Gradient Diffusion Strip Research Articles

P-2117. ETEST® Aztreonam-Avibactam (AZA) for Antimicrobial Susceptibility Testing of Enterobacterales: Performance Results From a Muticenter Study

Abstract Background AZA is a combination of Aztreonam, a monobactam not hydrolyzed by metallo-β-lactamases, and Avibactam, a novel non-β-lactam β-lactamase inhibitor with an extended spectrum that protects Aztreonam for hydrolysis by Ambler class A, class C, and some class D serine β-lactamases. Aztreonam-Avibactam is developed by Pfizer for the treatment of infections caused by Gram-negative bacteria, including metallo-β lactamase producers. This study evaluated the performance of ETEST® AZA (bioMérieux Marcy-L’Etoile, France), a new gradient diffusion strip, for determining antimicrobial susceptibility of Enterobacterales compared to the CLSI/ISO-20776-2 broth microdilution reference method (BMD). Methods A population of 650 (clinical and challenge) Enterobacterales isolates (including 150 Escherichia coli; 118 Klebsiella pneumoniae; 48 Klebsiella oxytoca; 67 Enterobacter cloacae complex; 50 Citrobacter freundii complex; 30 Citrobacter koseri; 32 Klebsiella aerogenes; 30 Morganella morganii; 32 Proteus mirabilis; 30 Providencia stuartii ; 30 Proteus vulgaris ; 33 Serratia marcescens) were tested at 4 clinical trial sites, comparing ETEST® AZA and the BMD reference method. Results were analyzed using preliminary EUCAST breakpoints (Enterobacterales: ≤ 8mg/L (S), > 8 mg/L (R)) and preliminary FDA breakpoints (Enterobacterales: ≤ 8mg/L (S), ≥ 16 mg/L (R)) provided by Pfizer. Results are presented as essential agreement (EA) and Bias for Worldwide registration and as essential (EA) and categorical (CA) agreements, Trend, very major (VME) and major (ME) error rates for US registration. Results Results are summarized in Table 1. ETEST® AZA performance for Enterobacterales met ISO acceptance criteria for EA (≥ 90%) and Bias ≤ ±30%, and FDA criteria for EA, CA (≥ 90%), ME (≤ 3%), VME (≤ 2%) and Trend ≤ ±30%. Conclusion Results of this multicenter trial support the accuracy of ETEST® AZA for determining MICs of Enterobacterales. As such, the ETEST® AZA test is considered to be equivalent to BMD. Disclosures Melanie L. Yarbrough, PhD, bioMerieux: Grant/Research Support|bioMerieux: Honoraria|Pattern Bioscience: Grant/Research Support|Shionogi: Advisor/Consultant

Candida auris MIC testing by EUCAST and clinical and laboratory standards institute broth microdilution, and gradient diffusion strips; to be or not to be amphotericin B resistant?

ObjectivesReported amphotericin B resistance rates for Candida auris vary considerably. This may reflect clinically relevant differences in susceptibility, technical issues with testing, or adoption of a clinical breakpoint that bisects the wild-type population. We compared reference methods and two gradient diffusion strips using a shared C. auris strain collection. MethodsForty C. auris strains from nine U.S. states and ≥3 clades were included. Fourteen MIC data sets were generated using European Committee on Antimicrobial Susceptibility Testing (EUCAST) E.Def 7.4, Clinical and Laboratory Standards Institute (CLSI) M27Ed4, Etest, and MIC gradient test strip (MTS, Liofilchem) MICs. MICs ≤1 mg/L were classified as susceptible. ResultsEUCAST and CLSI amphotericin B MIC testing were robust across the included method variables. The modal MIC was 1 mg/L, distributions unimodal and narrow with similar geometric mean (GM)-MICs (0.745–1.072); however, susceptibility classification varied (0–28% resistance). Gradient diffusion strip testing resulted in wider and bimodal distributions for 8/9 data sets. If adopting, per manufacturer's protocol, double inoculation for the Etest method, the modal MIC increased to 2–4 mg/L and resistance rates to 45–63% versus 25–30% with the single inoculation. The EUCAST, CLSI, Etest, and MTS strip MICs correlated to the optical density of drug-free control EUCAST wells, suggesting that some isolates grew better than others and that this was associated with MIC. Discussion: The EUCAST and CLSI MIC results were in close agreement, whereas the strip test showed wider and bimodal distributions with reader to reader and centre to centre variation. Our study adds to the concern for commercial MIC testing of amphotericin B against C. auris and suggests the current breakpoint leads to random susceptibility classification.

Evaluation of a customised Sensititre YeastOne plate containing isavuconazole for antifungal susceptibility testing in Singapore

This study evaluated the performance of a customised Sensititre YeastOne (SYO) plate including isavuconazole (YIT) against existing practice (comprising SYO YO10 plate and isavuconazole gradient strip) in order to streamline the workflow for antifungal susceptibility testing in a tertiary hospital in Singapore. A total of 101 (51 yeasts and 50 moulds) clinical isolates were included for analysis. Isolates included in the study were recovered from a variety of body sites and reflected the case mix encountered in daily practice. Antifungal susceptibility testing was performed using three methods: YO10, YIT and gradient diffusion strip (for isavuconazole only). Reproducibility, essential agreement (EA) and categorical agreement (CA) were calculated. When YO10 and YIT plates were compared, the reproducibility was 100% for eight common antifungals. The CA was >97% for all antifungals except for amphotericin B (89.4%), but this was attributed to seven isolates with minimum inhibitory concentrations bordering the wild-type (WT) cut-off. The EA obtained when testing isavuconazole using YIT versus gradient diffusion was 77.2% overall, 90.2% for yeasts and 64% for moulds. In conclusion, the YIT plate is suitable for antifungal susceptibility testing of yeasts in our laboratory. Its use for mould isolates needs to be monitored further.

Susceptibility evaluation of novel beta-lactam/beta-lactamase inhibitor combinations against carbapenem-resistant Klebsiella pneumoniae from bloodstream infections in hospitalized patients in Brazil

IntroductionNovel beta-lactam/beta-lactamase inhibitor (BIBLI) combinations are commercially available and have been used for treating carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. Continuous surveillance of susceptibility profiles and resistance mechanism identification are necessary to monitor the evolution of resistance within these agents. ObjectiveThe purpose of this study was to evaluate the susceptibility rates of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam in CRKP isolated from patients with bloodstream infections who underwent screening for a randomized clinical trial in Brazil. MethodsMinimum inhibitory concentrations (MICs) were determined for meropenem, ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam using the gradient diffusion strip method. Carbapenemase genes were detected by multiplex real-time polymerase chain reaction. Klebsiella pneumoniae carbapenemase (KPC)-producing isolates showing resistance to any BLBLI and New Delhi Metallo-beta-lactamase (NDM)-producing isolates with susceptibility to any BLBLI isolates were further submitted for whole-genome sequencing. ResultsFrom a total of 69 CRKP isolates, 39 were positive for blaKPC, 19 for blaNDM and 11 for blaKPC and blaNDM. KPC-producing isolates demonstrated susceptibility rates above 94 % for all BLBLIs. Two isolates with resistance to meropenem/vaborbactam demonstrated a Gly and Asp duplication at the porin OmpK36 as well as a truncated OmpK35. All NDM-producing isolates, including KPC and NDM coproducers, demonstrated susceptibility rates to ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam of 0 %, 9.1–21.1 % and 9.1–26.3 %, respectively. Five NDM-producing isolates that presented susceptibility to BLBLIs also had porin alterations ConclusionsThis study showed that, although high susceptibility rates to BLBLIs were found, KPC-2 isolates were able to demonstrate resistance probably as a result of porin mutations. Additionally, NDM-1 isolates showed susceptibility to BLBLIs in vitro.

Molecular epidemiology of carbapenem-resistant gram-negative bacilli in Ecuador

IntroductionCarbapenem-resistant gram-negative bacilli are a worldwide concern because of high morbidity and mortality rates. Additionally, the increasing prevalence of these bacteria is dangerous. To investigate the extent of antimicrobial resistance and prioritize the utility of novel drugs, we evaluated the molecular characteristics and antimicrobial susceptibility profiles of carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii in Ecuador in 2022.MethodsNinety-five clinical isolates of carbapenem non-susceptible gram-negative bacilli were collected from six hospitals in Ecuador. Carbapenem resistance was confirmed with meropenem disk diffusion assays following Clinical Laboratory Standard Institute guidelines. Carbapenemase production was tested using a modified carbapenemase inactivation method. Antimicrobial susceptibility was tested with a disk diffusion assay, the Vitek 2 System, and gradient diffusion strips. Broth microdilution assays were used to assess colistin susceptibility. All the isolates were screened for the blaKPC,blaNDM,blaOXA-48,blaVIM and blaIMP genes. In addition, A. baumannii isolates were screened for the blaOXA-23, blaOXA-58 and blaOXA-24/40 genes.ResultsCarbapenemase production was observed in 96.84% of the isolates. The blaKPC, blaNDM and blaOXA-48 genes were detected in Enterobacterales, with blaKPC being predominant. The blaVIM gene was detected in P. aeruginosa, and blaOXA-24/40 predominated in A. baumannii. Most of the isolates showed co-resistance to aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole. Both ceftazidime/avibactam and meropenem/vaborbactam were active against carbapenem-resistant gram-negative bacilli that produce serin-carbapenemases.ConclusionThe epidemiology of carbapenem resistance in Ecuador is dominated by carbapenemase-producing K. pneumoniae harbouring blaKPC. Extensively drug resistant (XDR) P. aeruginosa and A. baumannii were identified, and their identification revealed the urgent need to implement strategies to reduce the dissemination of these strains.

Efficacy of delafloxacin alone and in combination with cefotaxime against cefotaxime non-susceptible invasive isolates of Streptococcus pneumoniae.

We assessed the in vitro activity of delafloxacin and the synergy between cefotaxime and delafloxacin among cefotaxime non-susceptible invasive isolates of Streptococcus pneumoniae (CNSSP). A total of 30 CNSSP (cefotaxime MIC > 0.5 mg/L) were studied. Serotyping was performed by the Pneumotest-Latex and Quellung reaction. Minimum inhibitory concentrations (MICs) of delafloxacin, levofloxacin, penicillin, cefotaxime, erythromycin and vancomycin were determined by gradient diffusion strips (GDS). Synergistic activity of delafloxacin plus cefotaxime against clinical S. pneumoniae isolates was evaluated by the GDS cross method. Delafloxacin showed a higher pneumococcal activity than its comparator levofloxacin (MIC50, 0.004 versus 0.75 mg/L and MIC90, 0.047 versus >32 mg/L). Resistance to delafloxacin was identified in 7/30 (23.3%) isolates, belonging to serotypes 14 and 9V. Synergy between delafloxacin and cefotaxime was detected in 2 strains (serotypes 19A and 9V). Antagonism was not observed. Addition of delafloxacin increased the activity of cefotaxime in all isolates. Delafloxacin susceptibility was restored in 5/7 (71.4%) strains. CNSSP showed a susceptibility to delafloxacin of 76.7%. Synergistic interactions between delafloxacin and cefotaxime were observed in vitro among CNSSP by GDS cross method.

Development of gradient diffusion strips to identify the potentiating effect of antimicrobial compounds against multidrug-resistant Klebsiella pneumoniae strains

Objective. The aim of the study was to develop gradient diffusion strips with substances capable of potentiating the action of antibiotics for the treatment of infections caused by multidrug-resistant strains of K. pneumoniae. Materials and Methods. The substances azidothymidine and baicalin were used to evaluate the joint action of combinations of antibiotics with substances that potentiate their action. Determination of susceptibility to combinations of antibiotics (gentamicin, cefotaxime, ciprofloxacin and chloramphenicol) with the listed substances was carried out by a modified gradient-diffusion method (cross-test). We used gradient diffusion strips, which was made in the Saint-Petersburg Pasteur Institute. We investigated clinical multidrug-resistant K. pneumoniae strains (n = 20). The result of the joint action of a combination of two compounds was assessed by calculating the fractional inhibitory concentrations ΣFIC. Results. The values of MIC decreased eightfold when azidothymidine was combined with gentamicin and two times when combined with ciprofloxacin. MIC values were not reduced by the combination of azidothymidine with cefotaxime or chloramphenicol. It has been established that azidothymidine has antibacterial activity against strains of K. pneumoniae: MIC50 – 1 μg/ml and MIC90 – 2 μg/ml. The MIC50 and MIC90 values for baicalin were > 256 μg/ml. A synergistic antibacterial effect was detected when azidothymidine was combined with gentamicin (ΣFIC 0.33–0.50). An additive effect (ΣFIC 0.65–0.84) was detected when azidothymidine was combined with ciprofloxacin. Baikalin reduced the MIC value of cefotaxime by half, and the MIC50 values for gentamicin also decreased by half. Baicalin did not affect the susceptibility of multidrug-resistant K. pneumoniae strains to ciprofloxacin and chloramphenicol. Synergism was detected in the combination of baicalin and cefotaxime (ΣFIC 0.52 – 0.75 – additive effect). Conclusions. The development of gradient diffusion strips with azidothymidine and baicalin makes it possible to simplify methods for assessing the joint action of combinations of these substances with antibiotics and to avoid time-consuming preparatory steps. Our study demonstrated that azidothymidine exhibits synergistic activity in combination with gentamicin and ciprofloxacin and baicalin – in combination with cefotaxime. Further studies are needed to evaluate the potential use of these combinations in practical healthcare.

Aztreonam-avibactam synergy, a validation and comparison of diagnostic tools

IntroductionAntimicrobial resistance is a growing problem that necessitates the development of new therapeutic options. Cefiderocol and aztreonam (AT) are often the last active β-lactams for treating metallo-β-lactamases (MBL)-producing Gram-negative bacilli. In these difficult-to-treat bacterial strains, AT resistance is frequently attributed to the co-occurrence of other resistance mechanisms. In the case of β-lactamases they can often be inhibited by avibactam. In the present study, we evaluated the use of the double-disc synergy test (DDST) as a screening tool for the detection of synergy between AT-avibactam (ATA). We validated both the Gradient Diffusion Strips (GDSs) superposition method and the commercially available Liofilchem’s ATA GDS.Materials and methodsWe tested AT susceptibility in combination with ceftazidime-avibactam for 65 strains, including 18 Serine-β-Lactamase (SBL)- and 24 MBL-producing Enterobacterales, 12 MBL-producing P. aeruginosa, and 11 S. maltophilia isolates. Interpretation was done with EUCAST breakpoints (version 13.0), AT breakpoints being used for ATA. The accuracy and validity of the GDSs superposition method and ATA GDS were evaluated using an AT GDS applied on Mueller Hinton Agar plates supplemented with avibactam (MH-AV). A DDST was performed to screen for synergy between antibiotic combinations.ResultsUsing MH-AV, all SBL- and MBL-positive Enterobacterales were susceptible or susceptible at increased exposure to the combination AT-avibactam. In contrast, only 2 out of the 12 (17%) P. aeruginosa strains and 9/11 (82%) of the S. maltophilia strains were susceptible- or susceptible at increased exposure for the combination of AT-avibactam. The DDST detected all synergies, demonstrating a 100% sensitivity and 100% negative predictive value for all bacterial strains.ConclusionThe DDST is a sensitive tool for screening for antibiotic synergy. Unlike S. maltophilia and SBL- and MBL-positive Enterobacterales, most MBL-positive P. aeruginosa strains remain resistant to AT-avibactam. ATA GDS should be preferred for MIC determination of the AT-avibactam combination, while the GDSs superposition method can be used as an alternative to the commercial test.

Gradient diffusion strips for detecting azole resistance in Aspergillus fumigatus sensu lato.

Studies comparing gradient diffusion strips (GDSs) and the EUCAST E.Def 9.4 microdilution method are scarce, thwarted by a low number of isolates, and restricted to selected antifungal agents. We evaluated the performance of GDSs to detect azole resistance in A. fumigatus, including cryptic species. A. fumigatus sensu stricto (n=89) and cryptic species (n=52) were classified as susceptible or resistant to itraconazole, voriconazole, posaconazole and isavuconazole (EUCAST E.Def 9.4; clinical breakpoints v10). A. fumigatus sensu stricto azole-resistant isolates had the following cyp51A gene mutations: TR34 -L98H (n=24), G54R (n=5), TR46 -Y121F-T289A (n=1), F46Y-M172V-N248T-D255E-E427K (n=1), F165L (n=1) and cyp51A gene wild type (n=3). GDSs (ETEST®, bioMèrieux, Marcy-l'Etoile, France and Liofilchem®, Roseto degli Abruzzi, Italy) MICs were obtained by following the manufacturer's guidelines. For A. fumigatus sensu stricto, itraconazole MICs >1.5mg/L, voriconazole >0.38 mg/L, posaconazole >0.75 mg/L, and isavuconazole >0.5mg/L correctly separated resistant from susceptible isolates with two exceptions. Considering the aforementioned cut-off MICs, sensitivity/specificity values of GDSs to detect azole resistance were: itraconazole (97%/100%), voriconazole (97%/100%), posaconazole (97%/100%) and isavuconazole (93.3%/100%). For cryptic species isolates, voriconazole MICs >1mg/L and isavuconazole >0.75 mg/L separated resistant isolates from susceptible isolates with 15 and 27 exceptions, respectively. Considering the aforementioned cut-off MICs, sensitivity/specificity values were as follows: voriconazole (68.1%/100%) and isavuconazole (25%/100%). For itraconazole and posaconazole, it was not possible to establish cut-off values. We set tentative cut-off MIC values to correctly spot resistant Aspergillus fumigatus sensu stricto isolates using GDSs. The performance against cryptic species was poor.

In vitro killing of multidrug/extensively drug-resistant Pseudomonas aeruginosa by fosfomycin alone or in combination with antipseudomonal antibiotics

Pseudomonas aeruginosa is a leading cause of nosocomial infections. Given the constant rise in resistance, adequate therapy is increasingly demanding. Fosfomycin recently became an appealing treatment option of bacterial infections due to multidrug-resistant bacteria (MDR). So far, fosfomycin synergy with other antibiotics has been assessed in studies, but only a limited number focused on MDR P. aeruginosa and on the effect of these combinations on the duration of the postantibiotic effect (PAE). We investigated synergy of fosfomycin with an array of antipseudomonal antibiotics using gradient diffusion strip cross method and time-kill method, and their effect on the duration of PAE against 51 variously resistant P. aeruginosa isolates. The highest rate of synergy was observed for combination with ceftazidime (23.4%) and gentamicin (19.1%). The PAE of antibiotic combinations was superior to that of the drugs alone. Our findings indicate that fosfomycin combination therapy may be a valuable treatment alternative.

Comparison of Six Phenotypic Assays with Reference Methods for Assessing Colistin Resistance in Clinical Isolates of Carbapenemase-Producing Enterobacterales: Challenges and Opportunities.

The global escalation of severe infections due to carbapenemase-producing Enterobacterales (CPE) isolates has prompted increased usage of parenteral colistin. Considering the reported difficulties in assessing their susceptibility to colistin, the purpose of the study was to perform a comparative evaluation of six phenotypic assays—the colistin broth disc elution (CBDE), Vitek 2 Compact (bioMérieux SA, Marcy l’Etoile, France), the Micronaut MIC-Strip Colistin (Merlin Diagnostika GMBH, Bornheim-Hensel, Germany), the gradient diffusion strip Etest (bioMérieux SA, Marcy l’Etoile, France), ChromID Colistin R Agar (COLR) (bioMérieux SA, Marcy l’Etoile, France), and the Rapid Polymyxin NP Test (ELITechGroup, Signes, France)—versus the reference method of broth microdilution (BMD). All false resistance results were further assessed using population analysis profiling (PAP). Ninety-two nonrepetitive clinical CPE strains collected from two hospitals were evaluated. The BMD confirmed 36 (39.13%) isolates susceptible to colistin. According to the BMD, the Micronaut MIC-Strip Colistin, the CBDE, and the COLR medium exhibited category agreement (CA) of 100%. In comparison with the BMD, the highest very major discrepancy (VMD) was noted for Etest (n = 15), and the only false resistance results were recorded for the Rapid Polymyxin NP Test (n = 3). Only the PAP method and the Rapid Polymyxin NP Test were able to detect heteroresistant isolates (n = 2). Thus, there is an urgent need to further optimize the diagnosis strategies for colistin resistance.

Occurrence of multi-carbapenemases producers among carbapenemase-producing Enterobacterales and in vitro activity of combinations including cefiderocol, ceftazidime-avibactam, meropenem-vaborbactam, and aztreonam in the COVID-19 era.

PurposeTo evaluate the prevalence of multi-carbapenemase-producing Enterobacterales (EB) and the activity of cefiderocol (CFDC), meropenem-vaborbactam (MEV), ceftazidime-avibactam (CZA), and combinations of CZA plus aztreonam (ATM), MEV plus ATM and CFDC plus CZA against them.MethodsA collection of carbapenemase-producing EB clinical isolates (n = 1242) was investigated by lateral flow immunoassay NG-Test CARBA-5 and molecular testing. Cefiderocol MICs were determined using broth microdilution SensititreTM panel. MICs of CZA and MEV were determined by the gradient diffusion method. Antimicrobial synergy testing was performed using gradient diffusion strip crossing.ResultsKPC were the most frequent carbapenemases (83.2%), followed by VIM (9.2 %), OXA-48-like (4.3 %) and NDM enzymes (4.1%). Multi-carbapenemase producers were found in 10 (0.8%) isolates. Three combinations of two different carbapenemases were observed: KPC+VIM (n = 4), NDM+OXA-48-like (n = 4), and VIM+OXA-48-like (n = 2). CFDC showed potent activity against eight out of ten dual-carbapenemases producers, while resistance or reduced susceptibility was shown towards CZA and MEV. CFDC in combination with CZA showed no synergistic effects and only two additive effects on seven (87.5%) of the CFDC-susceptible strains. Conversely, CZA plus ATM and MEV plus ATM combinations were synergistic against all ATM-resistant strains regardless of dual-carbapenemases phenotype.ConclusionsThe occurrence of multi-carbapenemase producers is not uncommon in Northern Italy area. MEV in combination with ATM might be considered as a potential therapeutic option, alternative to CZA plus ATM. CFDC susceptibility testing and synergy evaluation of ATM-based combinations should be performed in the lab routine to evaluate the most in vitro active antimicrobial regimen.

Performance Evaluation of the Gradient Diffusion Strip Method and Disk Diffusion Method for Ceftazidime–Avibactam Against Enterobacterales and Pseudomonas aeruginosa: A Dual-Center Study

Objectives: Ceftazidime–avibactam is a novel synthetic beta-lactam + beta-lactamase inhibitor combination. We evaluated the performance of the gradient diffusion strip method and the disk diffusion method for the determination of ceftazidime–avibactam against Enterobacterales and Pseudomonas aeruginosa.Methods: Antimicrobial susceptibility testing of 302 clinical Enterobacterales and Pseudomonas aeruginosa isolates from two centers were conducted by broth microdilution (BMD), gradient diffusion strip method, and disk diffusion method for ceftazidime–avibactam. Using BMD as a gold standard, essential agreement (EA), categorical agreement (CA), major error (ME), and very major error (VME) were determined according to CLSI guidelines. CA and EA rate > 90%, ME rate < 3%, and VME rate < 1.5% were considered as acceptable criteria. Polymerase chain reaction and Sanger sequencing were performed to determine the carbapenem resistance genes of all 302 isolates.Results: A total of 302 strains were enrolled, among which 182 strains were from center 1 and 120 strains were from center 2. A percentage of 18.21% (55/302) of the enrolled isolates were resistant to ceftazidime–avibactam. The CA rates of the gradient diffusion strip method for Enterobacterales and P. aeruginosa were 100% and 98.65% (73/74), respectively, and the EA rates were 97.37% (222/228) and 98.65% (73/74), respectively. The CA rates of the disk diffusion method for Enterobacterales and P. aeruginosa were 100% and 95.95% (71/74), respectively. No VMEs were found by using the gradient diffusion strip method, while the ME rate was 0.40% (1/247). No MEs were found by using the disk diffusion method, but the VME rate was 5.45% (3/55). Therefore, all the parameters of the gradient diffusion strip method were in line with acceptable criteria. For 31 blaKPC, 33 blaNDM, 7 blaIMP, and 2 blaVIM positive isolates, both CA and EA rates were 100%; no MEs or VMEs were detected by either method. For 15 carbapenemase-non-producing resistant isolates, the CA and EA rates of the gradient diffusion strips method were 100%. Whereas the CA rate of the disk diffusion method was 80.00% (12/15), the VME rate was 20.00% (3/15).Conclusion: The gradient diffusion strip method can meet the needs of clinical microbiological laboratories for testing the susceptibility of ceftazidime–avibactam drugs. However, the VME rate > 1.5% (5.45%) by the disk diffusion method. By comparison, the performance of the gradient diffusion strip method was better than that of the disk diffusion method.

In vitro activity of cefiderocol against ceftazidime-avibactam susceptible and resistant KPC-producing Enterobacterales: cross-resistance and synergistic effects.

To assess the in vitro activity of cefiderocol (CFDC) against a collection of both ceftazidime-avibactam (CZA) susceptible and resistant KPC-producingEnterobacterales (KPC-EB) isolates. Secondly, to assess its synergistic activity in combination with different antibiotics. One hundred KPC-EB isolates were tested: 60 CZA susceptible and 40 CZA resistant. Among them, 17 pairs of CZA susceptible and resistant KPC-producing Klebsiella pneumoniae (KPC-Kp) isolates were collected from 17 distinct patients before and after CZA treatment, respectively. CFDC susceptibility was evaluated by both broth microdilution (lyophilized panels; Sensititre; Thermo Fisher) and disk diffusion testing. Results were interpreted using EUCAST breakpoints. Synergistic activity of CFDC in combination with CZA, meropenem-vaborbactam, imipenem, and amikacin against six characterized KPC-Kp strains, before and after acquisition of CZA resistance, was evaluated using gradient diffusion strip crossing method. CFDC resistance rate was significantly higher in CZA resistant EB subset than in the susceptible one (p < 0.001): 82.5% vs 6.7%. MIC50 and MIC90 values were 0.25 and 2mg/L, 8 and 64mg/L in CZA-susceptible and CZA-resistant subset, respectively. KPC-Kp isolates harboring KPC-D179Y or KPC-Δ242-GT-243 variants showed CFDC MICs ranging from 4 to 64mg/L. CFDC showed in vitro synergistic effect mostly with CZA, against both CZA susceptible and resistant isolates, resulting in a synergy rate of 66.7%. CZA resistance mechanisms in KPC-EB impair the in vitro activity of CFDC, often leading to co-resistance. CFDC in combination with the new β-lactamases inhibitors might represent a strategy to enhance its activity.

Broth Microdilution and Gradient Diffusion Strips vs. Reference Agar Dilution Method: First Evaluation for Clostridiales Species Antimicrobial Susceptibility Testing.

Antimicrobial susceptibility testing of anaerobes is challenging. Because MIC determination is recommended by both CLSI and EUCAST, commercial broth microdilution and diffusion strip tests have been developed. The reliability of broth microdilution methods has not been assessed yet using the agar dilution reference method. In this work, we evaluated two broth microdilution kits (MICRONAUT-S Anaerobes® MIC and Sensititre Anaerobe MIC®) and one gradient diffusion strip method (Liofilchem®) for antimicrobial susceptibility testing of 47 Clostridiales isolates (Clostridium, Clostridioides and Hungatella species) using the agar dilution method as a reference. The evaluation focused on comparing six antimicrobial molecules available in both microdilution kits. Analytical performances were evaluated according to the Food and Drug Administration (FDA) recommendations. Essential agreements (EA) and categorical agreements (CA) varied greatly according to the molecule and the evaluated method. Vancomycin had values of essential and categorical agreements above 90% for the three methods. The CA fulfilled the FDA criteria for three major molecules in the treatment of Gram-positive anaerobic infections (metronidazole, piperacillin/tazobactam and vancomycin). The highest rate of error was observed for clindamycin. Multicenter studies are needed to further validate these results.

In vitro synergy of ticarcillin/clavulanate in combination with aztreonam and ceftolozane/tazobactam against SPM-1-producing Pseudomonas aeruginosa strains

Minimal inhibitory concentrations (MICs) of ticarcillin/clavulanic acid (TLc), ceftolozane/tazobactam (C/T), and aztreonam (AT) were determined for 6 SPM-1-producing Pseudomonas aeruginosa (PSA) using Etest® strips and the synergistic effect of such antimicrobials against was evaluated by gradient diffusion strip crossing (GDSC) test. The fraction inhibitory concentration indexes (FICI) were calculated and showed a synergistic (n = 3) and additive (n = 2) effects of TLc + AT against SPM-1 producers, while TLc + C/T combination caused no effect. Average MIC reduction of TLc and AT by GDSC was 3-fold and 2-fold dilutions, respectively. Thus, TLc + AT might be a candidate as a combination therapy to treat SPM-1-producing PSA infections.

27. The Membrane Antimicrobial Peptide Defense (MadRS) System Orchestrates Resistance Against Antibiotics and Host Innate Immune Peptides in enterococcus Faecalis

BackgroundEnterococci have evolved a network of two component-signaling systems (TCS) to evade killing by cationic antimicrobial peptides (AP) of the host innate immune response. Activation of these pathways can also lead to cross-resistance to AP-like antibiotics such as daptomycin (DAP), complicating treatment of severe enterococcal infections. Our previous work implicated the MadRS (formerly YxdJK) TCS in resistance to DAP. In this work, we show that the MadRS system regulates a network of membrane defense proteins that provide targeted resistance to killing by LL-37.MethodsWe performed a global transcriptional analysis comparing wild type (WT) E. faecalis OG1RF and an OG1RF derivative possessing a mutated allele of the madS gene that activates the MadRS system (OG1RFmadSA202E). Selected genes identified in this analysis were knocked out using a CRISPR-cas9 gene editing system and confirmed by sequencing. Antibiotic minimum inhibitory concentrations (MIC) were determined using gradient diffusion strips. LL-37 killing assays were performed in triplicate in RPMI+5% LB broth. Caenorhabditis elegans assays were performed in triplicate, and representative results are shown.ResultsAnalysis of the MadRS regulon revealed a significant upregulation of a network of gene targets, including madLM (formerly yxdLM, involved in bacitracin resistance), madEFG, the dlt operon, salA, and EF2211 (Fig 1). The madSA202E allele was associated with increased bacterial survival on exposure to LL-37, while the madEFG deletion mutant was more susceptible to killing (Fig 2A). Deletion of madLM decreased bacitracin MICs, but did not alter survival on exposure to LL-37. In C. elegans, inactivation of the MadRS system by deletion of the madR response regulator resulted in attenuation as compared to WT OG1RF. The effect was reversed by complementing with wild-type madR on a plasmid (Fig 2B). The attenuation effect of the mutant was abolished in pmk-1 knockout worms, which lack the ability to produce innate immune peptides (Fig 2C).Figure 1 Figure 2ConclusionThe MadRS system plays an important role in mediating resistance to AP both in vitro and in vivo. The novel ABC-transporter MadEFG is likely to play a major role in preventing enterococcal killing by the human cathelicidin LL-37. Disclosures William R. Miller, MD, Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support)Shionogi (Advisor or Review Panel member) Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)

1600. Closing the gap on moxifloxacin breakpoints for Stenotrophomonas maltophilia

BackgroundMoxifloxacin (MOX) has in vitro activity against Enterobacterales and Stenotrophomonas maltophilia (SM). Although MOX commonly displays lower minimum inhibitory concentration (MIC)50/90 values against SM when compared to levofloxacin, there are currently no established MOX breakpoints for treatment of SM. The Clinical and Laboratory Standards Institute (CLSI) has established interpretive categories and MIC breakpoints for levofloxacin (S ≤2µg/ml) against SM. The US Food and Drug Administration and European Committee on Antimicrobial Susceptibility Testing provide MOX breakpoints for Enterobacterales with susceptible MICs represented at ≤ 2 µg/mL and ≤ 0.25 µg/mL, respectively. The purpose of this study was to evaluate MOX MIC distribution against SM strains recovered from clinical specimens.MethodsClinical samples from patients with suspected infection during calendar year 2018 and 2019 were processed in the microbiology lab of Wake Forest Baptist Medical Center. After incubation, SM colonies were identified by MALDI-TOF system. MOX susceptibility testing was performed for these clinical isolates by gradient diffusion strip methodologies. Results were displayed as MIC (µg/mL) without interpretation. MIC50/90 and susceptibility rates at potential breakpoints were calculated.ResultsA total of 211 isolates were tested, 112 from 2018 and 99 from 2019. MOX MIC50 and MIC90 for all isolates was 0.25 µg/mL and 2 µg/mL, respectively. The range of MIC distribution was ≤ 0.006 µg/mL to ≥ 64 µg/mL. Percent susceptibilities at incremental MICs, including established MOX breakpoints against Enterobacterales and established levofloxacin breakpoints against SM, are represented in Table 1. MIC distribution was plotted in Figure 1.Table 1. Susceptibility rates of S. maltophilia to moxifloxacin at theoretical breakpoints Figure 1. Moxifloxacin MIC Distribution against All S. maltophilia Isolates ConclusionWith no established breakpoint, these data represent one of the largest samples of MOX MICs against SM in the United States. Using the CLSI breakpoint for levofloxacin in SM (MIC of ≤2ug/ml) the overall susceptibility rate is 93%. This finding highlights the importance of performing susceptibility testing to this agent by the microbiology laboratory and the critical need for MOX breakpoints in SM.Disclosures Tyler J. Stone, PharmD, Paratek (Research Grant or Support) John Williamson, PharmD, Paratek (Research Grant or Support) Elizabeth Palavecino, MD, Paratek (Grant/Research Support)Paratek (Grant/Research Support)

In vitro synergy of ceftolozane/tazobactam in combination with fosfomycin or aztreonam against MDR Pseudomonas aeruginosa

Carbapenem-resistant Pseudomonas aeruginosa (CR-PSA) imposes great limitations on empirical therapeutic choices, which are further complicated by metallo-β-lactamase production. This study evaluated in vitro antimicrobial synergy of ceftolozane/tazobactam in combination with aztreonam and fosfomycin against MDR PSA. MICs were determined by broth microdilution and gradient strips. The effect of ceftolozane/tazobactam+aztreonam and ceftolozane/tazobactam+fosfomycin combinations were tested against 27 MDR PSA isolates carrying blaSPM-1 (n = 13), blaIMP (n = 4), blaVIM (n = 3), blaGES-1 (n = 2) and blaCTX-M-like (n = 2), and 3 isolates with no acquired β-lactamase production detected by gradient diffusion strip crossing (GDSC). Six genetically unrelated SPM-1-producing isolates were also evaluated by time-kill analysis (TKA). All CR-PSA isolates harbouring blaSPM-1, blaGES-1 and blaIMP-1 were categorized as resistant to ceftolozane/tazobactam, meropenem and fosfomycin, with 70% being susceptible to aztreonam. Synergism for ceftolozane/tazobactam+fosfomycin and ceftolozane/tazobactam+aztreonam combinations was observed for 88.9% (24/27) and 18.5% (5/27) of the isolates by GDSC, respectively. A 3- to 9-fold reduction in ceftolozane/tazobactam MICs was observed, depending on the combination. Ceftolozane/tazobactam+fosfomycin was synergistic by TKA against one of six SPM-1-producing isolates, with additional non-synergistic bacterial density reduction for another isolate. Aztreonam peak concentrations alone demonstrated a ≥3 log10 cfu/mL reduction against all six isolates, but all strains were within the susceptible range for the drug. No antagonism was observed. In the context of increasing CR-PSA and the genetic diversity of resistance mechanisms, new combinations and stewardship strategies may need to be explored in the face of increasingly difficult to treat pathogens.

Comparative Evaluation of the In Vitro Activities of WCK 5222 (Cefepime-Zidebactam) and Combination Antibiotic Therapies against Carbapenem-Resistant Pseudomonas aeruginosa

The in vitro activity of WCK 5222 (cefepime-zidebactam) was compared to that of several available combination therapies among 30 clinical carbapenem-resistant Pseudomonas aeruginosa (CRP) strains using gradient diffusion strips. The combinations included nonsusceptible β-lactams (cefepime, ceftolozane-tazobactam, and meropenem) with amikacin and fosfomycin. WCK 5222 MICs ranged from 2 to 32 mg/liter, and 97% were ≤16 mg/liter, while 105/146 (72%) combinations demonstrated inhibition below established susceptibility breakpoints. WCK 5222 monotherapy may be preferred over the combinations assessed for CRP infections.