C terminus of OprD deviated from the expected amino acid sequence after amino acid G388. In the other (ST309), the entire oprD gene was interrupted by an ISPa1328 insertion element after amino acid D43, rendering this porin nonfunctional. The poor inhibition by REL of the GES ß-lactamases (GES-2, -19, and -20; apparent Ki of 19 ± 2 µM, 23 ± 2 µM, and 21 ± 2 µM, respectively) within the isolates also contributed to the observed IMI/REL-resistant phenotype. Modeling of REL binding to the active site of GES-20 suggested that the acylated REL is positioned in an unstable conformation as a result of a constrained Ω-loop.
Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Aminoácidos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/uso terapêutico , Combinação de Medicamentos , Humanos , Imipenem/farmacologia , Imipenem/uso terapêutico , Testes de Sensibilidade Microbiana , Porinas/genética , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Estados Unidos , beta-Lactamases/metabolismoRESUMO
Previously, by targeting penicillin-binding protein 3, Pseudomonas-derived cephalosporinase (PDC), and MurA with ceftazidime-avibactam-fosfomycin, antimicrobial susceptibility was restored among multidrug-resistant (MDR) Pseudomonas aeruginosa. Herein, ceftazidime-avibactam-fosfomycin combination therapy against MDR P. aeruginosa clinical isolate CL232 was further evaluated. Checkerboard susceptibility analysis revealed synergy between ceftazidime-avibactam and fosfomycin. Accordingly, the resistance elements present and expressed in P. aeruginosa were analyzed using whole-genome sequencing and transcriptome profiling. Mutations in genes that are known to contribute to ß-lactam resistance were identified. Moreover, expression of blaPDC, the mexAB-oprM efflux pump, and murA were upregulated. When fosfomycin was administered alone, the frequency of mutations conferring resistance was high; however, coadministration of fosfomycin with ceftazidime-avibactam yielded a lower frequency of resistance mutations. In a murine infection model using a high bacterial burden, ceftazidime-avibactam-fosfomycin significantly reduced the P. aeruginosa colony-forming units (CFUs), by approximately 2 and 5 logs, compared with stasis and in the vehicle-treated control, respectively. Administration of ceftazidime-avibactam and fosfomycin separately significantly increased CFUs, by approximately 3 logs and 1 log, respectively, compared with the number at stasis, and only reduced CFUs by approximately 1 log and 2 logs, respectively, compared with the number in the vehicle-treated control. Thus, the combination of ceftazidime-avibactam-fosfomycin was superior to either drug alone. By employing a "mechanism-based approach" to combination chemotherapy, we show that ceftazidime-avibactam-fosfomycin has the potential to offer infected patients with high bacterial burdens a therapeutic hope against infection with MDR P. aeruginosa that lack metallo-ß-lactamases.
Assuntos
Antibacterianos/administração & dosagem , Compostos Azabicíclicos/administração & dosagem , Ceftazidima/administração & dosagem , Farmacorresistência Bacteriana Múltipla , Fosfomicina/administração & dosagem , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Combinação de Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Mutação , Infecções por Pseudomonas/microbiologia , Células-TroncoAssuntos
Infecções por Erysipelothrix/diagnóstico , Erysipelothrix/isolamento & purificação , Dedos/patologia , Dermatoses da Mão/microbiologia , Diagnóstico Diferencial , Infecções por Erysipelothrix/complicações , Eritema/etiologia , Febre/etiologia , Dedos/diagnóstico por imagem , Dedos/microbiologia , Dermatoses da Mão/patologia , Humanos , Imageamento Tridimensional , Masculino , Dor/etiologia , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
Enzymes are continually evolving in response to environmental pressures. In order to increase enzyme fitness, amino acid substitutions can occur leading to a changing function or an increased stability. These evolutionary drivers determine the activity of an enzyme and its success in future generations in response to changing conditions such as environmental stressors or to improve physiological function allowing continual persistence of the enzyme. With recent warning reports on antibiotic resistance and multidrug resistant bacterial infections, understanding the evolution of ß-lactamase enzymes, which are a large contributor to antibiotic resistance, is increasingly important. Here, we investigated a variant of the SHV ß-lactamase identified from a clinical isolate of Escherichia coli in 2011 (SHV-129, G238S-E240K-R275L-N276D) to identify the first instance of a global suppressor substitution in the SHV ß-lactamase family. We have used this enzyme to show that several evolutionary principles are conserved in different class A ß-lactamases, such as active site mutations reducing stability and requiring compensating suppressor substitutions in order to ensure evolutionary persistence of a given ß-lactamase. However, the pathway taken by a given ß-lactamase in order to reach its evolutionary peak under a given set of conditions is likely different. We also provide further evidence for a conserved stabilizing substitution among class A ß-lactamases, the back to consensus M182T substitution. In addition to expanding the spectrum of ß-lactamase activity to include the hydrolysis of cefepime, the amino acid substitutions found in SHV-129 provide the enzyme with an excess of stability, which expands the evolutionary landscape of this enzyme and may result in further evolution to potentially include resistance to carbapenems or ß-lactamase inhibitors.
Assuntos
Escherichia coli/genética , Família Multigênica , Supressão Genética , beta-Lactamases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Evolução Molecular , Humanos , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Alinhamento de Sequência , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/químicaRESUMO
Boronic acid transition state inhibitors (BATSIs) are competitive, reversible ß-lactamase inhibitors (BLIs). In this study, a series of BATSIs with selectively modified regions (R1, R2, and amide group) were strategically designed and tested against representative class A ß-lactamases of Klebsiella pneumoniae, KPC-2 and SHV-1. Firstly, the R1 group of compounds 1a to 1c and 2a to 2e mimicked the side chain of cephalothin, whereas for compounds 3a to 3c, 4a, and 4b, the thiophene ring was replaced by a phenyl, typical of benzylpenicillin. Secondly, variations in the R2 groups which included substituted aryl side chains (compounds 1a, 1b, 1c, 3a, 3b, and 3c) and triazole groups (compounds 2a to 2e) were chosen to mimic the thiazolidine and dihydrothiazine ring of penicillins and cephalosporins, respectively. Thirdly, the amide backbone of the BATSI, which corresponds to the amide at C-6 or C-7 of ß-lactams, was also changed to the following bioisosteric groups: urea (compound 3b), thiourea (compound 3c), and sulfonamide (compounds 4a and 4b). Among the compounds that inhibited KPC-2 and SHV-1 ß-lactamases, nine possessed 50% inhibitory concentrations (IC50s) of ≤ 600 nM. The most active compounds contained the thiopheneacetyl group at R1 and for the chiral BATSIs, a carboxy- or hydroxy-substituted aryl group at R2. The most active sulfonamido derivative, compound 4b, lacked an R2 group. Compound 2b (S02030) was the most active, with acylation rates (k2/K) of 1.2 ± 0.2 × 10(4) M(-1) s(-1) for KPC-2 and 4.7 ± 0.6 × 10(3) M(-1) s(-1) for SHV-1, and demonstrated antimicrobial activity against Escherichia coli DH10B carrying blaSHV variants and blaKPC-2 or blaKPC-3 and against clinical strains of Klebsiella pneumoniae and E. coli producing different class A ß-lactamase genes. At most, MICs decreased from 16 to 0.5 mg/liter.
Assuntos
Ácidos Borônicos/farmacologia , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Triazóis/farmacologia , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/efeitos dos fármacos , Ácidos Borônicos/química , Ceftazidima/química , Cefalotina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Testes de Sensibilidade Microbiana , Penicilinas/química , Relação Estrutura-AtividadeRESUMO
Around the world, Burkholderia spp. are emerging as pathogens highly resistant to ß-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A ß-lactamase PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A ß-lactamases in B. pseudomallei is largely undefined. Here, we performed site saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 to 64 mg/liter) when expressed in Escherichia coli. Surprisingly, quantitative immunoblotting showed that the steady-state protein levels of the C69F variant ß-lactamase were â¼4-fold lower than those of wild-type PenI (0.76 fg of protein/cell versus 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the relative amount of the C69F variant to greater than wild-type PenI levels. The C69F variant exhibits a branched kinetic mechanism for ceftazidime hydrolysis, suggesting there are two different conformations of the enzyme. When incubated with an anti-PenI antibody, one conformation of the C69F variant rapidly hydrolyzes ceftazidime and most likely contributes to the higher levels of ceftazidime resistance observed in cell-based assays. Molecular dynamics simulations suggest that the electrostatic characteristics of the oxyanion hole are altered in the C69F variant. When ceftazidime was positioned in the active site, the C69F variant is predicted to form a greater number of hydrogen-bonding interactions than PenI with ceftazidime. In conclusion, we propose "a new twist" for enhanced ceftazidime resistance mediated by the C69F variant of the PenI ß-lactamase based on conformational changes in the C69F variant. Our findings explain the biochemical basis of ceftazidime resistance in B. pseudomallei, a pathogen of considerable importance, and suggest that the full repertoire of conformational states of a ß-lactamase profoundly affects ß-lactam resistance.
Assuntos
Antibacterianos/farmacologia , Burkholderia pseudomallei/genética , Ceftazidima/farmacologia , Escherichia coli/genética , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , Burkholderia pseudomallei/efeitos dos fármacos , Domínio Catalítico/genética , Cristalografia por Raios X , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , beta-Lactamases/ultraestruturaRESUMO
ß-Lactamase enzymes (EC 3.5.2.6) are a significant threat to the continued use of ß-lactam antibiotics to treat infections. A novel non-ß-lactam ß-lactamase inhibitor with activity against many class A and C and some class D ß-lactamase variants, avibactam, is now available in the clinic in partnership with ceftazidime. Here, we explored the activity of avibactam against a variety of characterized isogenic laboratory constructs of ß-lactamase inhibitor-resistant variants of the class A enzyme SHV (M69I/L/V, S130G, K234R, R244S, and N276D). We discovered that the S130G variant of SHV-1 shows the most significant resistance to inhibition by avibactam, based on both microbiological and biochemical characterizations. Using a constant concentration of 4 mg/liter of avibactam as a ß-lactamase inhibitor in combination with ampicillin, the MIC increased from 1 mg/liter for blaSHV-1 to 256 mg/liter for blaSHV S130G expressed in Escherichia coli DH10B. At steady state, the k2/K value of the S130G variant when inactivated by avibactam was 1.3 M(-1) s(-1), versus 60,300 M(-1) s(-1) for the SHV-1 ß-lactamase. Under timed inactivation conditions, we found that an approximately 1,700-fold-higher avibactam concentration was required to inhibit SHV S130G than the concentration that inhibited SHV-1. Molecular modeling suggested that the positioning of amino acids in the active site of SHV may result in an alternative pathway of inactivation when complexed with avibactam, compared to the structure of CTX-M-15-avibactam, and that S130 plays a role in the acylation of avibactam as a general acid/base. In addition, S130 may play a role in recyclization. As a result, we advance that the lack of a hydroxyl group at position 130 in the S130G variant of SHV-1 substantially slows carbamylation of the ß-lactamase by avibactam by (i) removing an important proton acceptor and donator in catalysis and (ii) decreasing the number of H bonds. In addition, recyclization is most likely also slow due to the lack of a general base to initiate the process. Considering other inhibitor-resistant mechanisms among class A ß-lactamases, S130 may be the most important amino acid for the inhibition of class A ß-lactamases, perhaps even for the novel diazabicyclooctane class of ß-lactamase inhibitors.
Assuntos
Compostos Azabicíclicos/farmacologia , Farmacorresistência Bacteriana/fisiologia , Escherichia coli/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/química , Acilação , Ampicilina/farmacologia , Antibacterianos/farmacologia , Domínio Catalítico/efeitos dos fármacos , Ceftazidima/farmacologia , Combinação de Medicamentos , Escherichia coli/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , beta-Lactamases/efeitos dos fármacos , beta-Lactamases/ultraestruturaRESUMO
KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the ß-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 ß-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-ß-lactam ß-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 ß-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 ß-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV ß-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling. Escherichia coli DH10B carrying KPC-2 ß-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the k2/K ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M(-1) s(-1) to 1.2 M(-1) s(-1)). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent against E. coli DH10B strains possessing all of these variants.
Assuntos
Ampicilina/farmacologia , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , Escherichia coli/efeitos dos fármacos , beta-Lactamases/genética , Acilação , Substituição de Aminoácidos/genética , Antibacterianos/farmacologia , Cristalografia por Raios X , Combinação de Medicamentos , Escherichia coli/enzimologia , Escherichia coli/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Dinâmica Molecular , Resistência beta-Lactâmica/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/efeitos dos fármacos , beta-Lactamases/ultraestruturaRESUMO
Pseudomonas aeruginosa is a notoriously difficult-to-treat pathogen that is a common cause of severe nosocomial infections. Investigating a collection of ß-lactam-resistant P. aeruginosa clinical isolates from a decade ago, we uncovered resistance to ceftazidime-avibactam, a novel ß-lactam/ß-lactamase inhibitor combination. The isolates were systematically analyzed through a variety of genetic, biochemical, genomic, and microbiological methods to understand how resistance manifests to a unique drug combination that is not yet clinically released. We discovered that avibactam was able to inactivate different AmpC ß-lactamase enzymes and that blaPDC regulatory elements and penicillin-binding protein differences did not contribute in a major way to resistance. By using carefully selected combinations of antimicrobial agents, we deduced that the greatest barrier to ceftazidime-avibactam is membrane permeability and drug efflux. To overcome the constellation of resistance determinants, we show that a combination of antimicrobial agents (ceftazidime/avibactam/fosfomycin) targeting multiple cell wall synthetic pathways can restore susceptibility. In P. aeruginosa, efflux, as a general mechanism of resistance, may pose the greatest challenge to future antibiotic development. Our unexpected findings create concern that even the development of antimicrobial agents targeted for the treatment of multidrug-resistant bacteria may encounter clinically important resistance. Antibiotic therapy in the future must consider these factors.
Assuntos
Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Fosfomicina/farmacologia , Bactérias Gram-Negativas , Humanos , Testes de Sensibilidade MicrobianaRESUMO
OBJECTIVES: The objective of this study was to explore the activity of ceftazidime and ceftazidime/avibactam against a collection of isogenic strains of Escherichia coli DH10B possessing SHV and KPC ß-lactamases containing single amino acid substitutions in the Ω-loop (residues 164-179). METHODS: Ceftazidime and ceftazidime/avibactam MICs were determined by the agar dilution method for a panel of isogenic E. coli strains expressing SHV-1 and KPC-2 with amino acid substitutions at positions 164, 167, 169 or 179. Two KPC-2 ß-lactamase variants that possessed elevated MICs of ceftazidime/avibactam were selected for further biochemical analyses. RESULTS: Avibactam restored susceptibility to ceftazidime for all Ω-loop variants of SHV-1 with MICs <8 mg/L. In contrast, several of the Arg164 and Asp179 variants of KPC-2 demonstrated MICs of ceftazidime/avibactam >8 mg/L. ß-Lactamase kinetics showed that the Asp179Asn variant of KPC-2 demonstrated enhanced kinetic properties against ceftazidime. The Ki app, k2/K and koff of the Arg164Ala and Asp179Asn variant KPC-2 ß-lactamases indicated that avibactam effectively inhibited these enzymes. CONCLUSIONS: Several KPC-2 variants demonstrating ceftazidime resistance as a result of single amino acid substitutions in the Ω-loop were not susceptible to ceftazidime/avibactam (MICs >8 mg/L). We hypothesize that this observation is due to the stabilizing interactions (e.g. hydrogen bonds) of ceftazidime within the active site of variant ß-lactamases that prevent avibactam from binding to and inhibiting the ß-lactamase. As ceftazidime/avibactam is introduced into the clinic, monitoring for new KPC-2 variants that may exhibit increased ceftazidime kinetics as well as resistance to this novel antibiotic combination will be important.
Assuntos
Substituição de Aminoácidos , Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas Mutantes/metabolismo , beta-Lactamases/metabolismo , Combinação de Medicamentos , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Proteínas Mutantes/genética , beta-Lactamases/genéticaRESUMO
CMY-2 is a plasmid-encoded Ambler class C cephalosporinase that is widely disseminated in Enterobacteriaceae and is responsible for expanded-spectrum cephalosporin resistance. As a result of resistance to both ceftazidime and ß-lactamase inhibitors in strains carrying blaCMY, novel ß-lactam-ß-lactamase inhibitor combinations are sought to combat this significant threat to ß-lactam therapy. Avibactam is a bridged diazabicyclo [3.2.1]octanone non-ß-lactam ß-lactamase inhibitor in clinical development that reversibly inactivates serine ß-lactamases. To define the spectrum of activity of ceftazidime-avibactam, we tested the susceptibilities of Escherichia coli clinical isolates that carry bla(CMY-2) or bla(CMY-69) and investigated the inactivation kinetics of CMY-2. Our analysis showed that CMY-2-containing clinical isolates of E. coli were highly susceptible to ceftazidime-avibactam (MIC(90), ≤ 0.5 mg/liter); in comparison, ceftazidime had a MIC90 of >128 mg/liter. More importantly, avibactam was an extremely potent inhibitor of CMY-2 ß-lactamase, as demonstrated by a second-order onset of acylation rate constant (k2/K) of (4.9 ± 0.5) × 10(4) M(-1) s(-1) and the off-rate constant (k(off)) of (3.7 ± 0.4) × 10(-4) s(-1). Analysis of the reaction of avibactam with CMY-2 using mass spectrometry to capture reaction intermediates revealed that the CMY-2-avibactam acyl-enzyme complex was stable for as long as 24 h. Molecular modeling studies raise the hypothesis that a series of successive hydrogen-bonding interactions occur as avibactam proceeds through the reaction coordinate with CMY-2 (e.g., T316, G317, S318, T319, S343, N346, and R349). Our findings support the microbiological and biochemical efficacy of ceftazidime-avibactam against E. coli containing plasmid-borne CMY-2 and CMY-69.
Assuntos
Antibacterianos/farmacologia , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , Escherichia coli/efeitos dos fármacos , Resistência beta-Lactâmica/genética , beta-Lactamases/química , Sinergismo Farmacológico , Quimioterapia Combinada , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Ligação de Hidrogênio , Cinética , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Ligação Proteica , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
OBJECTIVE: The importance of infection prevention and control and healthcare epidemiology (IPC/HE) in healthcare facilities was highlighted during the COVID-19 pandemic. Infectious disease (ID) clinicians often hold leadership positions in IPC/HE teams; however, there is no standard for training or certification of ID physicians specializing in IPC/HE. We evaluated the current state of IPC/HE training in ID fellowship programs. DESIGN: A national survey of ID fellowship program directors was conducted to assess current IPC/HE training components in programs and plans for expanded offerings. SETTING AND PARTICIPANTS: All ID fellowship program directors in the United States and Puerto Rico. METHODS: Surveys were distributed using Research Electronic Data Capture (REDCap) to program directors in March 2023, with 2 reminder emails; the survey closed after 4 weeks. RESULTS: Of 166 program directors, 54 (32.5%) responded to the survey. Among respondent programs, 49 (90.7%) of 54 programs reported didactic training in IPC/HE averaging 4.4 hours over the course of the fellowship. Also, 18 (33.3%) of 54 reported a dedicated IPC/HE training track. Furthermore, 23 programs (42.6%) reported barriers to expanding training. There was support (n = 47, 87.0%) for formal IPC/HE certification from a professional society within the standard fellowship. CONCLUSIONS: Despite the COVID-19 pandemic highlighting the need for ID medical doctors with IPC/HE expertise, formal training in ID fellowship remains limited. Most program directors support formalization of IPC/HE training by a professional organization. Creation of standardized advanced curriculums for ID fellowship training in IPC/HE could be considered by the Society of Healthcare Epidemiology of America (SHEA) to grow, retain, and enhance the IPC/HE physician workforce.
Assuntos
COVID-19 , Doenças Transmissíveis , Humanos , Estados Unidos , Bolsas de Estudo , Pandemias/prevenção & controle , Educação de Pós-Graduação em Medicina , Atenção à Saúde , Inquéritos e QuestionáriosRESUMO
The past 10 years have brought paradigm-shifting changes to clinical microbiology. This paper explores the top 10 transformative innovations across the diagnostic spectrum, including not only state of the art technologies but also preanalytic and post-analytic advances. Clinical decision support tools have reshaped testing practices, curbing unnecessary tests. Innovations like broad-range polymerase chain reaction and metagenomic sequencing, whole genome sequencing, multiplex molecular panels, rapid phenotypic susceptibility testing, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry have all expanded our diagnostic armamentarium. Rapid home-based testing has made diagnostic testing more accessible than ever. Enhancements to clinician-laboratory interfaces allow for automated stewardship interventions and education. Laboratory restructuring and consolidation efforts are reshaping the field of microbiology, presenting both opportunities and challenges for the future of clinical microbiology laboratories. Here, we review key innovations of the last decade.
RESUMO
Mutations in the cohesin complex components (STAG2, RAD21, SMC1A, SMC3, and PDS5B) are recurrent genetic drivers in myelodysplastic neoplasm (MDS) and acute myeloid leukemia (AML). Whether the different cohesin subunit mutations share clinical characteristics and prognostic significance is not known. We analyzed 790 cohesin-mutant patients from the Dana-Farber Cancer Institute (DFCI) and the Munich Leukemia Laboratory (MLL), 390 of which had available outcome data, and identified subunit-specific clinical, prognostic, and genetic characteristics suggestive of distinct ontogenies. We found that STAG2 mutations are acquired at MDS stage and are associated with secondary AML, adverse prognosis, and co-occurrence of secondary AML-type mutations. In contrast, mutations in RAD21, SMC1A and SMC3 share features with de novo AML with better prognosis, and co-occurrence with de novo AML-type lesions. The findings show the heterogeneous nature of cohesin complex mutations, and inform clinical and prognostic classification, as well as distinct biology of the cohesin complex.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Coesinas , Leucemia Mieloide Aguda , Mutação , Síndromes Mielodisplásicas , Humanos , Proteínas Cromossômicas não Histona/genética , Proteínas de Ciclo Celular/genética , Prognóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Adulto , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas de Ligação a DNA/genética , Idoso de 80 Anos ou mais , Proteoglicanas de Sulfatos de CondroitinaRESUMO
Background: Bacillus cereus is a ubiquitous gram-positive rod-shaped bacterium that can cause sepsis and neuroinvasive disease in patients with acute leukemia or neutropenia. Methods: A single-center retrospective review was conducted to evaluate patients with acute leukemia, positive blood or cerebrospinal fluid test results for B cereus, and abnormal neuroradiographic findings between January 2018 and October 2022. Infection control practices were observed, environmental samples obtained, a dietary case-control study completed, and whole genome sequencing performed on environmental and clinical Bacillus isolates. Results: Five patients with B cereus neuroinvasive disease were identified. All patients had acute myeloid leukemia (AML), were receiving induction chemotherapy, and were neutropenic. Neurologic involvement included subarachnoid or intraparenchymal hemorrhage or brain abscess. All patients were treated with ciprofloxacin and survived with limited or no neurologic sequelae. B cereus was identified in 7 of 61 environmental samples and 1 of 19 dietary protein samples-these were unrelated to clinical isolates via sequencing. No point source was identified. Ciprofloxacin was added to the empiric antimicrobial regimen for patients with AML and prolonged or recurrent neutropenic fevers; no new cases were identified in the ensuing year. Conclusions: B cereus is ubiquitous in the hospital environment, at times leading to clusters with unrelated isolates. Fastidious infection control practices addressing a range of possible exposures are warranted, but their efficacy is unknown and they may not be sufficient to prevent all infections. Thus, including B cereus coverage in empiric regimens for patients with AML and persistent neutropenic fever may limit the morbidity of this pathogen.
RESUMO
Gram-negative bacteria harboring KPC-2, a class A ß-lactamase, are resistant to all ß-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A ß-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32â128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative ß-lactamase for more detailed analysis, we observed only a modest 25% increase in k(cat)/K(m) for ceftazidime (0.015â0.019 µm(-1) s(-1)). Employing pre-steady-state kinetics and mass spectrometry, we determined that acylation is rate-limiting for ceftazidime hydrolysis by KPC-2, whereas deacylation is rate-limiting in the R164S variant, leading to accumulation of acyl-enzyme at steady-state. CD spectroscopy revealed that a conformational change occurred in the turnover of ceftazidime by KPC-2, but not the R164S variant, providing evidence for a different form of the enzyme at steady state. Molecular models constructed to explain these findings suggest that ceftazidime adopts a unique conformation, despite preservation of Ω-loop structure. We propose that the R164S substitution in KPC-2 enhances ceftazidime resistance by proceeding through "covalent trapping" of the substrate by a deacylation impaired enzyme with a lower K(m). Future antibiotic design must consider the distinctive behavior of the Ω-loop of KPC-2.
Assuntos
Ceftazidima/química , beta-Lactamases/química , Sequência de Aminoácidos , Antibacterianos/química , Dicroísmo Circular , Hidrólise , Cinética , Klebsiella pneumoniae/enzimologia , Espectrometria de Massas/métodos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização por Electrospray/métodos , beta-Lactamases/metabolismoRESUMO
Objective: Screening for asymptomatic bacteriuria (ASB) is not recommended outside of patients undergoing invasive urological procedures and during pregnancy. Despite national guidelines recommending against screening for ASB, this practice is prevalent. We present outcomes from a quality-improvement intervention targeting patients undergoing cardiac artery bypass grafting surgery (CABG) at Massachusetts General Hospital, a tertiary-care hospital in Boston, Massachusetts, where preoperative testing checklists were modified to remove routine urinalysis and urine culture. This was a before-and-after intervention study. Methods: Prior to the intervention, screening for ASB was included in the preoperative check list for all patients undergoing CABG. We assessed the proportion of patients undergoing screening for ASB in the 6 months prior to and after the intervention. We estimated cost savings from averted laboratory analyses, and we evaluated changes in antibiotic prescriptions. We additionally examined the incidence of postoperative surgical-site infections (SSIs), central-line-associated bloodstream infections (CLABSIs), catheter-associated urinary tract infections (CAUTIs) and Clostridioides difficile infections (CDIs). Results: Comparing the pre- and postintervention periods, urinalyses decreased by 76.5% and urine cultures decreased by 87.0%, with an estimated cost savings of $8,090.38. There were 50% fewer antibiotic prescriptions for bacteriuria after the intervention. Conclusions: Removal of urinalysis and urine culture from preoperative checklists for cardiac surgery led to a statistically significant decrease in testing without an increase in SSIs, CLABSIs, CAUTIs, or CDI. Challenges identified included persistence of checklists in templated order sets in the electronic health record.
RESUMO
The authors describe infection prevention and control approaches to severe acute respiratory syndrome coronavirus 2 in the health care setting, including a review of the chain of transmission and the hierarchy of controls, which are cornerstones of infection control and prevention. The authors also discuss lessons learned from nosocomial transmission events.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/prevenção & controle , Atenção à Saúde , Humanos , Controle de InfecçõesRESUMO
To establish the feasibility of empirically testing crisis standards of care guidelines. DESIGN: Retrospective single-center study. SETTING: ICUs at a large academic medical center in the United States. SUBJECTS: Adult, critically ill patients admitted to ICU, with 27 patients admitted for acute respiratory failure due to coronavirus disease 2019 and 37 patients admitted for diagnoses other than coronavirus disease 2019. INTERVENTIONS: Review of electronic health record. MEASUREMENTS AND MAIN RESULTS: Many U.S. states released crisis standards of care guidelines with algorithms to allocate scarce healthcare resources during the coronavirus disease 2019 pandemic. We compared state guidelines that represent different approaches to incorporating disease severity and comorbidities: New York, Maryland, Pennsylvania, and Colorado. Following each algorithm, we calculated priority scores at the time of ICU admission for a cohort of patients with primary diagnoses of coronavirus disease 2019 and diseases other than coronavirus disease 2019 (n = 64). We assessed discrimination of 28-day mortality by area under the receiver operating characteristic curve. We simulated real-time decision-making by applying the triage algorithms to groups of two, five, or 10 patients. For prediction of 28-day mortality by priority scores, area under the receiver operating characteristic curve was 0.56, 0.49, 0.53, 0.66, and 0.69 for New York, Maryland, Pennsylvania, Colorado, and raw Sequential Organ Failure Assessment score algorithms, respectively. For groups of five patients, the percentage of decisions made without deferring to a lottery were 1%, 57%, 80%, 88%, and 95% for New York, Maryland, Pennsylvania, Colorado, and raw Sequential Organ Failure Assessment score algorithms, respectively. The percentage of decisions made without lottery was higher in the subcohort without coronavirus disease 2019, compared with the subcohort with coronavirus disease 2019. CONCLUSIONS: Inclusion of comorbidities does not consistently improve an algorithm's performance in predicting 28-day mortality. Crisis standards of care algorithms result in a substantial percentage of tied priority scores. Crisis standards of care algorithms operate differently in cohorts with and without coronavirus disease 2019. This proof-of-principle study demonstrates the feasibility and importance of empirical testing of crisis standards of care guidelines to understand whether they meet their goals.