First Study of Antimicrobial Activity of Ceftazidime-Avibactam and Ceftolozane-Tazobactam Against Pseudomonas aeruginosa Isolated from Patients with Urinary Tract Infection in Tehran, Iran.
ABSTRACT: PurposePseudomonas aeruginosa causes complicated and/or nosocomial UTI. These infections are usually associated with severe and multi-drug resistant P. aeruginosa isolates. As there is no study about the activity of novel antibiotics ceftazidime-avibactam (CZA) and ceftolozane-tazobactam (C/T) against P. aeruginosa isolates in Iran, we aimed to evaluate for the first time the efficacy of these agents against P. aeruginosa isolated from patients with UTI in Iran. Then, the genetic diversity of the resistant isolates was assayed.MethodsIn this study, a total of 200 P. aeruginosa isolates were collected from patients with UTI in Tehran, Iran. Disk diffusion and Minimum Inhibitory Concentration (MIC) methods were applied to determine the resistance of the isolates to CZA, C/T, and the other antibiotics. Extended-spectrum ?-lactamases (ESBLs) and Metallo Beta Lactamase (MBL) production were assayed by Combination disk diffusion test (CDDT). Polymerase chain reaction (PCR) was carried out to detect the resistance genes, including beta-lactamases and carbapenemases genes. Finally, genomic analysis of the isolates was performed using the Pulse field gel electrophoresis (PFGE).ResultsAmong the isolates, 16 (8%) were resistant to CZA and C/T that MIC confirmed it. The resistant isolates showed high resistance to the other classes of antibiotics. Among the resistant isolates, 31.2% and 75% were ESBL and MBL producers, respectively. The prevalence of blaOXA10, blaVIM, blaOXA48, blaOXA2, and blaCTX-M was 100%, 50%, 31.2%, 25%, and 12.5%. Furthermore, two isolates (12.5%) harbored blaPER and blaNDM genes. The resistant isolates were grouped into 14 distinct pulsotypes and two shared pulsotypes were found.ConclusionCeftazidime-avibactam and ceftolozane-tazobactam showed high activity against the P. aeruginosa isolated from patients with UTI in Iran. The low rate of resistance to the antibiotics is also alarming and should be considered to avoid further spreading of the antibiotic resistance among the P. aeruginosa and the other bacteria.
Project description:Ceftolozane-tazobactam is considered to be a last resort treatment for infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa Although, resistance to this antimicrobial have been described in vitro, development of resistance in vivo was rarely reported. Here, we described the evolution of resistance to ceftolozane-tazobactam of P. aeruginosa isolates recovered from the same patient during recurrent infections over 2.5 years.Antimicrobial susceptibility testing results showed that 24 of the 27 P. aeruginosa isolates recovered from blood (n=18), wound (n=2), pulmonary sample (n=1), bile (n=2) and stools (n=4) of the same patient were susceptible to ceftolozane-tazobactam and ceftazidime-avibactam but resistant to ceftazidime, piperacillin-tazobactam, imipenem and meropenem. Three clinical isolates acquired resistance to ceftolozane-tazobactam and ceftazidime-avibactam along with a partial restoration of piperacillin-tazobactam and carbapenems susceptibilities. Whole genome sequencing analysis reveals that all isolates were clonally related (ST-111) with a median of 24.9 single nucleotide polymorphisms (SNPs) (range 8-48). The ceftolozane-tazobactam and ceftazidime-avibactam resistance was likely linked to the same G183D substitution in the chromosome-encoded cephalosporinase.Our results suggest resistance to ceftolozane-tazobactam in P. aeruginosa might occur in vivo upon treatment through amino-acid substitution in the intrinsic AmpC leading to ceftolozane-tazobactam and ceftazidime-avibactam resistance accompanied by re-sensitization to piperacillin-tazobactam and carbapenems.
Project description:Multidrug-resistant (MDR) Pseudomonas aeruginosa is one of the main causes of morbidity and mortality in hospitalized patients and the leading cause of nosocomial infections. We investigated, here, two MDR P. aeruginosa clinical isolates from a hospitalized patient with differential antimicrobial resistance to ceftazidime/avibactam (CZA), ceftolozane/tazobactam (C/T), and piperacillin/tazobactam (P/T). Their assembled complete genomes revealed they belonged to ST235, a widespread MDR clone; and were isogenic with only a single nucleotide variant, causing G183D mutation in AmpC ?-lactamase, responsible for a phenotypic change from susceptible to resistant to CZA and C/T. Further epigenomic profiling uncovered two conserved DNA methylation motifs targeted by two distinct putative methyltransferase-containing restriction-modification systems, respectively; more intriguingly, there was a significant difference between the paired isolates in the pattern of genomic DNA methylation and modifications. Moreover, genome-wide gene expression profiling demonstrated the inheritable genomic methylation and modification induced 14 genes being differentially regulated, of which only toxR (downregulated), a regulatory transcription factor, had its promoter region differentially methylate and modified. Since highly expressed opdQ encodes an OprD porin family protein, therefore, we proposed an epigenetic regulation of opdQ expression pertinent to the phenotypic change of P. aeruginosa from resistant to susceptible to P/T. The disclosed epigenetic mechanism controlling phenotypic antimicrobial resistance deserves further experimental investigation.
Project description:Multidrug-resistant (MDR) Gram-negative organisms are a major health concern due to lack of effective therapy. Emergence of resistance to newer agents like ceftazidime-avibactam (CZA) further magnifies the problem. In this context, combination therapy of CZA with other antimicrobials may have potential in treating these pathogens. Unfortunately, there are limited data regarding these combinations. Therefore, the objective of this study was to evaluate CZA in combination with amikacin (AMK), aztreonam (AZT), colistin (COL), fosfomycin (FOS), and meropenem (MEM) against 21 carbapenem-resistant Klebsiella pneumoniae and 21 MDR Pseudomonas aeruginosa strains. The potential for synergy was evaluated via MIC combination evaluation and time-kill assays. All strains were further characterized by whole-genome sequencing, quantitative real-time PCR, and SDS-PAGE analysis to determine potential mechanisms of resistance. Compared to CZA alone, we observed a 4-fold decrease in CZA MICs for a majority of K. pneumoniae strains and at least a 2-fold decrease for most P. aeruginosa isolates in the majority of combinations tested. In both P. aeruginosa and K. pneumoniae strains, CZA in combination with AMK or AZT was synergistic (?2.15-log10 CFU/ml decrease). CZA-MEM was effective against P. aeruginosa and CZA-FOS was effective against K. pneumoniae Time-kill analysis also revealed that the synergy of CZA with MEM or AZT may be due to the previously reported restoration of MEM or AZT activity against these organisms. Our findings show that CZA in combination with these antibiotics has potential for therapeutic options in difficult to treat pathogens. Further evaluation of these combinations is warranted.
Project description:Metallo-?-lactamase (MBL)-producing Gram-negative bacteria are often extremely resistant, leading to a real therapeutic dead end. Here, we evaluated the in vitro and in vivo efficacy of aztreonam in combination with ceftazidime-avibactam, ceftolozane-tazobactam, or amoxicillin-clavulanate for the treatment of infections caused by MBL-producing Enterobacteriaceae, MBL-producing Pseudomonas aeruginosa, and extremely drug-resistant Stenotrophomonas maltophilia First, we report two clinical cases, namely, a urinary tract infection caused by an NDM-5-producing Escherichia coli isolate and a pulmonary infection caused by a S. maltophilia isolate efficiently treated with the association of aztreonam-ceftazidime-avibactam and aztreonam-amoxicillin-clavulanate, respectively. Then, a total of 50 MBL-producing Enterobacteriaceae isolates, 3 MBL-producing P. aeruginosa isolates, and 5 extremely drug-resistant S. maltophilia isolates were used to test aztreonam susceptibility in combination with ceftolozane-tazobactam, ceftazidime-avibactam, or amoxicillin-clavulanate. The Etest strip superposition method was used to determine the MICs of the aztreonam/inhibitor combinations. According to CLSI breakpoints, aztreonam susceptibility was fully restored for 86%, 20%, and 50% of the MBL-producing Enterobacteriaceae isolates when combined with ceftazidime-avibactam, ceftolozane-tazobactam, and amoxicillin-clavulanate, respectively. In P. aeruginosa, the aztreonam-ceftazidime-avibactam combination was the most potent, even though the reduction in MICs was at most 2-fold. With the 5 S. maltophilia isolates, aztreonam-ceftazidime-avibactam and aztreonam-amoxicillin-clavulanate were found to be equal (100% susceptibility). Overall, aztreonam-ceftazidime-avibactam was the most potent combination to treat infections caused by MBL producers compared with aztreonam-amoxicillin-clavulanate and aztreonam-ceftolozane-tazobactam. However, in many cases aztreonam-amoxicillin-clavulanate was found to be as efficient as aztreonam-ceftazidime-avibactam, offering the main advantage to be markedly cheaper. We also confirmed the validity of Etest superpositions as a very simple method to determine MICs of aztreonam combinations.
Project description:Background:Treatment of serious infections due to multidrug-resistant (MDR) Pseudomonas aeruginosa remains a challenge, despite the introduction of novel therapeutics. In this study, we report 2 extensively drug-resistant clinical isolates of sequence type (ST) 309 P aeruginosa resistant to all ?-lactams, including the novel combinations ceftolozane/tazobactam, ceftazidime/avibactam, and meropenem/vaborbactam. Methods:Isolates were sequenced using both short-read (Illumina) and long-read technology to identify resistance determinants, polymorphisms (compared with P aeruginosa PAO1), and reconstruct a phylogenetic tree. A pair of ?-lactamases, Guiana extended spectrum ?-lactamase (GES)-19 and GES-26, were cloned and expressed in a laboratory strain of Escherichia coli to examine their relative impact on resistance. Using cell lysates from E coli expressing the GES genes individually and in tandem, we determined relative rates of hydrolysis for nitrocefin and ceftazidime. Results:Two ST309 P aeruginosa clinical isolates were found to harbor the extended spectrum ?-lactamases GES-19 and GES-26 clustered in tandem on a chromosomal class 1 integron. The presence of both enzymes in E coli was associated with significantly elevated minimum inhibitory concentrations to aztreonam, cefepime, meropenem, ceftazidime/avibactam, and ceftolozane/tazobactam, compared with those expressed individually. The combination of ceftazidime/avibactam plus aztreonam was active in vitro and used to achieve cure in one patient. Phylogenetic analysis revealed ST309 P aeruginosa are closely related to MDR strains from Mexico also carrying tandem GES. Conclusions:The presence of tandem GES-19 and GES-26 is associated with resistance to all ?-lactams, including ceftolozane/tazobactam. Phylogenetic analysis suggests that ST309 P aeruginosa may be an emerging threat in the United States.
Project description:Pseudomonas aeruginosa produces a class C ?-lactamase (e.g., PDC-3) that robustly hydrolyzes early generation cephalosporins often at the diffusion limit; therefore, bacteria possessing these ?-lactamases are resistant to many ?-lactam antibiotics. In response to this significant clinical threat, ceftolozane, a 3' aminopyrazolium cephalosporin, was developed. Combined with tazobactam, ceftolozane promised to be effective against multidrug-resistant P. aeruginosa Alarmingly, ?-loop variants of the PDC ?-lactamase (V213A, G216R, E221K, E221G, and Y223H) were identified in ceftolozane/tazobactam-resistant P. aeruginosa clinical isolates. Herein, we demonstrate that the Escherichia coli strain expressing the E221K variant of PDC-3 had the highest minimum inhibitory concentrations (MICs) against a panel of ?-lactam antibiotics, including ceftolozane and ceftazidime, a cephalosporin that differs in structure largely in the R2 side chain. The k cat values of the E221K variant for both substrates were equivalent, whereas the Km for ceftolozane (341?±?64?µM) was higher than that for ceftazidime (174?±?20?µM). Timed mass spectrometry, thermal stability, and equilibrium unfolding studies revealed key mechanistic insights. Enhanced sampling molecular dynamics simulations identified conformational changes in the E221K variant ?-loop, where a hidden pocket adjacent to the catalytic site opens and stabilizes ceftolozane for efficient hydrolysis. Encouragingly, the diazabicyclooctane ?-lactamase inhibitor avibactam restored susceptibility to ceftolozane and ceftazidime in cells producing the E221K variant. In addition, a boronic acid transition state inhibitor, LP-06, lowered the ceftolozane and ceftazidime MICs by 8-fold for the E221K-expressing strain. Understanding these structural changes in evolutionarily selected variants is critical toward designing effective ?-lactam/?-lactamase inhibitor therapies for P. aeruginosa infections.IMPORTANCE The presence of ?-lactamases (e.g., PDC-3) that have naturally evolved and acquired the ability to break down ?-lactam antibiotics (e.g., ceftazidime and ceftolozane) leads to highly resistant and potentially lethal Pseudomonas aeruginosa infections. We show that wild-type PDC-3 ?-lactamase forms an acyl enzyme complex with ceftazidime, but it cannot accommodate the structurally similar ceftolozane that has a longer R2 side chain with increased basicity. A single amino acid substitution from a glutamate to a lysine at position 221 in PDC-3 (E221K) causes the tyrosine residue at 223 to adopt a new position poised for efficient hydrolysis of both cephalosporins. The importance of the mechanism of action of the E221K variant, in particular, is underscored by its evolutionary recurrences in multiple bacterial species. Understanding the biochemical and molecular basis for resistance is key to designing effective therapies and developing new ?-lactam/?-lactamase inhibitor combinations.
Project description:Acute pulmonary exacerbations (APE) involving Pseudomonas aeruginosa are associated with increased morbidity and mortality in cystic fibrosis (CF) patients. Drug resistance is a significant challenge to treatment. Ceftazidime-avibactam (CZA) demonstrates excellent in vitro activity against isolates recovered from CF patients, including drug-resistant strains. Altered pharmacokinetics (PK) of several beta-lactam antibiotics have been reported in CF patients. Therefore, this study sought to characterize the PK of CZA and perform target attainment analyses to determine the optimal treatment regimen. The PK of CZA in 12 adult CF patients administered 3 intravenous doses of 2.5 g every 8 h infused over 2 h were determined. Population modeling utilized the maximum likelihood expectation method. Monte Carlo simulations determined the probability of target attainment (PTA). An exposure target consisting of the cumulative percentage of a 24-h period that the free drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (fT>MIC) was evaluated for ceftazidime (CAZ), and an exposure target consisting of the cumulative percentage of a 24-h period that the free drug concentration exceeds a 1-mg/liter threshold concentration (fT>1 mg/liter) was evaluated for avibactam (AVI). Published CAZ and CZA MIC distributions were incorporated to evaluate cumulative response probabilities. CAZ and AVI were best described by one-compartment models. The values of total body clearance (CL; CAZ CL, 7.53 ± 1.28 liters/h; AVI CL, 12.30 ± 1.96 liters/h) and volume of distribution (V; CAZ V, 18.80 ± 6.54 liters; AVI V, 25.30 ± 4.43 liters) were broadly similar to published values for healthy adults. CZA achieved a PTA (fT>MIC, 50%) of >0.9 for MICs of ?16 mg/liter. The overall likelihood of a treatment response was 0.82 for CZA, whereas it was 0.42 for CAZ. These data demonstrate improved pharmacodynamics of CZA in comparison with those of CAZ and provide guidance on the optimal dosing of CZA for future studies. (This study has been registered at ClinicalTrials.gov under registration no. NCT02504827.).
Project description:Strains of Pseudomonas aeruginosa with deficiencies in DNA mismatch repair have been studied in the context of chronic infection, where elevated mutational rates ("hypermutation") may facilitate the acquisition of antimicrobial resistance. Whether P. aeruginosa hypermutation can also play an adaptive role in the more dynamic context of acute infection remains unclear. In this work, we demonstrate that evolved mismatch repair deficiencies may be exploited by P. aeruginosa to facilitate rapid acquisition of antimicrobial resistance in acute infection, and we directly document rapid clonal succession by such a hypermutating lineage in a patient. Whole-genome sequencing (WGS) was performed on nine serially cultured blood and respiratory isolates from a patient in whom ceftazidime-avibactam (CZA) resistance emerged in vivo over the course of days. The CZA-resistant clone was differentiated by 14 mutations, including a gain-of-function G183D substitution in the PDC-5 chromosomal AmpC cephalosporinase conferring CZA resistance. This lineage also contained a substitution (R656H) at a conserved position in the ATPase domain of the MutS mismatch repair (MMR) protein, and elevated mutational rates were confirmed by mutational accumulation experiments with WGS of evolved lineages in conjunction with rifampin resistance assays. To test whether MMR-deficient hypermutation could facilitate rapid acquisition of CZA resistance, in vitro adaptive evolution experiments were performed with a mutS-deficient strain. These experiments demonstrated rapid hypermutation-facilitated acquisition of CZA resistance compared with the isogenic wild-type strain. Our results suggest a possibly underappreciated role for evolved MMR deficiency in facilitating rapid adaptive evolution of P. aeruginosa in the context of acute infection.IMPORTANCE Antimicrobial resistance in bacteria represents one of the most consequential problems in modern medicine, and its emergence and spread threaten to compromise central advances in the treatment of infectious diseases. Ceftazidime-avibactam (CZA) belongs to a new class of broad-spectrum beta-lactam/beta-lactamase inhibitor combinations designed to treat infections caused by multidrug-resistant bacteria. Understanding the emergence of resistance to this important new drug class is of critical importance. In this work, we demonstrate that evolved mismatch repair deficiency in P. aeruginosa, an important pathogen responsible for significant morbidity and mortality among hospitalized patients, may facilitate rapid acquisition of resistance to CZA in the context of acute infection. These findings are relevant for both diagnosis and treatment of antimicrobial resistance emerging in acute infection in the hypermutator background and additionally have implications for the emergence of more virulent phenotypes.
Project description:A clinical Pseudomonas aeruginosa isolate resistant to all β-lactams, including ceftolozane-tazobactam and carbapenems, was recovered. It belonged to sequence type 235 and produced the extended-spectrum β-lactamase (ESBL) GES-6 differing from GES-1 by two amino acid substitutions (E104K and G170S). GES-6 possessed an increased hydrolytic activity toward carbapenems and to ceftolozane and a decreased susceptibility to β-lactamase inhibitors compared to GES-1, except for avibactam. We show here that resistance to ceftolozane-tazobactam may occur through acquisition of a specific ESBL in P. aeruginosa but that ceftazidime-avibactam combination remains an effective alternative.
Project description:The variability of carbapenem-resistant Pseudomonas aeruginosa strains (CRPA) isolated from urine and respiratory samples in a large microbiological laboratory, serving several health care settings, and from effluents of two wastewater treatment plants (WWTP) from the same region was assessed by PFGE typing and by resistance to 10 antibiotics. During the 12-month period altogether 213 carbapenem-resistant P. aeruginosa isolates were cultured and distributed into 65 pulsotypes and ten resistance profiles. For representatives of all 65 pulsotypes 49 different MLSTs were determined. Variability of clinical and environmental strains was comparable, 130 carbapenem-resistant P. aeruginosa obtained from 109 patients were distributed into 38 pulsotypes, while 83 isolates from WWTPs were classified into 31 pulsotypes. Only 9 pulsotypes were shared between two or more settings (hospital or WWTP). Ten MLST were determined for those prevalent pulsotypes, two of them (ST111 and ST235) are among most successful CRPA types worldwide. Clinical and environmental carbapenem-resistant P. aeruginosa strains differed in antibiotic resistance. The highest proportion of clinical isolates was resistant to piperacillin/tazobactam (52.3%) and ceftazidime (42.3%). The highest proportion of environmental isolates was resistant to ceftazidime (37.1%) and ciprofloxacin (35.5%). The majority of isolates was resistant only to imipenem and/or meropenem. Strains with additional resistances were distributed into nine different patterns. All of them included clinically relevant strains, while environmental strains showed only four additional different patterns.