Mandy Wootton

Imipenem-relebactam (I/R) is a beta-lactam/beta-lactamase inhibitor combination given with cilastatin that is indicated for the treatment of complicated urinary tract infections, including pyelonephritis and complicated intra-abdominal infections in patients. Recently, a new gradient diffusion method for antimicrobial susceptibility testing of this antibiotic was developed for Pseudomonas aeruginosa (ETEST IRPA) as an alternative to broth microdilution (BMD). Our objective was to evaluate the performance of the I/R ETEST in comparison to BMD across three external and one internal study sites for 437 (77 challenge, 360 clinical) P. aeruginosa isolates. Using the U.S. Food and Drug Administration (FDA) interpretive breakpoints, ETEST IRPA achieved a 97.7% (427/437) essential agreement (EA), 91.1% (398/437) categorical agreement (CA), 0% very major errors (VMEs), 0.9% (3/329) major errors (MEs), and 8.2% (36/437) minor errors (mEs). Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) interpretive breakpoints, ETEST IRPA achieved a 97.7% (427/437) EA, 93.8% (410/437) CA, 12% (13/108) VME, and 4.2% (14/329) ME. Both bias and trending were in alignment with regulatory requirements. Reproducibility testing on 10 strains showed 99.6% (269/270) of results were within ±1 doubling dilution of the modal minimum inhibitory concentration (MIC). MIC ₅₀ and MIC ₉₀ values for imipenem were 8 and 32, respectively, in comparison to 1 and 8 with the addition of relebactam as determined by ETEST. In conclusion, ETEST IRPA provides both accurate and reproducible results for performing antimicrobial susceptibility testing of P. aeruginosa when interpreted with both FDA and EUCAST criteria. IMPORTANCE: Antimicrobial susceptibility testing is key to proper treatment of bacterial infections. Broth microdilution is the gold standard for antimicrobial susceptibility testing; however, it can be time-consuming and requires high levels of technical expertise. This study evaluates a new gradient diffusion method for antimicrobial susceptibility testing of imipenem-relebactam against Pseudomonas aeruginosa . The findings support the use of imipenem-relebactam gradient diffusion as an accurate and reproducible method of antimicrobial susceptibility testing against Pseudomonas aeruginosa .

The Burkholderia cepacia complex (BCC) is comprised of 24 species of gram-negative bacteria that cause opportunistic infections. While antimicrobial susceptibility testing (AST) has historically been used to guide treatment for BCC infections, recent work highlighting problems with AST for these organisms led the Clinical and Laboratory Standards Institute (CLSI) to remove disk diffusion and minimal inhibitory concentration (MIC) breakpoints for BCC from its M100 standards document. Epidemiological cut-off values (ECVs) may be helpful to clinicians in the absence of breakpoints, as they may be used to determine whether an isolate has a wild-type or non-wild-type phenotype. Here, we present an analysis of BCC ECVs for ceftazidime (CAZ), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), and trimethoprim-sulfamethoxazole (TMP-SMX). ECVs were calculated using MIC data from 3 previous studies and 3 independent laboratories for 1,896 BCC isolates. ECVs were 16 μg/mL for CAZ, 8 μg/mL for LVX, 16 μg/mL for MEM, and 8 μg/mL for MIN. The ECV for TMP-SMX varied depending on the analysis from 2, 8, and 16 μg/mL and therefore could not be reliably established. Challenges with establishing ECVs for BCC include limitations with the pooled MIC data set, broad MIC distributions, and high ECVs that are above the obsolete susceptible MIC breakpoints. These challenges limit the clinical utility of ECVs for these organisms and support the removal of ECVs from the CLSI M100 standards document. IMPORTANCE: The Burkholderia cepacia complex is a group of bacterial species that cause difficult-to-treat opportunistic infections. Recently, clinical breakpoints, which are used to determine whether organisms are susceptible to certain antimicrobials, were removed from Clinical and Laboratory Standards Institute (CLSI) standards for these organisms due to problems with antimicrobial susceptibility testing performance. Clinicians are now faced with the challenge of how to treat these complex infections without clinical breakpoints. Here, we determine epidemiological cut-off values (ECVs) for relevant antimicrobials for the B. cepacia complex. While we established ECVs for ceftazidime, levofloxacin, meropenem, and minocycline, we were unable to determine a reliable ECV for trimethoprim-sulfamethoxazole. Additionally, we encountered significant challenges in our analyses, including data limitations for these organisms and high ECVs that are not clinically useful. These challenges limit the practical use of these ECVs in helping guide treatment and support the removal of ECVs from the CLSI M100 standards document.

Objectives: A subset of clinical Pseudomonas aeruginosa isolates from the INVICTUS I study was selected to compare antimicrobial susceptibility performance for disc diffusion and broth microdilution of ceftolozane/tazobactam and five comparator antipseudomonal agents. Methods: Overall, 159 non–cystic fibrosis isolates were chosen from INVICTUS I that had the smallest zone diameters from disc testing to ceftolozane/tazobactam, including isolates that were close to the breakpoints. The isolates were tested across 14 centres in the UK. Susceptibility results were interpreted using EUCAST 2026 criteria. Results: Discrepancy analysis showed combined error rates (very major and major errors) highest for ceftolozane/tazobactam, ceftazidime and piperacillin/tazobactam (15.7%, 14.5% and 12.6%, respectively); when adjusted to remove isolates close to the breakpoint for ceftolozane/tazobactam and piperacillin/tazobactam there were still remarkably high error rates (10.6% and 7.2%, respectively). Essential agreement and bias analysis were not performed as MICs were being compared with zone diameters. Discussion: Our findings highlight substantial challenges in performing and interpreting susceptibility testing for these two important antipseudomonal β-lactam/β-lactamase inhibitors, which are commonly employed as either first-line empirical therapy (piperacillin/tazobactam) or for serious and MDR infection (ceftolozane/tazobactam). A review of disc testing for these agents may be warranted, and diagnostic laboratories may wish to review the technical limitations and put in place mitigating measures.

The British National Formulary and the NICE provide empirical treatment guidance for the management of various infections, including recommendations on antibiotic doses. In parallel, and for over 15 years, EUCAST has published details of the doses upon which their antimicrobial susceptibility breakpoints are predicated, termed here ‘breakpoint-defining doses’. Recently, differences between these dosing recommendations have attracted attention, particularly since EUCAST introduced its ‘susceptible, increased exposure’ category of susceptibility. We discuss where and why these differences exist and how to reconcile them.

The Burkholderia cepacia complex (BCC) is comprised of 24 species of Gram-negative bacteria that cause opportunistic infections. While antimicrobial susceptibility testing (AST) has historically been used to guide treatment for BCC infections, recent work highlighting problems with AST for these organisms led the Clinical and Laboratory Sciences Institute (CLSI) to remove disk diffusion (DD) and minimal inhibitory concentration (MIC) breakpoints for BCC from its M100 standards document. Epidemiological cut-off values (ECVs) may be helpful to clinicians in the absence of breakpoints, as they may be used to determine whether an isolate has a wild-type or non-wild- type phenotype. Here we present an analysis of BCC ECVs for ceftazidime (CAZ), levofloxacin (LVX), meropenem (MEM), minocycline (MIN), and trimethoprim-sulfamethoxazole (TMP-SMX). ECVs were calculated using MIC data from 3 previous studies and 3 independent laboratories for 1,896 BCC isolates. ECVs were 16 μg/ml for CAZ, 8 μg/ml for LVX, 16 μg/ml for MEM, and 8 μg/ml for MIN. The ECV for TMP-SMX varied depending on the analysis from 2 μg/ml, 8 μg/ml, and 16 μg/ml and therefore could not be reliably established. Challenges with establishing ECVs for BCC include limitations with the pooled MIC dataset, broad MIC distributions, and high ECVs that are above the obsolete susceptible MIC breakpoints. These challenges limit the clinical utility of ECVs for these organisms and supported removal of ECVs from the CLSI M100 standards document.

Aztreonam-avibactam (AZA) is a new monobactam/beta-lactamase inhibitor combination that recently received regulatory approval in the United States (U.S.) and Europe. In this study, we evaluated the performance of ETEST AZA, a gradient diffusion method, compared to reference broth microdilution for 650 challenge ( n = 77) and clinical ( n = 573) Enterobacterales isolates across four study sites. Using the U.S. Food and Drug Administration (FDA) interpretive breakpoints for ETEST AZA, challenge isolates demonstrated a 97.3% (72/74) essential agreement (EA), 93.2% (69/74) categorical agreement (CA), 0% very major errors (VME) and major errors (ME), and 6.8% (5/74) minor errors (mE). Clinical isolates demonstrated a 95.5% (504/528) EA, 98.7% (521/528) CA, 0% VME, 0.2% (1/516) ME, and 1.1% (6/528) mE. Overall, the combined set achieved a 95.7% (576/602) EA and 98.0% (590/602) CA, with 0% VME, 0.2% (1/577) ME, and 1.8% (11/602) mE. Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) interpretive breakpoints, challenge isolates demonstrated a 96.1% (74/77) EA, 93.5% (72/77) CA, and 31.3% (5/16) VMEs, while clinical isolates demonstrated a 94.1% (539/573) EA and 99.7% (571/573) CA including 7.7% (1/13) VME and 0.2% (1/560) ME. The combined set demonstrated a 94.3% (613/650) EA, 98.9% (643/650) CA, 0.2% (1/573) MEs, but 20.7% (6/21) VMEs due to the lack of an intermediate breakpoint that is addressed as a limitation in the European package insert. Both bias and trending were in alignment with regulatory requirements. In reproducibility testing of 10 on-scale strains, 100% (270/270) of the results were within ±1 doubling dilution of the modal minimal inhibitory concentration. In conclusion, ETEST AZA provides accurate, reproducible susceptibility results that meet regulatory requirements for many Enterobacterales species when interpreted using FDA or EUCAST criteria. IMPORTANCE: Aztreonam-avibactam (AZA) is a novel monobactam/β-lactamase inhibitor combination approved for clinical use in the United States and Europe in 2025 and 2024, respectively. It is indicated for the treatment of multidrug-resistant Gram-negative bacteria that produce multiple classes of β-lactamases. Until recently, antimicrobial susceptibility testing for this combined agent relied on broth disk elution or microdilution using ceftazidime–avibactam and aztreonam combined. In this study, we evaluated the performance of the bioMérieux ETEST AZA gradient diffusion strip as a more streamlined method for clinical microbiology laboratories. Testing of 650 Enterobacterales isolates demonstrated that ETEST AZA met regulatory performance criteria using interpretive breakpoints from both the FDA and the European Committee on Antimicrobial Susceptibility Testing. Overall, our findings indicate that the ETEST AZA demonstrates acceptable performance for many Enterobacterales isolates and represents a practical alternative for implementing AZA susceptibility testing in clinical microbiology laboratories.

Objectives: To assess the adherence of Ghanaian healthcare laboratories to AST standards and to assess discrepancies between breakpoint-defining dosages and recommended doses in Ghanaian National Standard Treatment Guidelines (NSTGs). Methods: Nineteen laboratories were issued electronic questionnaires regarding AST methods and guidance. NSTG doses and breakpoint-defining dosages were compared to determine the applicability of AST standards. Results: Eleven of the 19 laboratories responded. Traditional biochemical species identification was the most common method among surveyed laboratories, with disc diffusion being the main AST method. All laboratories reported using CLSI standards. Major quality assurance issues identified regarding AST testing included the use of outdated standards, failure to consistently determine inoculum density, irregular use of quality control strains, and agent limitations on antibiotic disc panels. Discrepancies in definitions for multidrug resistance and the testing of such isolates were also identified. When comparing recommended NSTG doses to breakpoint-defining dosages, the EUCAST standard had a higher number of doses available for review (30/30, 100% versus 14/30, 47%) and a higher percentage of full agreement (19/30, 63% versus 6/30, 20%) compared with CLSI. Discrepancies between breakpoint-defining and recommended dosages were found for meropenem, ceftriaxone, and amoxicillin-clavulanic acid oral/iv, leading to potential underdosing. Conclusions: This study highlights the need for rigorous adherence to AST standards and outlines considerations for the adoption of AST standards in a low-middle-income country.

EUCAST introduced ‘breakpoints in brackets’ to warn against the use of specified agents without additional therapeutic measures (EUCAST. Guidance document “EUCAST breakpoints in brackets”. 2021. Available at: www.mic.eucast.org.). Rifampicin is rarely used as monotherapy for Staphylococcus aureus infections, yet its breakpoints remain unbracketed. Here, we explore whether this remains appropriate. Although frequent emergent resistance largely precludes rifampicin’s use as monotherapy, this does not meet EUCAST’s core criterion for bracketing—that an agent is inherently ineffective as monotherapy.

Rising rates of multidrug-resistant Klebsiella infections necessitate a comprehensive understanding of the major strains and plasmids driving spread of resistance elements. Here, we analysed 540 clinical, screen and environmental Klebsiella isolates recovered from across Wales between 2007 and 2020 using combined short- and long-read sequencing approaches. We identified resistant clones that have spread within and between hospitals including the high-risk strain sequence type (ST)307, which acquired the bla _OXA-244 carbapenemase gene on a pOXA-48-like plasmid. We found evidence that this strain, which caused an acute outbreak largely centred on a single hospital in 2019, had been circulating undetected across South Wales for several years prior to the outbreak. In addition to clonal transmission, our analyses revealed evidence for substantial plasmid spread, mostly notably involving bla _KPC-2 and bla _OXA-48-like (including bla _OXA-244) carbapenemase genes that were found among many species and strain backgrounds. Two thirds (20/30) of the bla _KPC-2 genes were carried on the Tn4401a transposon and associated with IncF plasmids. These were mostly recovered from patients in North Wales, reflecting an outward expansion of the plasmid-driven outbreak of bla _KPC-2-producing Enterobacteriaceae in North-West England. A total of 92.1 % (105/114) of isolates with a bla _OXA-48-like carbapenemase carried the gene on a pOXA-48-like plasmid. While this plasmid family is highly conserved, our analyses revealed novel accessory variation including integrations of additional resistance genes. We also identified multiple independent deletions involving the tra gene cluster among pOXA-48-like plasmids in the ST307 outbreak lineage. These resulted in loss of conjugative ability and signal adaptation of the plasmids to carriage by the host strain. Altogether, our study provides, to our knowledge, the first high resolution view of the diversity, transmission and evolutionary dynamics of major resistant clones and plasmids of Klebsiella in Wales, and forms an important basis for ongoing surveillance efforts. This article contains data hosted by Microreact.