European Committee On Antimicrobial Susceptibility Testing Breakpoints Research Articles

Ceftazidime/avibactam serum concentration in patients on ECMO.

The use of extracorporeal membrane oxygenation (ECMO) may alter blood levels of several drugs, including antibiotics, leading to under dosing of these drugs and thus to potential treatment failure. No data exist on pharmacokinetics of new antimicrobial, in particular ceftazidime/avibactam. We therefore perform this study to evaluate ceftazidime/avibactam blood levels in ECMO patients and find factors associated with underdosing. Retrospective observational study of patients on ECMO having received ceftazidime/avibactam and in whom trough blood levels of ceftazidime and avibactam were available. Main outcome measurement was the number of patients with ceftazidime and avibactam blood levels above predefined cut-off values, derived from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for Enterobacteriaceae and Pseudomonas aeruginosa, namely 8 mg/L for ceftazidime and 4 mg/L for avibactam, and explored factors associated with underdosing. Twenty-three ceftazidime/avibactam trough levels were available in 14 ECMO patients, all of them having received veno-venous ECMO for SARS-CoV-2-associated pneumonia. Although ceftazidime levels were above 8 mg/L in all except one patient, nine (39%) of the avibactam dosages were below 4 mg/L. Increased renal clearance (creatinine clearance > 130 mL/min) was the main factor associated with under dosing, since 7 out of the 10 dosages below the predefined cut-offs were measured in patients with this condition. In ECMO patients receiving ceftazidime/avibactam, ceftazidime and avibactam serum levels are above EUCAST breakpoints in most cases, justifying the use of normal dosing in ECMO patients. Increased renal clearance may lead to ceftazidime and avibactam under dosing.

In vitro Activity of Dalbavancin and Fourteen Other Antimicrobial Agents against Toxigenic Clostridioides difficile Clinical Isolates in a Greek Tertiary-Care Hospital

Objective: Clostridioides difficile is a major cause of healthcare-associated diarrhea worldwide. For years, metronidazole and vancomycin were considered the standard treatment for C. difficile infection. However, they are increasingly being associated with treatment failure and recurrence. In this study, we investigated the in vitro activity of dalbavancin and fourteen other antimicrobials against 155 toxigenic C. difficile isolates originating from patients with C. difficile-associated diarrhea. Materials and Methods: Antimicrobial susceptibility was evaluated by the MIC Test Strip, and the results were interpreted using both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. Results: C. difficile isolates were fully susceptible to metronidazole, vancomycin, amoxicillin/clavulanate, piperacillin/tazobactam, and tigecycline. All isolates were dalbavancin susceptible by the CLSI breakpoint (≤0.25 μg/mL) compared with 97.4% susceptibility by the EUCAST breakpoint (≤0.125 μg/mL). Dalbavancin demonstrated significantly lower MIC50 and MIC90 values compared to vancomycin (0.047 vs. 0.38 and 0.125 vs. 0.5, respectively, p < 0.001). Resistance rates to penicillin, ampicillin, cefoxitin, imipenem, meropenem, clindamycin, moxifloxacin, chloramphenicol, and tetracycline were 20%, 14.2%, 100%, 75.5%, 0.6%, 51%, 36.1%, 3.2%, and 14.8%, respectively. Multidrug-resistant phenotypes were detected among 41.3% of the isolates. Conclusion: Dalbavancin exhibited potent activity against the isolates tested. As C. difficile is an important healthcare-associated pathogen, continued surveillance is required to monitor for development of resistance.

2162. In Vitro Antimicrobial Activity of Ceftobiprole against Streptococcus pneumoniae Isolates from the United States (2016–2020)

Abstract Background Ceftobiprole (BPR) is an advanced-generation cephalosporin approved in Europe and many non-European countries for the treatment of community-acquired pneumonia and non–ventilator-associated hospital-acquired pneumonia in adults caused by indicated species, including Streptococcus pneumoniae (SPN). This study evaluated the in vitro antimicrobial activity of BPR and comparator agents against recent SPN isolates causing lower respiratory tract infections in patients at medical centers in the United States (US). Methods A total of 2,793 SPN isolates from 32 US medical centers (2016–2020) were collected from patients with lower respiratory tract infections. Isolates were tested for antimicrobial susceptibility using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. MIC interpretations for BPR and comparators utilized European Committee on Antimicrobial Susceptibility Testing (EUCAST) or CLSI criteria, respectively. The EUCAST BPR susceptibility breakpoint for SPN was 0.5 mg/L. Results BPR inhibited 99.5% of all SPN isolates at the EUCAST breakpoint (MIC50/90, 0.015/0.25 mg/L). The MIC50/90 values were stable over all 5 surveillance years. The following comparator agents displayed lower activity against the isolate set: clindamycin (85.5% susceptible [S]), erythromycin (53.2% S), penicillin (63.2% S using oral breakpoints), tetracycline (79.4% S), and trimethoprim-sulfamethoxazole (72.8% S). BPR maintained excellent in vitro activity against each of the isolate subsets resistant to these comparator antimicrobials (95.4–98.8% S to BPR depending on the comparator antimicrobial; 95.4% of the penicillin-resistant isolates were S to BPR). This SPN isolate set was also 99.4% S to levofloxacin, 97.4% S to ceftriaxone (using non-meningitis breakpoints), and 100% S to linezolid and vancomycin. Conclusion BPR exhibited potent in vitro antimicrobial activity against recent SPN clinical isolates collected in US medical centers (2016–2020). These data indicate that BPR merits further investigation as a potential option for the treatment of lower respiratory tract infections caused by SPN in the US. Disclosures Leonard R. Duncan, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|CorMedix: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Jennifer Smart, PhD, Basilea Pharmaceutica International Ltd, Allschwil, Switzerland: Stocks/Bonds Mark E. Jones, PhD, Astellas Pharma Global Development, Inc: Support for the present publication|Basilea Pharmaceutica International Ltd: Employee of Basilea Pharmaceutica International Ltd|Basilea Pharmaceutica International Ltd: Stocks/Bonds Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Entasis: Grant/Research Support|GSK: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support

Assessment of cefiderocol disk diffusion versus broth microdilution results when tested against Acinetobacter baumannii complex clinical isolates.

The aim of this study was to evaluate the correlation between inhibitory zones and minimum inhibitory concentrations (MICs) when testing cefiderocol against Acinetobacter baumannii complex using disk diffusion and the broth microdilution (BMD) method according to the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Four-hundred and sixty-eight non-duplicated A. baumannii complex clinical isolates were collected from 56 hospitals of the China Antimicrobial Surveillance Network from 2019 to 2021. BMD using iron-depleted cation-adjusted Mueller-Hinton broth (ID-CAMHB) and standard disk diffusion methods were performed according to CLSI guidelines. Results were interpreted according to the CLSI and EUCAST breakpoints. Categorical agreement (CA), minor error (mE), major error (ME), and very major error (VME) were calculated for disk diffusion methods. The susceptibilities of all A. baumannii complex isolates by BMD were 98.7% (CLSI) and 97.6% (EUCAST). For all A. baumannii complex isolates, CA was 98.1% (CLSI) and 97.0% (EUCAST), with 0.9% (CLSI) and 1.9% (EUCAST) of VME, respectively. For the carbapenem-susceptible A. baumannii complex, the CA was 100%, with no mE or VME using both CLSI and EUCAST breakpoints. For carbapenem-resistant A. baumannii complex, CA was 97.5% (CLSI) and 96.2% (EUCAST), with 1.1% (CLSI) and 2.5% (EUCAST) of VME, respectively. Regarding the difficult-to-treat resistance A. baumannii complex isolates, CA was 97.6% (CLSI) and 95.7% (EUCAST), with 1.2% (CLSI) and 3.1% (EUCAST) of VME, respectively. Cefiderocol disk diffusion was difficult to assess in this study. Very few isolates were resistant to cefiderocol by BMD using CLSI breakpoint, and these were categorized as susceptible with the disk diffusion test. This study did, however, show that the main proportion of A. baumannii isolates were susceptible to cefiderocol by BMD, including carbapenem-resistant A. baumannii. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii is a major global health concern due to its high prevalence and limited treatment options. Cefiderocol is the only novel Food and Drug Administration (FDA)-approved β-lactam agent for the salvage treatment of carbapenem-resistant A. baumannii infection. Currently, a commercial automated susceptibility testing panel of cefiderocol is unavailable. Both the preparation of iron-depleted cation-adjusted Mueller-Hinton broth and the performance of broth microdilution are cumbersome in routine microbiology laboratories. A disk diffusion method is convenient for cefiderocol antimicrobial susceptibility testing, but limited data are available specifically for A. baumannii clinical isolates. Moreover, the Clinical and Laboratory Standards Institute published revisions to the A. baumannii cefiderocol disk diffusion breakpoints in 2022. Hence, we evaluated the performance of cefiderocol disk diffusion compared with the reference BMD against A. baumannii clinical isolates, especially those with cefiderocol zone diameters ≤ 14 mm.

The Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Aspergillus Section Flavi from Three French Hospitals.

(1) Background: Aspergillus flavus is a cosmopolitan mold with medical, veterinary, and agronomic concerns. Its morphological similarity to other cryptic species of the Flavi section requires molecular identification techniques that are not routinely performed. For clinical isolates of Aspergillus section Flavi, we present the molecular identification, susceptibility to six antifungal agents, and clinical context of source patients. (2) Methods: One hundred forty fungal clinical isolates were included in the study. These isolates, recovered over a 15-year period (2001-2015), were identified based on their morphological characteristics as belonging to section Flavi. After the subculture, sequencing of a part of the β-tubulin and calmodulin genes was performed, and resistance to azole antifungals was screened on agar plates containing itraconazole and voriconazole. Minimum inhibitory concentrations were determined for 120 isolates by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. (3) Results: Partial β-tubulin and calmodulin sequences analysis showed that 138/140 isolates were A. flavus sensu stricto, 1 isolate was A. parasiticus/sojae, and 1 was A. nomiae. Many of the isolates came from samples collected in the context of respiratory tract colonization. Among probable or proven aspergillosis, respiratory infections were the most frequent, followed by ENT infections. Antifungal susceptibility testing was available for isolates (n = 120, all A. flavus ss) from one hospital. The MIC range (geometric mean MIC) in mg/L was 0.5-8 (0.77), 0.5-8 (1.03), 0.125-2 (0.25), 0.03-2 (0.22), 0.25-8 (1.91), and 0.03-0.125 (0.061) for voriconazole, isavuconazole, itraconazole, posaconazole, amphotericin B, and caspofungin, respectively. Two (1.67%) isolates showed resistance to isavuconazole according to current EUCAST breakpoints with MICs at 8 mg/L for isavuconazole and voriconazole. One of these two isolates was also resistant to itraconazole with MIC at 2 mg/L. (4) Conclusions: The present characterization of a large collection of Aspergillus belonging to the Flavi section confirmed that A. flavus ss is the predominant species. It is mainly implicated in respiratory and ENT infections. The emergence of resistance highlights the need to perform susceptibility tests on section Flavi isolates.

Antimicrobial resistance and its detection in Staphylococcus saprophyticus urinary isolates

The aim of this study was to describe the antibiotic susceptibility of clinical Staphylococcus saprophyticus isolates collected prospectively from urine specimens over a 2-month period from September to October 2022 at a single centre in Melbourne, Australia. Species identification was performed by MALDI-TOF MS. All isolates underwent phenotypic antibiotic susceptibility testing by disc diffusion using European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) guidelines and VITEK2, and mecA polymerase chain reaction. A total of 302 S.saprophyticus isolates from 298 patients were included in this study. Most specimens (91.1%) were referred by community general practitioners from non-hospitalised patients. Antimicrobial resistance to non-β-lactam antibiotics was uncommon; trimethoprim susceptibility was 97%; trimethoprim/sulfamethoxazole, 98%; nitrofurantoin, 100%; and ciprofloxacin, 100% (100% ciprofloxacin susceptible, increased exposure by EUCAST breakpoints). Methicillin resistance (by mecA detection) was the most common form of urinary antibiotic resistance at 5.6%. VITEK2 susceptibility testing for methicillin resistance had a poor specificity of 61.8% (95% CI 55.8-67.4%) compared to mecA detection. These findings indicate that empiric antibiotic recommendations of trimethoprim, trimethoprim/sulfamethoxazole, and nitrofurantoin for treatment of urinary S.saprophyticus remain appropriate.

Antifungal Susceptibility Pattern of Candida glabrata from a Referral Center and Reference Laboratory: 2012-2022.

The prevalence of invasive candidiasis caused by non-Candida albicans has rapidly increased. Candida glabrata (Nakaseomyces glabrata) is an important pathogen associated with substantial mortality. Our study examined the antifungal temporal susceptibility of C. glabrata and cross-resistance/non-wild-type patterns with other azoles and echinocandins. Laboratory data of all adult patients with C. glabrata isolated from clinical specimens at the Mayo Clinic, Rochester, from 2012 to 2022 were collected. Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used. We obtained 1046 C. glabrata isolates from 877 patients. Using CLSI and EUCAST breakpoints, 187 (17.9%) isolates and 256 (24.5%) isolates were fluconazole-resistant, respectively. Focusing on C. glabrata bloodstream infections, fluconazole-resistance ranged from 16 to 22%. Among those 187 fluconazole-resistant isolates, 187 (100%) and 184 (98.4%) isolates were also voriconazole and posaconazole non-wild-type, respectively, with 97 (51.9%) isolates deemed non-wild type for itraconazole. The fluconazole susceptibility pattern has not changed over the past decade. The proportion of fluconazole-resistant C. glabrata is relatively high, which could be due to the complexity of patients and fluconazole exposure. Itraconazole appears to be a compelling step-down therapy for fluconazole-resistant C. glabrata, given the high proportion of wild-type isolates. Further research to examine clinical outcomes is warranted.

In vitro activity of eravacycline and comparators against Gram-negative and Gram-positive bacterial isolates collected from patients globally between 2017 and 2020

To evaluate eravacycline (ERV) activity against Gram-negative and Gram-positive bacteria collected between 2017 and 2020 from worldwide locations. MIC determinations were performed using Clinical and Laboratory Standards Institute (CLSI) broth microdilution methodology. ERV and tigecycline susceptibility was interpreted using United States Food and Drug Administration (FDA) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. Comparator susceptibility was interpreted using CLSI and EUCAST breakpoints. ERV MIC90 was 0.5 μg/mL against 12,436 Enterobacteriaceae isolates, which only increased to 1 μg/mL against multidrug-resistant (MDR) isolates (n=2931 (23.6%). Similar activity was shown against 1,893 Acinetobacter baumannii (MIC90 1 μg/mL) and 356 Stenotrophomonas maltophilia (MIC90 2 μg/mL). ERV was more active against Gram-positive bacteria: 415 Streptococcus pneumoniae (MIC90 0.008 μg/mL), 273 S. anginosus group (MIC90 0.015 μg/mL), 1,876 Enterococcus faecalis and 1,724 E. faecium (MIC90 2 μg/mL), 2,158 Staphylococcus aureus and 575 S. saprophyticus (MIC90 0.12 μg/mL), 1,143 S. epidermidis and 423 S. haemolyticus (MIC90 0.25 μg/mL). ERV MIC90 against methicillin-resistant staphylococci and vancomycin-resistant enterococci was similar to susceptible strains. However, ERV susceptibility varied between EUCAST or FDA against staphylococci, especially S. epidermidis (91.5% vs 47.2%), and vancomycin-resistant E. faecalis (98.3% vs 76.5%). This study reaffirms ERV's consistent broad-spectrum activity that has been evaluated since 2003. ERV remains a key agent for the treatment of bacterial infections, including resistant isolates, but urgent re-assessment of clinical breakpoints is required for staphylococci and enterococci.

Interpretation of Antimicrobial Susceptibility Testing Using European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) Breakpoints: Analysis of Agreement.

Breakpoints provided by European Committee on Antimicrobial Susceptibility Testing (EUCAST) are now being used in many countries. This study was planned to ascertain the agreement in antimicrobial susceptibility using the Clinical and Laboratory Standards Institute (CLSI) and EUCAST breakpoints during the Kirby-Bauer disk diffusion method. This was aprospective observational study. Clinical isolates belonging to the family Enterobacteriaceae recovered between January and December, 2022, were included in the analysis. The diameter of the zone of inhibition of the 14 antimicrobials (viz. amoxicillin/clavulanic acid, cefazolin, ceftriaxone, cefuroxime, cefixime, aztreonam, meropenem, gentamicin, amikacin, ciprofloxacin, levofloxacin, norfloxacin, trimethoprim/sulfamethoxazole and fosfomycin)was analysed. Antimicrobial susceptibility was interpreted using CLSI 2022 and EUCAST 2022 guidelines. Results:Susceptibility data from a total of 356 isolates showed a slight increase in the percentage of resistant isolates with most of the drugs using EUCAST guidelines. The level of agreement varied from almost perfect to slight. For two drugs, i.e., fosfomycin and cefazolin, the agreement was least among the drug analysed (kappa (κ) value < 0.5, p < 0.001). For Ceftriaxone and Aztreonam, with EUCAST, susceptible (S) isolates would have been categorised in the newly redefined "I" category. It would have indicated the use of higher dosages of drugs. Conclusion: Changein the breakpoints impacts the interpretation of the susceptibility. It can also lead to a change in the dosage of the drug used for treatment. Therefore, there is an urgent need to see the impact of recent modifications "I" category of EUCAST on the clinical outcome and usage of antimicrobials.

Genomic analysis of Fosfomycin resistance in multi-drug resistant uropathogens and comparison of in-vitro susceptibility methods uropathogens.

Urinary tract infection is one of the most common bacterial infections causing high morbidity and mortality. The alarming rise of multidrug-resistant uropathogens worldwide forced the clinician to rethink the old drugs like Fosfomycin for its therapeutic management. Our objective was to compare agar dilution, disc diffusion and E-test method for antimicrobial susceptibility testing of Fosfomycin against different drug-resistant uropathogens. Consecutive 181 uropathogens were tested for Fosfomycin susceptibility using agar dilution, disc diffusion and E-test. Results were interpreted using Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. Whole genome sequencing analysis was done on the 4 XDR/PDR Fosfomycin resistant Klebsiella pneumoniae isolates. Escherichia coli was found as the most common (62.4%) uropathogen followed by Klebsiella pneumoniae (21%). Considering agar dilution as the gold standard, 6.1% of isolates were resistant to Fosfomycin. Following CLSI breakpoints, the susceptibility of Escherichia coli, Klebsiella pneumoniae, other Enterobacterales and Pseudomonas aeruginosa were 92.9%, 92.1%, 100%, 100%; whereas using EUCAST breakpoints the susceptibility rates were 85.7%, 86.9%, 92.9%, and 100%, respectively. The essential agreement, categorical agreement, major error, and very major error for E-test/disc diffusion for all the organisms were 91.2%/Not Applicable, 95%/93.9%, 1.8%/4.7%, 9.1%/9.1%, respectively. Whole-genome sequencing showed mutation UhpT gene as well as the presence of plasmid-mediated fosA5 or fosA6 genes conferring Fosfomycin resistance. This result supports very low resistance of Enterobacterales against Fosfomycin; hence should be considered a valuable option to treat multidrug-resistant uropathogens. Disc diffusion was observed to be a convenient method for Fosfomycin susceptibility testing compared to agar dilution.

Global update on the in vitro activity of tigecycline and comparators against isolates of Acinetobacter baumannii and rates of resistant phenotypes (2016–2018)

ObjectivesThis study presents 2016–2018 in vitro antimicrobial activity data and rates of resistant phenotypes for clinical isolates of Acinetobacter baumannii from Africa/Middle East, Asia/South Pacific, Europe, Latin America, and North America. MethodsA total of 4320 A. baumannii isolates were collected across all regions between 2016 and 2018. The in vitro antimicrobial activities of amikacin, colistin, levofloxacin, meropenem, and tigecycline were determined using the broth microdilution methodology of the Clinical and Laboratory Standards Institute. MICs were interpreted using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (version 11.0). Rates of subsets that were resistant to amikacin, colistin, levofloxacin, and meropenem, according to EUCAST breakpoints, are also presented. ResultsIn each region, tigecycline and colistin were active against isolates of A. baumannii (MIC90 values, 1 or 2 mg/L) and the lowest rate of resistance was to colistin (1.2%–7.3%). The rates of resistance to the panel of agents were generally lower among A. baumannii from North America (1.3%–42.7%), compared with the other regions. Fewer than 11% of meropenem-resistant A. baumannii were also resistant to colistin. The rates of amikacin-, levofloxacin- and meropenem-resistant A. baumannii were lowest in North America and mostly higher in Africa/Middle East and Latin America. ConclusionIn each geographical region, tigecycline and colistin maintained good in vitro antimicrobial activity against isolates of A. baumannii, including antimicrobial-resistant subsets. The higher rates of meropenem-resistant isolates, particularly in Africa/Middle East and Latin America, require continued monitoring because of the scarcity of effective treatment options.

Antimicrobial susceptibility testing of clinical isolates of Gram-negative bacilli collected in Morocco by the ATLAS Global Surveillance Program from 2018 to 2020

To report reference method antimicrobial susceptibility testing results for recent clinical isolates of Gram-negative bacilli from Morocco. CLSI (Clinical and Laboratory Standards Institute) broth microdilution antimicrobial susceptibility testing was performed by a central laboratory for isolates of Enterobacterales (n=810), Pseudomonas aeruginosa (n=321), and Acinetobacter baumannii (n=191) collected in 2018-2020 by three hospital laboratories in Morocco. MICs were interpreted using both CLSI (2021) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) (2021) breakpoints. Molecular testing for β-lactamase genes was performed on isolates meeting defined screening criteria. Most isolates of Enterobacterales were susceptible (CLSI/EUCAST breakpoints) to amikacin (98.0%/96.2%), ceftazidime-avibactam (94.8%/94.8%), and meropenem (92.5%/94.2%). Of Enterobacterales isolates eligible for β-lactamase gene screening (n=210), 174 were ESBL-positive, 40 were metallo-β-lactamase-positive (all NDM), 39 were serine carbapenemase-positive (all OXA); and 7 isolates carried both OXA-48 and NDM-1. Amikacin (89.1%/89.1%) and ceftazidime-avibactam (88.2%/88.2%) were the most active agents tested against P. aeruginosa. Applying CLSI and EUCAST breakpoints, MDR rates were 21.9% and 29.3% for Enterobacterales and 18.4% and 21.8% for P. aeruginosa. Susceptible rates for amikacin, ceftazidime-avibactam, and meropenem were 93.2%/89.5%, 77.4%/82.3%, and 67.8%/80.2% for MDR Enterobacterales and 50.8%/57.1%, 40.7%/45.7%, and 27.1/32.9% for MDR P. aeruginosa. ≥70% of A. baumannii isolates were resistant to all agents tested (except colistin, EUCAST breakpoints only) including amikacin and meropenem. Newer β-lactam/β-lactamase inhibitor combinations such as ceftazidime-avibactam warrant testing and reporting for Enterobacterales and P. aeruginosa in Morocco given the presence of significant resistance to first-line β-lactams and fluoroquinolones, pervasive ESBLs and carbapenemases, and toxicity concerns associated with some second-line agents.

Evaluation of EUCAST Direct Rapid Antimicrobial Susceptibility Test Method in Blood Culture Bottles with Positive Signal

Early reporting of the antibiotic susceptibility testing (AST) results is essential for the survival of sepsis patients. In 2019, European Committee on Antimicrobial Susceptibility Testing (EUCAST) published a proposal to detect antimicrobial susceptibility from positive blood culture bottles with a rapid antimicrobial susceptibility test (RAST) method in a maximum of eight hours. In this study, it was aimed to evaluate the EUCAST RAST method in blood culture bottles that resulted with positive signal in BacT/ALERT (bioMérieux, France) blood culture system and that showed gram-negative bacteria with single morphology with Gram stain. The study was conducted prospectively between April 2019 and November 2019. Ninety blood culture bottles that we detected single gram negative bacteria morphology by Gram stain were tested according to the EUCAST RAST method, The isolates obtained from the blood culture bottles were studied with the EUCAST disk diffusion method and the Vitek 2 Compact (bioMerieux, France) automated system. The results obtained with RAST were compared with the results of these methods. The turn around time of the RAST method was recorded. Categorical agreement of the RAST method with conventional methods and the very major error rates were determined. Of the 14 isolates not yet covered by the EUCAST HADT method, 12 were determined to be other Enterobacterales members and two as other non-fermentatives. Two isolates were detected with the same morphological characteristics in Gram stain of the blood culture bottle and the same antibiotic susceptibility profile, but with different identification results. These sixteen isolates were excluded from the study. In this study the susceptibility of 74 isolates were determined according to the EUCAST breakpoint tables, of which 31 were Klebsiella pneumoniae, 35 were Escherichia coli, four were Acinetobacter baumannii and four were Pseudomonas aeruginosa. According to the evaluation periods of EUCAST RAST; the susceptibility profile was reported for nine (12%) of E.coli at four hours, eight (11%) at six hours, 18 (24%) at eight hours; three (4%) of K.pneumoniae at four hours, 16 (21%) at six hours, 12 (16%) at eight hours; three of P.aeruginosa (4%) at six hours, one (1%) at eight hours; two of A.baumannii (2%) at six hours and two (2%) at eight hours. The categorical aggrement of the RAST method was 91.8% with the automated system and 96.8% with the disc diffusion method. Very major errors of RAST method compared to the automated system were detected for piperacillin-tazobactam (17.7%), ceftazidime (11.6%) and meropenem (5.6%); and when compared to the disc diffusion method, for cefotaxime (5.7%) and meropenem (6.7%). Our results have shown that EUCAST RAST method can practicaly be performed in routine laboratories to report early results with a low cost. Because of the very major errors it is necessary to confirm the results with the standard methods.

Surveillance of ceftobiprole against Gram-positive and Gram-negative clinical isolates from 2018 from different European territories

Ceftobiprole is approved for the treatment of hospital-acquired and community-acquired pneumonia in 17 different European countries and is currently undergoing clinical trials in the USA. In this study, isolates were collected from hospital laboratories from 15 European countries during 2018 as part of an ongoing post-marketing surveillance study. Minimum inhibitory concentrations (MICs) were determined using European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution methodology and were interpreted using 2019 EUCAST breakpoints. Ceftobiprole was active (MIC, ≤2 mg/L) against 100% and 98.9% of methicillin-susceptible Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from 2018, respectively. Only six MRSA isolates (1.1%) were resistant to ceftobiprole and originated from four countries. Against Streptococcus pneumoniae, ceftobiprole was active (MIC, ≤0.5 mg/L) against 98.7% of isolates. Overall, 75.6% of Enterobacterales were susceptible, although isolate numbers in certain countries were notably low. In addition, based on non-species-related pharmacokinetic/pharmacodynamic breakpoints, 63.2% of Pseudomonas aeruginosa isolates were susceptible to ceftobiprole. Data for ceftobiprole for isolates from 2018 are very similar to studies performed on isolates from earlier years, showing that susceptibility to ceftobiprole has remained high.

In vitro activity of the novel siderophore cephalosporin, cefiderocol, in Gram-negative pathogens in Europe by site of infection

ObjectivesWe assessed the activity of the novel siderophore cephalosporin, cefiderocol and selected other antibacterial agents against Gram-negative bacterial isolates in Europe. MethodsIsolates were obtained between 2013 and 2018 from European countries participating in the SIDERO-WT and SIDERO-Proteeae multinational surveillance studies. Isolates were categorised by infection site, focusing on bloodstream infections, hospital-acquired/ventilator-associated bacterial pneumonia (HABP/VABP), complicated intra-abdominal infections and complicated urinary tract infections. Cefiderocol activity was compared with ceftazidime–avibactam, ceftolozane–tazobactam, colistin and meropenem using standard susceptibility testing methods. European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used to interpret susceptibility data. ResultsIsolates (n = 20 911) were collected from 145 sites in 24 countries in Europe, the highest proportion (34%) being from patients with HABP/VABP. Enterobacterales (66.6% of isolates) were more frequent than glucose non-fermenting species (33.4%) overall, with some differences between infection sites. Across all infection sites, the MIC50/MIC90 for cefiderocol was ≤0.5/≤2 mg/L for Enterobacter spp., ≤0.25/<2 mg/L for Klebsiella spp., 0.12/2 mg/L for Acinetobacter spp., ≤0.25/1 mg/L for Pseudomonas aeruginosa and ≤0.12/≤0.5 mg/L for Stenotrophomonas maltophilia. Across all infection sites, cefiderocol MICs were ≤2 mg/L for ≥96% of Enterobacter spp., ≥95% of Klebsiella spp., ≥90% of Acinetobacter spp. and ≥99% of Pseudomonas aeruginosa and Stenotrophomonas maltophilia isolates. Cefiderocol maintained high activity in carbapenem-resistant isolates, and the difference in activity between carbapenem-resistant (percentage susceptibility at EUCAST breakpoint: E. coli 77.8%, Klebsiella spp. 69.2%, Pseudomonas aeruginosa 97.5%, Acinetobacter spp. 90.7%, Stenotrophomonas maltophilia 99.6%) and carbapenem-susceptible (percentage susceptibility at EUCAST breakpoint: E. coli 99.4%, Klebsiella spp. 98.0%, Pseudomonas aeruginosa 99.7%, Acinetobacter spp. 94.9%) isolates was lower for cefiderocol than other agents. ConclusionsCefiderocol had excellent activity against all Gram-negative species, independent of key infection site and carbapenem MIC. Cefiderocol is a useful addition to the therapeutic options available for these difficult-to-treat infections.

In vitro activity of ceftaroline against bacterial pathogens isolated from patients with skin and soft tissue and respiratory tract infections in the Middle East and Africa: AWARE global surveillance programme 2015–2018

ObjectivesTo report antimicrobial susceptibility testing surveillance data for ceftaroline and comparative agents from the AWARE global surveillance programme for bacterial pathogens causing skin and soft tissue infections (SSTIs) and lower respiratory infections (RTIs) in Middle East and African countries from 2015 to 2018. MethodsPathogens were identified by MALDI-TOF/MS. Antimicrobial susceptibility testing was performed using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. MICs were interpreted by both CLSI (M100, 2020) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) (v 10.0, 2020) breakpoints. ResultsAll MSSA (n = 1125) and 93.9% of MRSA (n = 1235) were susceptible to ceftaroline (MIC ≤ 1 μg/mL, CLSI and EUCAST). The maximum ceftaroline MIC observed for MRSA was 2 μg/mL; no ceftaroline-resistant MRSA were identified among SSTI (CLSI and EUCAST) and RTI (CLSI) isolates. All isolates of β-haemolytic Streptococcus (n = 324), and penicillin-susceptible (PSSP) and -intermediate Streptococcus pneumoniae (PISP; n = 369) were susceptible to ceftaroline. Rates of susceptibility to ceftaroline for penicillin-resistant S. pneumoniae (penicillin MIC ≥ 2 μg/mL; n = 175), and β-lactamase-negative (BLNHI; n = 224) and β-lactamase-positive Haemophilus influenzae (n = 49) were 99.4%, 98.7%, and 98.0% (CLSI) and 92.6%, 98.2%, and 83.7% (EUCAST), respectively. Rates of susceptibility to ceftaroline for ESBL-negative Escherichia coli (n = 442), Klebsiella pneumoniae (n = 381), and Klebsiella oxytoca (n = 103) were 92.1%, 93.2%, and 96.1%, respectively. ConclusionCeftaroline-resistant isolates of MRSA causing SSTIs were not identified in Middle East and African countries in 2015–2018 using recently revised CLSI (in 2019) or EUCAST (in 2018) breakpoint criteria. Common bacterial pathogens causing SSTIs (Staphylococcus aureus, β-haemolytic Streptococcus) and lower RTIs (PSSP, PISP, BLNHI) demonstrated no resistance or low levels of resistance (0–1.8%) to ceftaroline.

Evaluation of Intravenous Fosfomycin Disodium Dosing Regimens in Critically Ill Patients for Treatment of Carbapenem-Resistant Enterobacterales Infections Using Monte Carlo Simulation.

There are limited intravenous fosfomycin disodium (IVFOS) dosing regimens to treat carbapenem-resistant Enterobacterales (CRE) infections. This study aimed to use Monte Carlo simulation (MCS) for evaluation of IVFOS dosing regimens in critically ill patients with CRE infections. The dosing regimens in critically ill patients with various creatinine clearance were evaluated with MCS using minimum inhibitory concentration (MIC) distributions of fosfomycin against CRE clinical isolates in Thailand and the 24 h area under the plasma drug concentration–time curve over the minimum inhibitory concentration (AUC0-24/MIC) of ≥21.5 to be a target for IVFOS. The achieved goal of the probability of target attainment (PTA) and a cumulative fraction of response (CFR) were ≥90%. A total of 129 non-duplicated CRE clinical isolates had MIC distributions from 0.38 to >1024 mg/L. IVFOS 8 g every 8 h, 1 h, or 4 h infusion, could achieve approximately 90% PTA of AUC0-24/MIC target to treat CRE infections with MICs ≤ 128 mg/L. According to PTA target, an IVFOS daily dose to treat carbapenem-resistant Escherichia coli based on Clinical Laboratory Standards Institute (CLSI) breakpoints for urinary tract infections and one to treatment for CRE infections based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were 16 g/day and 8 g/day, respectively. All dosing regimens of IVFOS against CRE achieved CFR ≤ 70%. This study proposes the IVFOS dosing regimens based on CLSI and EUCAST breakpoints for the treatment of CRE infections. However, further clinical studies are needed to confirm the results of these findings.

In Vitro Activities of Antimicrobials Against Toxigenic Clostridioides difficile Isolates Obtained in a University Training and Research Hospital in Turkey

Clostridioides difficile, a gram-positive, anaerobic, spore forming bacillus known as Clostridium difficile according to the previous taxonomy, is the most important agent of antibiotic-associated diarrhea. C.difficile infections have become a major health problem for many countries. The rate of antimicrobial resistant C.difficile isolates is rapidly increasing all around the world. Yet there is limited data on this subject in our country. The aim of this study was to determine the antimicrobial susceptibility profiles of C.difficile strains isolated from stool samples in Marmara University Pendik Training and Research Hospital Microbiology Laboratory. A total of 93 toxigenic C.difficile, defined by serological and molecular techniques, were included in this study. Antimicrobial susceptibility profiles of isolates were determined by using agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI; M11-A7). The following antimicrobials commonly used for the treatment of C.difficile infections or applied previously in C.difficile epidemiological studies were tested: metronidazole, vancomycin, meropenem, ceftriaxone, ampicillin-sulbactam, clindamycin, erythromycin, moxifloxacin, tetracycline, doxycycline, tigecycline and linezolid. The minimum inhibitory concentration (MIC) results were interpreted according to the breakpoints described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoints recommended by CLSI were applied for ceftriaxone, clindamycin, tetracycline and moxifloxacin since there were no EUCAST breakpoints for these antimicrobials. MIC50 and MIC90 values were determined for three antimicrobials (linezolid, erythromycin, doxycycline) whose breakpoints were not described by EUCAST or CLSI guidelines. All isolates were susceptible to metronidazole, vancomycin, ampicillin-sulbactam, meropenem and tetracycline. Susceptibility to ceftriaxone, clindamycin and moxifloxacin was found in 58.1%, 35.5% and 20.4% of the isolates, respectively. MIC50 and MIC90 values of tigecycline, erythromycin linezolid, doxycycline were 0.125-0.25 mg/L, 1-2 mg/L, 2-2 mg/L, 0.062- 0.125 mg/L, respectively. This study shows the current antimicrobial susceptibility patterns of C.difficile isolates in our hospital and will also be the reference data for clinical laboratories in our country where anaerobic culture and susceptibility tests are not performed in routine practice. In conclusion, two main antimicrobial agents commonly used in the treatment of C.difficile infections, metronidazole and vancomycin, seem to be effective. However, high resistance rates against to the certain tested antimicrobials highlight the need for further surveillance to monitor the emergence of resistance.

Antimicrobial Susceptibility of Pathogenic Gram-positive Anaerobic Cocci: Data of a University Hospital in Turkey

Gram-positive anaerobic cocci (GPAC), a large group of anaerobic bacteria, are the members of the normal microbiota that colonizes the skin and mucosal surfaces of the human body. However, in case of a wound or when the host becomes immunocompromised, GPAC can cause invasive and most frequently mixed infections. GPAC are the second most frequently isolated bacteria in anaerobic infections. Although the studies are limited, GPAC have been reported to develop resistance to antimicrobial drugs. The resistance of the pathogens to the antimicrobials and improper therapy can cause poor clinical outcomes. Therefore, monitoring of the resistance trends of regional clinically important anaerobic bacteria periodically is recommended. In our study, we aimed to determine the antimicrobial susceptibility profiles of clinically important GPAC. A total of 100 non-duplicated pathogenic GPAC isolates were collected from Marmara University Hospital between 2013 and 2015. The isolates were identified by using conventional methods, "matrix-assisted laser desorption ionization-time of flight mass spectrometry system (MALDI-TOF MS)" (VITEK MS; v3.0, bioMerieux, France) and 16S rRNA gene sequencing. Antimicrobial susceptibility test was carried out by the agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The following antimicrobials were tested: penicillin, amoxicillin/ clavulanic acid (AMC), cefoxitin, meropenem, clindamycin, erythromycin, tetracycline, tigecycline, chloramphenicol, moxifloxacin and metronidazole. The minimum inhibitory concentration (MIC) results were interpreted according to the breakpoints described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoints recommended by CLSI for cefoxitin, tetracycline and moxifloxacin, and breakpoint recommended by Food and Drug Administration (FDA) for tigecycline were used since there were no EUCAST breakpoints for these antimicrobials. MIC50 and MIC90 values were determined for erythromycin since the breakpoint was not described by EUCAST, CLSI or FDA guidelines. The identification results showed that the strains (n= 100) consisted of five different GPAC genus; Parvomonas (40%), Finegoldia (34%), Peptoniphilus (14%), Peptostreptococcus (10%) and Anaerococcus (1%). All of the organisms were susceptible to meropenem, tigecycline and metronidazole. The isolates were highly susceptible to penicillin, AMC, cefoxitin, and chloramphenicol, since the resistance rates against these antimicrobials were 5% or less. The resistance rates against clindamycin, tetracycline and moxifloxacin were 14%, 31% and 24%, respectively. In total, 11% of the isolates were multidrug resistant. Metronidazole and tigecycline displayed high in vitro activity against GPAC and both are appropriate antimicrobials for the selection of empiric therapy. The effectiveness of meropenem was also found high, but it was observed that this antimicrobial would be more appropriate to use in the treatment of severe mixed infections accompanied by other microorganisms with the resistance potential. Detection of penicillin and AMC resistant isolates, which are frequently used in the treatment of GPAC infection, requires periodic monitoring of the antimicrobial susceptibility patterns of GPAC. The high rates of resistance against clindamycin, tetracycline and moksifloxacin indicated that these antimicrobials should not be used for empirical treatment of infections without prior antimicrobial susceptibility testing. This study is one of the largest susceptibility studies specifically carried out on GPAC to date in Turkey. We believe that our results will provide good surveillance data both for our hospital and our country.

Tigecycline susceptibility of multidrug-resistant Acinetobacter baumannii from intensive care units in the western Balkans.

Tigecycline can be effective to treat infections of carbapenem-resistant Acinetobacter baumannii (CRAB) however, no interpretive criteria have been approved so far. The objectives of this study were to evaluate the proportion of CRAB isolates and to compare gradient test with a broth microdilution (BMD) method for tigecycline susceptibility testing of A. baumannii.This study included 349 multidrug-resistant (MDR) Acinetobacter spp. collected from Serbia, Montenegro, Bosnia and Herzegovina in 2016 and 2017. Antibiotic susceptibility testing was performed by disk diffusion, VITEK2, gradient, ComASP Colistin. Tigecycline susceptibilities were interpreted according to breakpoints of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Food and Drug Administration (FDA).Majority of the tested isolates were CRAB (92.8%). Tigecycline MIC50/MIC90 values were 4/8 μg/mL by BMD and 0.5/4 μg/mL by gradient test. Essential agreement for BMD and gradient test amounted to 65.1%. With EUCAST breakpoints, categorical agreement (CA) was achieved in 38% isolates. Major discordance (MD-false susceptibility/resistance) and minor discordance (mD-false categorization involving intermediate results) were observed in 10% and 57% A. baumannii, respectively. With FDA breakpoints, CA, MD and mD were observed in 44%, 16% and 47% isolates, respectively. Colistin resistance was 2.1%.The study highlights a high proportion of CRAB and several discordances between BMD and gradient test which may lead to inappropriate therapy.