Disk Diffusion Breakpoints Research Articles

Evaluation of piperacillin-tazobactam disks using contemporary Enterobacterales isolates suggests the need for disk potency optimization.

In this article, we address major gaps in contemporary data for piperacillin-tazobactam (TZP) susceptibility testing and evaluate the performance of disk diffusion. TZP is the most common empiric broad-spectrum agent against Gram-negative pathogens and is used as a carbapenem-sparing regimen. OXA-1 β-lactamases drive global Enterobacterales TZP resistance and raise MICs to the clinical breakpoints, making susceptibility testing challenging. In 2022, CLSI revised the Enterobacterales TZP MIC breakpoints. Due to the lack of contemporary correlates, disk diffusion breakpoints were revised using outdated historical data from 1991 and 2003 and yielded unacceptable error rates. Additionally, there is a lack of global consensus on disk potency. The EUCAST TZP disks contain 30 μg piperacillin and 6 μg tazobactam. The CLSI disks, established after TZP approval in 1979 and prior to the widespread prevalence of ESBLs, contain 100 μg piperacillin and 10 μg tazobactam. Here, we evaluate disk diffusion using the 100/10 and 30/6 μg TZP disks with 100 contemporary Enterobacterales isolates, including 40 harboring blaOXA-1. We conducted a disk development study to determine if an alternative potency might address accuracy issues with TZP susceptibility testing. We demonstrate that decreasing TZP potency improves the performance of disk diffusion and improves separation between susceptible and non-susceptible isolates, particularly those harboring OXA-1, but no disk yielded optimal results. The alternative 20/5 μg disk yielded the lowest errors using CLSI MIC breakpoints and the best separation between susceptible isolates and isolates harboring blaOXA-1. Our study addresses an unmet need, shows that further optimization of the TZP disk potency is possible, and provides clinical laboratories with a better understanding of the performance of TZP disks using contemporary, challenging isolates. A larger, multicenter study is needed for further optimization but has been limited by a lack of funding for an off-patent antimicrobial. Our struggles in accessing funding underscore the frequent challenge with AST for older but heavily used antimicrobials.

Real-world performance of susceptibility testing for cefiderocol: insights from a prospective multicentre study on Gram-negative bacteria.

Cefiderocol is a novel siderophore-conjugated cephalosporin developed for the treatment of multidrug-resistant Gram-negative bacterial (GNB) infections. However, the current gold standard for cefiderocol susceptibility testing, broth microdilution (BMD) using iron-depleted cation-adjusted Mueller-Hinton broth, presents challenges for many microbiology laboratories. In this study, we evaluate the real-world performance of disc diffusion (DD) and a commercial BMD method (ComASP®) to test cefiderocol susceptibility in a series of isolates collected prospectively from severely ill patients in a multicentre study. The susceptibilities of 1472 isolates (632 Enterobacterales, 532 Pseudomonas aeruginosa, 84 Acinetobacter spp. and 224 Stenotrophomonas maltophilia) collected in 60 Spanish hospitals were analysed following the EUCAST 2023 and 2024 criteria. We assessed the performance of DD (cefiderocol 30 μg disc, Liofilchem) and a commercial BMD method (ComASP® Cefiderocol, Liofilchem). A total of 1408 and 1450 isolates were susceptible by DD and ComASP® BMD, respectively. Overall, the agreement between both methods was 96.9%. Forty-four isolates were resistant by DD but susceptible by ComASP® BMD, and two were susceptible by DD but resistant by ComASP® BMD (Acinetobacter baumannii isolates). Adoption of the updated 2024 EUCAST DD breakpoints and areas of technical uncertainty (ATUs) led to a decrease in susceptibility among Enterobacterales (95.3% versus 92.6%). DD is a straightforward, rapid and accessible method for routine determination of cefiderocol susceptibility in real-world practice. ComASP® BMD shows a high agreement with DD in susceptible isolates and may help to resolve DD interpretability concerns in isolates with susceptibility results within the ATU, but caution is warranted when testing resistant isolates.

Current trend of biapenem susceptibility and disc diffusion breakpoints in Enterobacterales and Pseudomonas aeruginosa

IntroductionBiapenem has been recently approved by the Drug Controller General of India for the treatment of complicated urinary tract infections (cUTI). However, there are no assessment studies that evaluate the in-vitro activity of biapenem against contemporary ESBL-producing Indian Enterobacterales isolates. To determine the activity of biapenem against contemporary ESBLs and/or OXA-1/ampC producing Enterobacterales and Pseudomonas aeruginosa isolates. MethodologyIsolates were tested for susceptibility to biapenem and its comparators using the broth microdilution method. Presence of ESBLs (SHV, TEM, CTX-M) genes, OXA-1, and ampC genes (ACC, ACT, DHA, CIT/CMY, FOX) using multiplex PCR. ResultsAgainst ESBL with OXA-1 and/or ampC-producing E. coli, ESBL-K. pneumoniae, and cephalosporin-resistant P. aeruginosa, biapenem showed in-vitro activity similar to that of meropenem. Overall, a biapenem disc concentration of 10 μg provided no error rates for testing E. coli, K. pneumoniae, and P. aeruginosa isolates. ConclusionIt is more accurate to test biapenem at a 10 μg disc concentration and apply more stringent disc diffusion breakpoints for interpretation.

Possible impact of revisions in disc diffusion breakpoints for aminoglycosides and piperacillin/tazobactam in the 33rd edition of CLSI M100 document on clinical reporting and use in Indian settings with low susceptibility

PurposeThe study explores the impact of significant interpretative breakpoint changes for aminoglycosides and piperacillin-tazobactam in Enterobacterales and Pseudomonas aeruginosa, considering PK/PD, clinical data, and susceptibility on clinical reporting and use. ProcedureBetween January 2021 and June 2023, a total of 189,583 samples were processed for bacterial pathogens and antimicrobial susceptibility testing was performed using disc diffusion method/VITEK® 2 Compact system/broth microdilution. WHONET software was utilised to capture and analyse the changes in the interpretation of disc diffusion method, following updates to CLSI M100 documents in comparison to previous editions. Antimicrobial consumption data was collected and interpreted as DDD/100 bed days using AMC tool software. Here, we present data for 13,615 members of Order Enterobacterales and 1793 Pseudomonas aeruginosa isolates. FindingEnterobacterales exhibited a significant susceptibility drop of 14.7% for gentamicin and 21.7% for amikacin. Pseudomonas aeruginosa showed an increase in isolates with intermediate tobramycin susceptibility, from 0.6% to 29.7%, with relatively minor changes in piperacillin-tazobactam interpretation. ConclusionThe changes indicate a shift toward increased 'resistance' and 'intermediate susceptibility' for these antibiotics, emphasizing the need for cautious use and leveraging PK/PD knowledge for improved antibiotic utilization, patient outcomes, and antimicrobial stewardship.

Evaluation of Antibiotics by Disk Diffusion and Minimum Inhibitory Concentration Breakpoints in Urinary Tract Infections

Antibiotic resistance (ABR) has made it more challenging to treat uropathogenic organisms. It is impossible to compromise antimicrobial susceptibility testing (AST), which is essential and has a significant impact on infection treatment strategies. Although labor-intensive and technically challenging for everyday laboratory use, the agar dilution technique is appropriate for monitoring and assessing novel antimicrobials. Objective: To determine the minimum inhibitory concentration (MIC), Agar dilution technique and disk diffusion as susceptibility test methodologies. Methods: This study was carried out at Khyber Girls Medical College (KGMC) Peshawar. Keeping in view the Clinical and Laboratory Standards Institute (CLSI) guidelines AST was executed. BIOMÉRIEUX® API® kits and gram staining were utilized for identification of bacteria. The disk diffusion was performed using Thermo ScientificTM OxoidTM antibiotic discs of Co-trimoxazole, Levofloxacin, Nitrofurantoin and Fosfomycin. The MIC and zone of inhibitions for disk diffusion were noted according to the CLSI protocol. Results: 158 culture positive samples were isolated out of 680 total received. Esherechia Coli (E. coli) (74.1%) being the most isolated organism. In comparison of disk diffusion and agar dilution, categorical agreement for Levofloxacin, Cotrimoxazole, Nitrofurantoin and Fosfomycin were (82.28%, 72.15 %, 87.97% and 82.28%) respectively. Kappa coefficients of (0.64, 0.43, 0.57 and 0.37) (p < 0.0001) were calculated for Levofloxacin, Co-trimoxazole, Nitrofurantoin, and Fosfomycin respectively, revealing considerable level of agreement for these antibiotics. Conclusions: It was concluded that Agar dilution is more precise than disk diffusion but being more labor intensive and technical. Disk diffusion can still produce significantly accurate results with less resource consumption.

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.

Cutibacterium Acnes (Formerly Propionibacterium Acnes ): Friend or Foe?

Cutibacterium acnes protects skin homeostasis. The species has three subspecies, and associations between C. acnes subsp. acnes and acne, C. acnes subsp. defendens and prostate cancer, and C. acnes subsp. elongatum and progressive macular hypomelanosis have recently been suggested. Different phylotypes/clonal complexes may cause prosthetic joint and other infections, and virulence factors such as fimbriae, biofilms, multidrug-resistance plasmids, porphyrin, Christie-Atkins-Munch-Petersen factors and cytotoxicity contribute to infections. Isolates are subtyped by multiplex PCR or multi- or single-locus sequence typing; however, these methods could be better synchronized. Resistance of acneic strains to macrolides (25.0-73.0%), clindamycin (10.0-59.0%) and tetracyclines (up to 37.0%) is worrisome, but susceptibility testing is now facilitated by European Committee on Antimicrobial Susceptibility Testing disk diffusion breakpoints. New therapeutic approaches include sarecycline, antimicrobial peptides and bacteriophages.

Usefulness of EUCAST rapid antibiotic susceptibility breakpoints and screening cut-off values directly from blood cultures for the inference of β-lactam resistance mechanisms in Enterobacterales.

Reducing the turnaround time for reporting antimicrobial susceptibility testing (AST) results is important for adjusting empirical treatments and may impact clinical outcomes of septic patients, particularly in settings with high antimicrobial resistance. Disc diffusion could be useful for inferring β-lactam resistance mechanisms. To evaluate the usefulness of EUCAST rapid AST (RAST) disc diffusion breakpoints for the screening of resistance mechanisms (sRAST) and interpretive reading of resistance phenotypes to infer ESBL and carbapenemases production in Enterobacterales. Blood cultures were artificially spiked with Enterobacterales clinical isolates with well-characterized β-lactam resistance mechanisms (n = 93), WT phenotypes (n = 26) and ATCC strains (n = 8). AST was performed by disc diffusion directly from blood cultures and inhibition zones were manually measured at 4, 6 and 8 h. To infer the presence of resistance mechanisms, EUCAST RAST breakpoints and screening cut-off values (sRAST) combined with the double-disc synergy test (DDS) for ESBLs or aztreonam susceptibility for carbapenemases detection were used. DDS together with sRAST detected all ESBL producers as early as at 4 h incubation. Cefotaxime was the antibiotic with the highest discriminatory power. The suspicion of carbapenemase production by sRAST at 8 h was possible in 73% of Klebsiella pneumoniae and in 100% of Escherichia coli carbapenemase-producing isolates. Phenotypic analysis improves the detection of some low hydrolytic carbapenemases (OXA-48 or KPC-3 mutants). Early detection of β-lactam resistance mechanisms directly from positive blood cultures was possible using sRAST together with the interpretive reading of antibiotic resistance phenotypes. Some carbapenemase types such as OXA-48 might be difficult to infer. Screening-positive isolates should be confirmed using an alternative technique.

Evaluation and comparison of antibiotic susceptibility profiles of Streptomyces spp. from clinical specimens revealed common and region-dependent resistance patterns

Notwithstanding the fact that streptomycetes are overlooked in clinical laboratories, studies describing their occurrence in disease and potential pathogenicity are emerging. Information on their species diversity in clinical specimens, aetiology and appropriate therapeutic treatment is scarce. We identified and evaluated the antibiotic susceptibility profile of 84 Streptomyces clinical isolates from the Czech Republic. In the absence of appropriate disk diffusion (DD) breakpoints for antibiotic susceptibility testing (AST) of Streptomyces spp., we determined DD breakpoints by correlation with the broth microdilution method and by the distribution of zone diameters among isolates. Correlation accuracy was high for 9 antibiotics, leading to the establishment of the most valid DD breakpoints for Streptomyces antibiotic susceptibility evaluation so far. Clinical strains belonged to 17 different phylotypes dominated by a cluster of strains sharing the same percentage of 16S rRNA gene sequence identity with more than one species (S. albidoflavus group, S. hydrogenans, S. resistomycificus, S. griseochromogenes; 70% of isolates). AST results showed that Streptomyces exhibited intrinsic resistance to penicillin, general susceptibility to amikacin, gentamycin, vancomycin and linezolid, and high percentage of susceptibility to tetracyclines and clarithromycin. For the remaining antibiotics, AST showed inter- and intra-species variations when compared to available literature (erythromycin, trimethoprim-sulfamethoxazole), indicating a region-dependent rather than species-specific patterns.

Susceptibility of Tigecycline against Carbapenem Resistant Enterobacteriaceae

Background: Enterobacteriaceae, a large family of Gram-negative bacteria, are one of the commonest etiological agents causing serious bacterial infections to humans. Carbapenems are the group of antibiotics with a broad spectrum of antimicrobial action. Infections caused due to Carbapenem resistant Enterobacteriaceae (CRE) are a huge challenge for existing medical practice. Therefore, this project aimed to find out the antimicrobial susceptibility pattern of Tigecycline against CRE. Methods: This cross-sectional study with non-probability consecutive sampling was done at Ziauddin Hospital Microbiology Laboratory from 15th August 2017 to 15th April 2018. Accordingly, 151 isolates of CRE were collected from cultures of blood, respiratory tract, wound pus and other body fluids. The growth inhibition zones were measured following the Food and Drug Administration (FDA) disk diffusion breakpoint criteria. Frequencies and percentages were computed for gender, microorganism, and antimicrobial susceptibility. Chi-squared test was applied and p≤ 0.05 was considered as statistically significant. Results: Klebsiella species were most commonly isolated pathogen, 67.5% (n=102) followed by Escherichia coli (E. coli) 23.2% (n=35), Enterobacter 7.3% (n=11) and Serratia species 2% (n=3). Tigecycline was 97% (34 /35) sensitive for E. coli, 86.3% (88/102) for Klebsiella species), 91% (10/11) for Enterobacter species, and 100% for Serratia species. Klebsiella species showed the highest rate of resistance to tigecycline i.e., 13.7% of the total Klebsiella isolates. Conclusion: Among the Enterobacteriaceae family, Klebsiella species have the greatest ability to acquire resistance. Tigecycline showed good activity against isolates of CRE recovered from infections of skin, soft tissue, intra-abdomen, lower respiratory tract and blood stream. Keywords: Carbapenems; beta-Lactamases; Anti-Bacterial Agents; Tigecycline; Klebsiella; Escherichia coli.

Correlation between Broth Microdilution and Disk Diffusion Results when Testing Ceftazidime-Avibactam against a Challenge Collection of Enterobacterales Isolates: Results from a Multilaboratory Study.

We assessed the ceftazidime-avibactam disk diffusion breakpoints that provide the lowest discrepancy error rates by testing an Enterobacterales isolate collection with ceftazidime-avibactam MIC values near the breakpoints. Isolates (n = 112) were susceptibility tested by broth microdilution and disk diffusion methods in 3 laboratories. Current disk diffusion breakpoints (≥21/≤20 mm for susceptible/resistant) provided the lowest error rates, but confirmatory MIC testing is indicated for isolates with inhibition zones of 20 to 22 mm.

What's New from the CLSI Subcommittee on Antimicrobial Susceptibility Testing M100, 29th Edition

The 29th edition of Clinical and Laboratory Standards Institute (CLSI) document M100, Performance Standards for Antimicrobial Susceptibility Testing, was published in January 2019. This edition of the document was corrected in January, February, and May 2019 and revised in March and July 2019. Categorical interpretations were redefined to accommodate the adoption of a susceptible–dose-dependent (SDD) category using off-label drug dosages for Enterococcus faecium treated with daptomycin and methicillin-resistant Staphylococcus aureus (MRSA) treated with ceftaroline. Additional revisions included updated breakpoints for disk diffusion and minimum inhibitory concentrations (MICs) for Enterobacteriaceae treated with meropenem-vaborbactam; investigational intended use of cefiderocol breakpoints for Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter spp., and Stenotrophomonas maltophilia; fluoroquinolone disk diffusion and MIC breakpoints for Enterobacteriaceae and P. aeruginosa; and MIC breakpoints for azithromycin to be used with Neisseria gonorrhoeae. A review of these changes and the rationale supporting the changes are presented here, along with the new and updated recommendations, clarifications, and other less significant changes to the M100 document.

2162. Comparison of Plazomicin Disk Diffusion vs. Gradient Diffusion Susceptibility Testing Results Against Drug-Resistant Clinical Enterobacteriaceae Isolates

BackgroundPlazomicin, a novel aminoglycoside, is active against carbapenem-resistant Enterobacteriaceae (EB) and is not inhibited by most aminoglycoside modifying enzymes that affect gentamicin and tobramycin. We investigated the activity of plazomicin against resistant EB clinical isolates and compared disk diffusion (DD) vs. gradient diffusion (GD) results.MethodsEB isolates that were carbapenem resistant and/or resistant to both gentamicin and tobramycin were retrieved from the UW Health clinical isolate repository. Each isolate was tested against plazomicin using both DD (MAST Group Ltd. Plazomicin disk 30 µg) and GD (Liofilchem Plazomicin MIC Test Strip 0.16–256 µg/mL) methods according to manufacturer instructions and using FDA clinical breakpoints for interpretation.Results51 isolates were tested: 21 E. coli, 9 P. mirabilis, 7 E. cloacae, 6 K. pneumoniae, 3 K. oxytoca, 3 S. marcescens, 1 K. aerogenes, and 1 C. freundii. Specimen sites included: 29 blood, 8 urine, 8 soft tissue or bone, 5 intra-abdominal, and 1 sputum. Previous phenotypic AST results demonstrated 19 (37%) were CRE, of which 5 were also gentamicin and tobramycin resistant, and 32 (63%) were tobramycin and gentamicin resistant but carbapenem susceptible. Plazomicin zone diameters and minimal inhibitory concentrations (MIC) for all isolates are shown in the figure (data jittered to show frequency). There was a significant correlation between increased MIC and smaller zone diameters (Pearson coefficient −0.443, P = 0.001). However, while all 51 isolates were susceptible by DD breakpoints, only 46 (92%) were susceptible by GD breakpoints. All 5 discordant results were P. mirabilis which had an MIC of 4 µg/mL (intermediate) but zone diameters of 20–21 mm (susceptible).ConclusionConcordance between plazomicin DD and GD susceptibility was only 92%. All 5 discordant results were P. mirabilis. Surveillance studies demonstrate >80% of P. mirabilis have MIC of 2–4 mg/L. Given the DD breakpoint is 16 mm, our data suggest DD was overly active in our sample set. Comparison of DD and GD to reference broth microdilution against a larger set of isolates is warranted to determine which method is optimal; however, our data suggest DD may result in categorical errors for P. mirabilis. DisclosuresAlexander Lepak, MD, Paratek Pharmaceuticals: Research Grant; Tetraphase Pharmaceuticals: Research Grant.

Evaluation of the Revised Ceftaroline Disk Diffusion Breakpoints When Testing a Challenge Collection of Methicillin-Resistant Staphylococcus aureus Isolates

We assessed ceftaroline disk diffusion breakpoints for Staphylococcus aureus when applying revised Clinical and Laboratory Standards Institute (CLSI) ceftaroline MIC breakpoints. Disk-MIC correlation was evaluated by testing a challenge collection (n = 158) of methicillin-resistant S. aureus (MRSA) isolates composed of 106 randomly selected isolates plus 52 isolates with decreased susceptibility to ceftaroline (MIC, 1 to 16 μg/ml). Disk diffusion was performed with 30-μg disks and Mueller-Hinton agar from 2 manufacturers each. Revised CLSI susceptible (S)/susceptible dose-dependent (SDD)/resistant (R) MIC breakpoints of ≤1/2 to 4/≥8 μg/ml were applied. The disk breakpoints that provided the lowest error rates were CLSI S/R breakpoints of ≥25 mm/≤19 mm, with no very major (VM) or major (Ma) errors and with minor (Mi) error rates of 0.0% for ≥2 doubling dilutions above the I or SDD (≥I + 2), 22.1% for I or SDD plus or minus 1 doubling dilution (I ± 1), and 2.3% for ≤2 doubling dilutions below the I or SDD ≤I - 2 (overall Mi error rate, 16.5%). No mutation in the penicillin-binding protein 2a (PBP2a) was observed in 5 of 15 isolates with a ceftaroline MIC of 2 μg/ml; 3 of 11 isolates with a ceftaroline MIC of 1 μg/ml exhibited mutations in the penicillin-binding domain (PBD; 1 isolate) or in the non-PBD (2 isolates). All isolates except 1, with a ceftaroline MIC of ≥4 μg/ml, showed ≥1 mutation in the PBD and/or non-PBD. In summary, results from the disk diffusion method showed a good correlation with those from the reference broth microdilution method. Our results also showed that the ceftaroline MIC distribution of isolates with no mutations in the PBP2a goes up to 4 μg/ml, and reference broth microdilution and disk diffusion methods do not properly separate wild-type from non-wild-type isolates.

Relative performance of antimicrobial susceptibility assays on clinical Escherichia coli isolates from animals

The assessment of antimicrobial resistance in bacteria derived from animals is often performed using the disc diffusion assay. However broth-microdilution is the preferred assay for national antimicrobial resistance surveillance programs. This study aimed to evaluate the accuracy of disc diffusion relative to broth-microdilution across a panel of 12 antimicrobials using data from a collection of 994 clinical Escherichia coli isolates from animals. Disc diffusion performance was evaluated by diagnostic sensitivity, specificity, likelihood ratio pairs and receive-operating characteristic (ROC) analysis. Data was dichotomised using CLSI susceptible and resistant clinical breakpoints. In addition, disc diffusion breakpoints produced using diffusion Breakpoint Estimation Testing Software (dBETS) were evaluated. Analysis revealed considerable variability in performance estimates for disc diffusion susceptible and resistant breakpoints (AUC ranges: 0.78–0.99 and 0.92–1.0, respectively) across the panel of antimicrobials. Ciprofloxacin, tetracycline, and ampicillin estimates were robust across both breakpoints, whereas estimates for several antimicrobials including amoxicillin-clavulanic acid, cefoxitin and gentamicin were less favourable using susceptible breakpoints. Overall performance estimates were moderately improved when dBETS susceptible breakpoints were applied. For most antimicrobials, disc diffusion was accurate at predicting resistance of clinical E. coli from animals that could otherwise be determined by broth-microdilution. While disc diffusion is suboptimal for assessing the proportion of fully susceptible isolates for some drugs, sensitivity and specificity estimates provided here allow for the use of standard formula to correct this. For this reason, disc diffusion has applicability in national surveillance provided the performance of the assay is taken into account.

Analysis of complement gene variants in the clinical laboratory: Comparison of Next-Generation Sequencing (NGS) and Sanger methods

The assessment of antimicrobial resistance in bacteria derived from animals is often performed using the disc diffusion assay. However broth-microdilution is the preferred assay for national antimicrobial resistance surveillance programs. This study aimed to evaluate the accuracy of disc diffusion relative to broth-microdilution across a panel of 12 antimicrobials using data from a collection of 994 clinical Escherichia coli isolates from animals. Disc diffusion performance was evaluated by diagnostic sensitivity, specificity, likelihood ratio pairs and receive-operating characteristic (ROC) analysis. Data was dichotomised using CLSI susceptible and resistant clinical breakpoints. In addition, disc diffusion breakpoints produced using diffusion Breakpoint Estimation Testing Software (dBETS) were evaluated. Analysis revealed considerable variability in performance estimates for disc diffusion susceptible and resistant breakpoints (AUC ranges: 0.78–0.99 and 0.92–1.0, respectively) across the panel of antimicrobials. Ciprofloxacin, tetracycline, and ampicillin estimates were robust across both breakpoints, whereas estimates for several antimicrobials including amoxicillin-clavulanic acid, cefoxitin and gentamicin were less favourable using susceptible breakpoints. Overall performance estimates were moderately improved when dBETS susceptible breakpoints were applied. For most antimicrobials, disc diffusion was accurate at predicting resistance of clinical E. coli from animals that could otherwise be determined by broth-microdilution. While disc diffusion is suboptimal for assessing the proportion of fully susceptible isolates for some drugs, sensitivity and specificity estimates provided here allow for the use of standard formula to correct this. For this reason, disc diffusion has applicability in national surveillance provided the performance of the assay is taken into account.

Revisit of fluoroquinolone and azithromycin susceptibility breakpoints for Salmonella enterica serovar Typhi.

In recent years, increase in occurrence of fluoroquinolone (FQ)-resistant S almonella Typhi isolates has caused considerable inconvenience in selecting appropriate antimicrobials for treatment of typhoid. The World Health Organization (WHO) recommends azithromycin for the empirical treatment option of uncomplicated typhoid. The CLSI updated the breakpoints of disc diffusion (DD) and MIC results of FQs and azithromycin for Salmonella Typhi in 2015, but DD breakpoints of ofloxacin and levofloxacin were not included. In this study, the inhibition zone diameters and MICs of nalidixic acid, ciprofloxacin, ofloxacin, levofloxacin and azithromycin were determined in Salmonella Typhi Kolkata isolates (n =146) over a 16-year period (1998 to 2013) and the data were compared with the available CLSI breakpoints. Very major error and major error (ME) of FQs were not observed in the study isolates, but the minor error of ciprofloxacin (15.8 %) and ME of azithromycin (3.5 %) exceeded the acceptable limit. A positive correlation between MICs of FQ and mutations in the quinolone-resistance-determining region (QRDR) showed the reliability of MIC results to determine FQ susceptibility of Salmonella Typhi (n =74). Isolates showing decreased ciprofloxacin susceptibility (MIC 0.125-0.5 µg ml-1) were likely to have at least one mutation in the QRDR region. The results on DD breakpoints of ofloxacin (resistant, ≤15 mm; intermediate, 16-24 mm, and susceptible, ≥25 mm) and levofloxacin (resistant, ≤18 mm; intermediate, 19-27 mm, and susceptible, ≥28 mm) corroborated those of earlier studies. In view of the emerging FQ- and azithromycin-resistant Salmonella Typhi isolates, DD and MIC breakpoints of those antimicrobials should be revisited routinely.

Gentamicin in vitro activity and tentative gentamicin interpretation criteria for the CLSI and calibrated dichotomous sensitivity disc diffusion methods for Neisseria gonorrhoeae.

XDR Neisseria gonorrhoeae imposes the threat of untreatable gonorrhoea. Gentamicin is considered for future treatment; however, no interpretation criteria for the CLSI and calibrated dichotomous sensitivity (CDS) disc diffusion (DD) techniques are available for N. gonorrhoeae. We investigated the in vitro gentamicin activity by MIC and DD methods, proposed DD breakpoints and determined DD ranges for 10 international quality control (QC) strains. Gentamicin susceptibility of 333 N. gonorrhoeae isolates, including 323 clinical isolates and 10 QC strains, was determined. MIC determination (Etest) and DD methods (CLSI and CDS) were performed. The relationship between MIC, inhibition zone diameter and annular radius was determined by linear regression analysis and the correlation coefficient (r) was calculated. Gentamicin MICs for the QC strains were within published ranges. Of the 323 clinical isolates, according to published breakpoints 75.9%, 23.5% and 0.6% were susceptible, intermediately susceptible and resistant, respectively. Based on error minimization with MICs of ≤4, 8-16 and ≥32 mg/L, breakpoints proposed are susceptible ≥16 mm, intermediately susceptible 13-15 mm and resistant ≤12 mm for the CLSI method and susceptible ≥6 mm, less susceptible 3-5 mm and resistant ≤2 mm for the CDS technique. Low resistance to gentamicin was identified and gentamicin might be a future treatment option for gonorrhoea. Tentative gentamicin zone breakpoints were defined for two DD methods and QC ranges for 10 international reference strains were established. Our findings suggest that in resource-poor settings where MIC testing is not a feasible option, the DD methods can be used to indicate gentamicin resistance.

Results from the Survey of Antibiotic Resistance (SOAR) 2011-14 in the Democratic Republic of Congo, Ivory Coast, Republic of Senegal and Kenya.

To assess antibiotic susceptibility of community-acquired respiratory tract isolates from Ivory Coast, Kenya, Democratic Republic of Congo (DRC) and Senegal in 2011-14. Bacterial isolates were collected and MICs determined using Etest(®) for all antibiotics except erythromycin, for which testing was by disc diffusion. Susceptibility was assessed using CLSI, EUCAST and pharmacokinetic/pharmacodynamic (PK/PD) breakpoints. For macrolide interpretation, CLSI breakpoints were adjusted for incubation in CO2. Susceptibility to penicillin (using CLSI oral or EUCAST breakpoints) was low among isolates of Streptococcus pneumoniae from the DRC and Kenya (17.4% and 19%, respectively) but higher among isolates from the Ivory Coast (70%) and Senegal (85.7%). Penicillin susceptibility using CLSI iv breakpoints was higher in all countries, but still only 69.6% in the DRC. Macrolide susceptibility (based on CLSI erythromycin disc diffusion breakpoints) was also low in Kenya (∼65%) but 87%-100% elsewhere. Haemophilus influenzae were only collected in the DRC and Senegal, with β-lactamase prevalence of 39% and 4%, respectively. Furthermore, β-lactamase-negative ampicillin-resistant (BLNAR) isolates were found in DRC (four isolates, 17%), but only two isolates were found in Senegal (by EUCAST definition). Amoxicillin/clavulanic acid in vitro susceptibility was 73.9% in the DRC and 100% in Senegal based on CLSI breakpoints, but this reduced to 65.2% in the DRC when BLNAR rates were considered. Clarithromycin susceptibility was >95% in both countries. There was considerable variability in antibiotic susceptibility among the African countries participating in the surveillance programme. Thus, continued surveillance is necessary to track future changes in antibiotic resistance. Use of EUCAST versus CLSI breakpoints showed profound differences for cefaclor and ofloxacin against S. pneumoniae, with EUCAST showing lower susceptibility.

Pefloxacin as a Surrogate Marker for Fluoroquinolone Susceptibility for Salmonella typhi: Problems and Prospects.

Pefloxacin as a Surrogate Marker for Fluoroquinolone Susceptibility for Salmonella typhi: Problems and Prospects.