Variability of Beta-Lactam Broth Microdilution for Pseudomonas aeruginosa

Author(s):  
A. A. Bhalodi ◽  
N. Oppermann ◽  
S. A. Campeau ◽  
R. M. Humphries

Antimicrobial susceptibility testing for Pseudomonas aeruginosa is critical to determine suitable treatment options. Commercial susceptibility tests are typically calibrated against the reference method, broth microdilution (BMD). Imprecision of minimum inhibitory concentrations (MICs) obtained by BMD for the same isolate on repeat testing is known to exist. Factors that impact the extent of variability include concentration of the inoculum, operator effects, contents of the media, inherent strain properties, and the testing process or materials. We evaluated the variability of BMD for anti-pseudomonal beta-lactams (aztreonam, cefepime, ceftazidime, meropenem, piperacillin-tazobactam, ceftazidime-avibactam, ceftolozane-tazobactam) tested against a collection of P. aeruginosa isolates. Multiple replicate BMD tests were performed and MICs were compared to assess reproducibility, including the impact of the inoculum and operator. Overall, essential agreement (EA) was ≥ 90% for all beta-lactams tested. Absolute agreement (AA) was as low as 70% for some beta-lactams. Variability from the inoculum and operators impacted the reproducibility of MICs. Piperacillin-tazobactam exhibited the highest degree of variability with 74% AA and 94%% EA. The implications of MIC variability are extensive as the MIC is essential for multiple facets of microbiology, such as the development of new compounds and susceptibility tests, dose optimization and pharmacokinetic/pharmacodynamic (PK/PD) targets for individual patients.

Author(s):  
Natália Conceição ◽  
Wellington Francisco Rodrigues ◽  
Kessys Lorrânya Peralta de Oliveira ◽  
Lucas Emanuel Pinheiro da Silva ◽  
Laís Rezende Cardoso de Souza ◽  
...  

Abstract This study aimed to evaluate the accuracy of disk diffusion and Etest methods, compared to that of the broth dilution reference method for identifying beta-lactam susceptibilities of Penicillin-Resistant, Ampicillin-Susceptible Enterococcus faecalis (PRASEF) isolates. Fifty-nine PRASEF and 15 Penicillin-Susceptible, Ampicillin-Susceptible E. faecalis (PSASEF) clinical nonrepetitive isolates were evaluated. The effectiveness of five beta-lactams (ampicillin, amoxicillin, imipenem, penicillin, and piperacillin) was tested. All antimicrobial susceptibility tests were performed and interpreted according to the Clinical and Laboratory Standards Institute guidelines. Interpretative discrepancies, such as essential agreement, categorical agreement, and errors, were assessed. The acceptability was ≥ 90% for both categorical agreement and essential agreement. Etest proved to be an accurate method for testing beta-lactam susceptibilities of the emerging PRASEF isolates, disk diffusion presented poor performance, particularly for imipenem and piperacillin.


2010 ◽  
Vol 4 (04) ◽  
pp. 239-242 ◽  
Author(s):  
Supriya Upadhyay ◽  
Malay Ranjan Sen ◽  
Amitabha Bhattacharjee

Introduction: Infections caused by Pseudomonas aeruginosa are difficult to treat as the majority of isolates exhibit varying degrees of beta-lactamase mediated resistance to most of the beta-lactam antibiotics. It is also not unusual to find a single isolate that expresses multiple β-lactamase enzymes, further complicating the treatment options. Thus the present study was designed to investigate the coexistence of different beta-lactamase enzymes in clinical isolates of P. aeruginosa. Methodology: A total of 202 clinical isolates of P. aeruginosa were tested for the presence of AmpC beta-lactamase, extended spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) enzyme. Detection of AmpC beta-lactamase was performed by disk antagonism test and a modified three-dimensional method, whereas detection of ESBL was done by the combined disk diffusion method per Clinical and Laboratory Standards Institute (CLSI) guidelines and MBL were detected by the Imipenem EDTA disk potentiation test. Results: A total of 120 (59.4%) isolates were confirmed to be positive for AmpC beta-lactamase. Among them, 14 strains (7%) were inducible AmpC producers. Co-production of AmpC along with extended spectrum beta-lactamase and metallo beta-lactamase was reported in 3.3% and 46.6% isolates respectively. Conclusion: The study emphasizes the high prevalence of multidrug resistant P. aeruginosa producing beta-lactamase enzymes of diverse mechanisms. Thus proper antibiotic policy and measures to restrict the indiscriminative use of cephalosporins and carbapenems should be taken to minimize the emergence of this multiple beta-lactamase producing pathogens.


Antibiotics ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 67 ◽  
Author(s):  
Ahmed Zikri ◽  
Kamal El Masri

Infections, with multidrug-resistant Pseudomonas aeruginosa, are a major concern in the pediatric intensive care unit, especially in immunocompromised patients. Some of these strains are resistant to all beta-lactams, including carbapenems, leaving very limited treatment options remaining. These options include aminoglycosides and colistin, both of which have poor pharmacokinetic profiles with significant toxicities. Newer beta-lactam/beta-lactamase inhibitor combinations offer additional novel options to treat such infections, given their good pharmacokinetic profiles and activity against multi-drug resistant strains. Ceftolozane/tazobactam is a novel cephalosporin/beta-lactamase inhibitor combination approved in 2014. The drug demonstrates good activity against multidrug-resistant P. aeruginosa strains, including those resistant to all other antibiotics. Ceftolozane/tazobactam is currently approved in adult patients 18 years and older only. There are very limited data on its pharmacokinetic profile and clinical utility in the pediatric population. We report the use of ceftolozane/tazobactam to successfully treat pneumonia caused by multidrug-resistant P. aeruginosa in a pediatric patient with combined immunodeficiency syndrome.


1997 ◽  
Vol 41 (1) ◽  
pp. 148-155 ◽  
Author(s):  
S K Spangler ◽  
M R Jacobs ◽  
P C Appelbaum

Agar dilution MIC methodology was used to test the activities of GV 118819X (sanfetrinem), ampicillin, amoxicillin, amoxicillin-clavulanate, cefpodoxime, loracarbef, levofloxacin, clarithromycin, ceftriaxone, imipenem, and vancomycin against 53 penicillin-susceptible, 84 penicillin-intermediate and 74 penicillin-resistant pneumococci isolated in the United States. GV 118819X was the most active oral beta-lactam, with MIC at which 50% of the isolates were inhibited (MIC50)/MIC90 values of 0.008/0.03, 0.06/0.5, and 0.5/1.0 micrograms/ml against penicillin-susceptible, -intermediate, and -resistant stains, respectively. Amoxicillin and amoxicillin in the presence of clavulanate (2:1) were the second most-active oral beta-lactams, followed by ampicillin and cefpodoxime; loracarbef was not active against penicillin-intermediate and -resistant strains. Clarithromycin was most active against penicillin-susceptible strains but was less active against intermediate and resistant stains. All pneumococcal stains were inhibited by ceftriaxone and imipenem at MICs of < or = 4.0 and < or = 1.0 micrograms/ml, respectively. The activities of levofloxacin and vancomycin were unaffected by penicillin susceptibility. Time-kill studies of three penicillin-susceptible, three penicillin-intermediate, and three penicillin-resistant pneumococci showed that all compounds, at the broth microdilution MIC, yielded 99.9% killing of all strains after 24 h. Kinetic patterns of all oral beta-lactams, ceftriaxone, and vancomycin were similar relative to the MIC, with 90% killing of all strains first observed after 12 h. However, killing by amoxicillin-clavulanate, imipenem, and levofloxacin was slightly faster and that by clarithromycin was slower than that by the above-described drugs. At 2 x the MIC, more strains were killed earlier than was the case at the MIC, but the pattern seen at the MIC prevailed. When MICs and kill kinetics were combined, sanfetrinem was the most active oral antipneumococcal agent in this study.


2021 ◽  
Vol 8 ◽  
Author(s):  
Rutan Zhang ◽  
Ismael A. Barreras Beltran ◽  
Nathaniel K. Ashford ◽  
Kelsi Penewit ◽  
Adam Waalkes ◽  
...  

Methicillin-resistant S. aureus (MRSA) are resistant to beta-lactams, but synergistic activity between beta-lactams and glycopeptides/lipopeptides is common. Many have attributed this synergy to the beta-lactam-glycopeptide seesaw effect; however, this association has not been rigorously tested. The objective of this study was to determine whether the seesaw effect is necessary for synergy and to measure the impact of beta-lactam exposure on lipid metabolism. We selected for three isogenic strains with reduced susceptibility to vancomycin, daptomycin, and dalbavancin by serial passaging the MRSA strain N315. We used whole genome sequencing to identify genetic variants that emerged and tested for synergy between vancomycin, daptomycin, or dalbavancin in combination with 6 beta-lactams with variable affinity for staphylococcal penicillin binding proteins (PBPs), including nafcillin, meropenem, ceftriaxone, ceftaroline, cephalexin, and cefoxitin, using time-kills. We observed that the seesaw effect with each beta-lactam was variable and the emergence of the seesaw effect for a particular beta-lactam was not necessary for synergy between that beta-lactam and vancomycin, daptomycin, or dalbavancin. Synergy was more commonly observed with vancomycin and daptomycin based combinations than dalbavancin in time-kills. Among the beta-lactams, cefoxitin and nafcillin were the most likely to exhibit synergy using the concentrations tested, while cephalexin was the least likely to exhibit synergy. Synergy was more common among the resistant mutants than the parent strain. Interestingly N315-D1 and N315-DAL0.5 both had mutations in vraTSR and walKR despite their differences in the seesaw effect. Lipidomic analysis of all strains exposed to individual beta-lactams at subinhibitory concentrations suggested that in general, the abundance of cardiolipins (CLs) and most free fatty acids (FFAs) positively correlated with the presence of synergistic effects while abundance of phosphatidylglycerols (PGs) and lysylPGs mostly negatively correlated with synergistic effects. In conclusion, the beta-lactam-glycopeptide seesaw effect and beta-lactam-glycopeptide synergy are distinct phenomena. This suggests that the emergence of the seesaw effect may not have clinical importance in terms of predicting synergy. Further work is warranted to characterize strains that don’t exhibit beta-lactam synergy to identify which strains should be targeted with combination therapy and which ones cannot and to further investigate the potential role of CLs in mediating synergy.


Author(s):  
Olga Lomovskaya ◽  
Debora Rubio-Aparicio ◽  
Kirk Nelson ◽  
Dongxu Sun ◽  
Ruslan Tsivkovski ◽  
...  

QPX7728 is an ultra-broad-spectrum beta-lactamase inhibitor with potent inhibition of key serine and metallo beta-lactamases. QPX7728 enhances the potency of multiple beta-lactams in beta-lactamase producing Enterobacterales and Acinetobacter spp. In this study we evaluated the in vitro activity of QPX7728 (8 μg/ml) combined with multiple beta-lactams against clinical isolates of Pseudomonas aeruginosa with varying beta-lactam resistance mechanisms. Seven-hundred-ninety clinical isolates were included in this study; 500 isolates, termed a “representative panel”, were selected to be representative the MIC distribution of meropenem (MEM), ceftazidime-avibactam (CAZ-AVI), and ceftolozane-tazobactam (TOL-TAZ) resistance for clinical isolates according to 2017 SENTRY surveillance data (representative panel). An additional 290 selected isolates (“challenge panel”), that were either non-susceptible to MEM or were resistant to TOL-TAZ or CAZ-AVI were also tested; 61 strains carried metallo beta-lactamases (MBLs), 211 strains were defective in the carbapenem porin OprD and 185 strains had the MexAB-OprM efflux pump overproduced based on a phenotypic test. Against the representative panel, susceptibility for all QPX7728/beta-lactam combinations was >90%. For the challenge panel, QPX-ceftolozane (TOL) was the most active combination (78.6% susceptible) followed by equipotent QPX-piperacillin (PIP) and QPX-cefepime (FEP), restoring susceptibility in 70.3% of strains (CLSI breakpoints for the beta-lactam compound alone). For MBL-negative strains, QPX-TOL and QPX-FEP restored the MIC values to susceptibility rates in ∼90% and ∼80% of strains, respectively, vs 68-70% for QPX-MEM and QPX-PIP and 63-65% for TOL-TAZ and CAZ-AVI. For MBL-positive strains, QPX-PIP restored the MIC to susceptibility values for ∼70% of strains vs 2-40% for other combinations. Increased efflux and impaired OprD had varying effect on QPX7728 combination depending on the partner beta-lactam tested. QPX7728 enhanced the potency of multiple beta-lactams against P. aeruginosa, with varying results according to the beta-lactamase production and other intrinsic resistance mechanisms.


Author(s):  
Rafael Cantón ◽  
◽  
Elena Loza ◽  
Ricardo M. Arcay ◽  
Emilia Cercenado ◽  
...  

Objective. To analyse the susceptibility to ceftolozane-tazobactam and comparators in Enterobacterales and Pseudomonas aeruginosa isolates recovered from intraabdominal (IAI), urinary (UTI), respiratory (RTI) and bloodstream infection (BSI) in the SMART (Study for Monitoring Antimicrobial Resistance Trends) study. Methods. The susceptibility of 5,351 isolates collected in 11 Spanish hospitals (2016-2018) were analysed (EUCAST-2020 criteria) by broth microdilution and were phenotypically studied for the presence of extended-spectrum beta-lactamases (ESBL). Ceftolozane-tazobactam and/or carbapenem resistant isolates were genetically characterized for ESBL and carbapenemases. Results. Escherichia coli was the most frequent pathogen (49.3% IAI, 54.9% UTI, 16.7% RTI and 50% BSI), followed by Klebsiella pneumoniae (11.9%, 19.1%, 13.1% and 15.4%, respectively). P. aeruginosa was isolated in 9.3%, 5.6%, 32% and 9%, respectively. The frequency of isolates with ESBLs (2016-2017) was: 30.5% K. pneumoniae, 8.6% E. coli, 2.3% Klebsiella oxytoca and 0.7% Proteus mirabilis. Ceftolozane-tazobactam was very active against non-ESBL-(99.3% susceptible) and ESBL-(95.2%) producing E. coli being less active against K. pneumoniae (98% and 43.1%, respectively) isolates. CTX-M-15 was the most prevalent ESBL in E. coli (27.5%) and K. pneumoniae (51.9%) frequently associated with OXA-48-like carbapenemase. Overall, 93% of P. aeruginosa isolates were susceptible to ceftolozane-tazobactam, preserving this activity (>75%) in isolates resistant to other beta-lactams except in those resistant to meropenen or ceftazidime-avibactam. GES-5, PER-1, VIM-1/2 were the most prevalent enzymes in isolates resistant to ceftolozane-tazobactam. Conclusions. Ceftolozane-tazobactam showed high activity rates against isolates recovered in the SMART study although it was affected in K. pneumoniae and P. aeruginosa isolates with ESBL and/or carbapenemases.


Author(s):  
Olga Lomovskaya ◽  
Debora Rubio-Aparicio ◽  
Ruslan Tsivkovski ◽  
Jeff Loutit ◽  
Michael Dudley

QPX7728 is a cyclic boronate ultra-broad-spectrum beta-lactamase inhibitor, with potent activity against both serine and metallo beta-lactamases. QPX7728 can be delivered systemically by the IV or oral route of administration. Oral β-lactam antibiotics alone or in combination with QPX7728 were evaluated for 1) sensitivity to hydrolysis by various common beta-lactamases and inhibition of hydrolysis by QPX7728; 2) the impact of non-beta-lactamase-mediated resistance mechanisms on potency of beta-lactams; and 3) in vitro activity against a panel of clinical strains producing diverse beta-lactamases. The carbapenem tebipenem had stability for many serine beta-lactamases from all molecular classes followed by cephalosporin ceftibuten. Addition of QPX7728 to tebipenem, ceftibuten and mecillinam completely reversed beta-lactamase-mediated resistance in cloned beta-lactamases from serine and metallo enzyme classes; the degree of potentiation of other beta-lactams varied according to the beta-lactamase produced. Tebipenem, ceftibuten and cefixime had the lowest MICs against laboratory strains with various combinations of beta-lactamases and the intrinsic drug-resistance mechanisms of porin and efflux mutations. There was a high degree of correlation between potency of various combinations against cloned beta-lactamases and efflux/porin mutants and the activity against clinical isolates, showing the importance of both inhibition of beta-lactamase along with minimal impact of general intrinsic resistance mechanisms affecting the beta-lactam. Tebipenem and ceftibuten appeared to be the best beta-lactam antibiotics when combined with QPX7728 for activity against Enterobacterales that produce serine or metallo beta-lactamases.


2017 ◽  
Vol 56 (3) ◽  
Author(s):  
Romney M. Humphries ◽  
Janet A. Hindler ◽  
Paul Magnano ◽  
Annie Wong-Beringer ◽  
Robert Tibbetts ◽  
...  

ABSTRACT The performance characteristics of the ceftolozane-tazobactam (C-T) Etest (bioMérieux, Marcy l'Etoile, France), MIC test strips (MTS; Liofilchem, Italy), and disk diffusion (Hardy, Santa Ana, CA) were evaluated for a collection of 308 beta-lactam-resistant isolates of Pseudomonas aeruginosa recovered from three institutions in Los Angeles, CA. Reference testing was performed by the reference broth microdilution (rBMD) method. MIC and disk results were interpreted using Clinical and Laboratory Standards Institute breakpoints. Overall, 72.5% of the isolates were susceptible to C-T by rBMD. Etest and disk diffusion demonstrated acceptable performance, whereas MTS yielded a greater than acceptable percentage of minor errors. Categorical agreement was 96.8% for Etest, 87.0% for MTS, and 92.9% for disk diffusion. No very major errors were observed by any test, and no major errors (ME) were observed by Etest or disk diffusion. Two ME (0.9% of susceptible isolates) were observed by MTS. The incidence of minor errors was 3.2%, 12.3%, and 7.1% for Etest, MTS, and disk diffusion, respectively. Essential agreement (EA) for Etest was excellent, at 97.7%, whereas the MICs obtained by MTS tended to be 1 to 2 dilutions higher than those obtained by rBMD, with an EA of 87.0%.


2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S580-S581
Author(s):  
Robert K Flamm ◽  
Michael A Pfaller ◽  
Paul G Ambrose ◽  
David Andes ◽  
John S Bradley ◽  
...  

Abstract Background In 2015 USCAST, the National Advisory Committee for the United States (US) to EUCAST, produced a report (Version 1.0) on their website (www.uscast.org) re-evaluating fluoroquinolone (FQ) breakpoint interpretive criteria (IC) based on analysis of current microbiology and pharmacokinetic/pharmacodynamic (PK/PD) data. EUCAST initiated a consultative process using USCAST analyses in an effort to update FQ IC, released in 2017. CLSI formed an ad-hoc working group in late 2015 to review the USCAST FQ document and formulate questions about content. In 2018, USCAST released V1.3 of the FQ document and CLSI subsequently published updated FQ MIC IC in the M100-S29 (2019) document. This study evaluated the impact on susceptibility (S) rates for US surveillance data that these IC changes created. Methods Clinical isolates (reference broth microdilution MIC) from 2016–2018 US SENTRY Program were analyzed for S based on current and previous IC. FQ results for ciprofloxacin (CIP), levofloxacin (LEV), and moxifloxacin (MOX) were evaluated. Benchmark S comparison data for meropenem, cefepime, piperacillin–tazobactam and delafloxacin (new FQ) were also included. Results S rates for Enterobacteriaceae (ENT;Figure) were reduced by 3.8/3.7% for CIP/LEV (CLSI) and 2.3/2.5% (EUCAST). MOX-S rate vs. ENT declined 5.7% (EUCAST). Although reductions in S occurred for most organism groups, K. pneumoniae (6.0/5.5% for CIP/LEV [CLSI] and 4.0/4.2% [EUCAST]) and S. marcescens (7.4/4.1% for CIP/LEV [CLSI] and 4.1/5.0% [EUCAST]) reductions were among the largest changes. For Pseudomonas aeruginosa (PSA), CIP-S decreased 6.8% and LEV-S 10.1% (CLSI); but potential for false-S results remain using CLSI IC (5 pathogens). Conclusion USCAST’s comprehensive analyses of FQ IC in 2015 led to revised breakpoints for most organism/drug combinations among ENT and PSA compared with those being used before. USCAST analysis was most influenced by PK/PD in vivo data as current clinical outcomes data by MIC was limited. Awareness and interactions (both formal and informal) among breakpoint setting organizations has modified FQ ICs which are lower than previously recommended, and although not perfectly harmonized in time and detail, this represents a successful model. Disclosures All authors: No reported disclosures.


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