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Amoxicillin-clavulanate (AMC) is among the most frequently prescribed antibiotics globally. It has broad antibacterial activity against gram-positive, gram-negative, and anaerobic bacteria and has been used to treat infections caused by a broad range of pathogens. AMC breakpoints against Enterobacterales were initially set in the 1980s. However, since that time, increases in antibiotic resistance, advances in pharmacokinetic/pharmacodynamic analyses, and publication of additional clinical data prompted a reassessment by the Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing. Based on this contemporary reappraisal, the CLSI retained the Enterobacterales breakpoints but revised comments regarding dosing associated with use of the AMC breakpoints in the 2022 supplement of M100. This viewpoint provides insight into the CLSI breakpoint reevaluation process and summarizes the data and rationale used to support these revisions to the AMC Enterobacterales breakpoint., Competing Interests: Potential conflicts of interest. N. N. reports support from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (under award K23AI159396); honoraria from Astellas Pharma and Beckman Coulter; and research contracts with Merck and Shionogi Inc. E. W. reports honoraria from AbbVie Inc, Astellas Pharma, Ferring Pharmaceuticals, Melinta Therapeutics, Shionogi Inc, and Venatorx Pharmaceuticals. C. G. G. is immediate past-chair of the European Committee on Antimicrobial Susceptibility Testing. A. J. M. serves as a scientific advisor for Cepheid, DayZero Diagnostics and OpGen; and previously served as a scientific advisor for Merck, Qpex Biopharma, Venatorx Pharmaceuticals, and Melinta Therapeutics. N. M. M. reports research contracts from Cepheid, Inc and Abbott Molecular paid to his institution outside the submitted work; honoraria from the American Society for Clinical Laboratory Science; and serving as a member of the American Society for Clinical Laboratory Science Board of Directors and as a member of the Microbiology Resource Committee for the College of American Pathologists. All authors serve as unpaid volunteers for the Clinical and Laboratory Standards Institute. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Antimicrobial stewardship (AMS) programmes are established across the world to treat infections efficiently, prioritize patient safety, and reduce the emergence of antimicrobial resistance. One of the core elements of AMS programmes is guidance to support and direct physicians in making efficient, safe and optimal decisions when prescribing antibiotics. To optimize and tailor AMS, we need a better understanding of prescribing physicians' experience with AMS guidance., Objectives: To explore the prescribing physicians' user experience, needs and targeted improvements of AMS guidance in hospital settings., Methods: Semi-structured interviews were conducted with 36 prescribing physicians/AMS guidance users from hospital settings in Canada, Germany, Israel, Latvia, Norway and Sweden as a part of the international PILGRIM trial. A socioecological model was applied as an overarching conceptual framework for the study., Results: Research participants were seeking more AMS guidance than is currently available to them. The most important aspects and targets for improvement of AMS guidance were: (i) quality of guidelines; (ii) availability of infectious diseases specialists; and (iii) suitability of AMS guidance to department context., Conclusions: Achieving prudent antibiotic use not only depends on individual and collective levels of commitment to follow AMS guidance but also on the quality, availability and suitability of the guidance itself. More substantial commitment from stakeholders is needed to allocate the required resources for delivering high-quality, available and relevant AMS guidance to make sure that the prescribers' AMS needs are met., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Objectives: Treatment options for urinary tract infections (UTIs) caused by ESBL-producing Enterobacterales are limited. Moreover, evidence to support therapeutic decisions is lacking. This study assessed current treatment strategies and patient and pathogen characteristics in relation to clinical and microbiological outcomes., Methods: Patients with UTI caused by ESBL-producing Enterobacterales were prospectively recruited by investigators at 15 infectious disease hospital departments. Data were collected on patient characteristics, treatments, clinical and microbiological cure 10-14 days after the end of treatment, and relapse within 3 months. Bacterial isolates were subjected to MIC determination and WGS., Results: In total, 235 patients (107 febrile UTI, 128 lower UTI) caused by Escherichia coli (n = 223) and Klebsiella spp. (n = 12) were included. Clinical and microbiological cure rates were 83% and 64% in febrile UTI, and 79% and 65% in lower UTI. Great variability in treatments was observed, especially in oral therapy for febrile UTI. No difference was seen in clinical outcomes with piperacillin/tazobactam (n = 28) compared with carbapenems (n = 41). Pivmecillinam was frequently used in lower UTI (n = 62), and was also associated with high clinical cure rates when used as initial therapy (10/10) or follow-up (7/8) for febrile UTI. Recurrent infection, diabetes mellitus and urogenital disease were associated (P < 0.05) with clinical failure and relapse. In E. coli, ST131 was significantly associated with relapse, and haemolysin with microbiological failure or relapse., Conclusions: Antibiotic treatments were highly variable. Patient and pathogen factors were identified as potential determinants of disease presentation and outcomes and may prove useful to guide individualized treatment and follow-up., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Background: Therapeutic drug monitoring (TDM) has been suggested to optimize antimicrobial target attainment, typically using 100%T>MIC, in β-lactam treatment in the ICU. The MIC parameter used in this equation is mostly the worst case scenario MIC (MICWCS)-the highest MIC the empirical treatment should cover. However, the impact of the MIC parameter used in pharmacokinetic/pharmacodynamic calculations has been poorly investigated., Objectives: To assess the influence of target attainment rates for two different MIC parameters using actual MICs of the causative pathogens as the primary reference., Methods: In a Swedish multicentre study of target attainment for 138 ICU patients treated with β-lactams, the causative pathogen was isolated and subjected to reference MIC testing. Whenever the strain belonged to the WT distribution, we assigned it to the category MICECOFF (epidemiological cut-off value). In the calculations we compared the MICECOFF and the MICWCS., Results: The proportion of patients with target attainment failure for all antibiotics using 100%T>MIC was 45% (95% CI, 37%-53%) for MICWCS and 23% (95% CI, 16%-31%) for MICECOFF. When the target 50%T>4×MIC was used, corresponding attainment failures were 57% (95% CI, 49%-66%) and 25% (95% CI, 17%-32%) for MICWCS and MICECOFF, respectively., Conclusions: MICWCS can overestimate target attainment failure. The use of MICWCS could be one reason for the difficulties in establishing a relationship between target failure and mortality in other studies. Based on findings herein, the MICECOFF, which is based on the MIC of the causative pathogen, should be considered a more suitable alternative. When no pathogen is detected, the MICECOFF of likely pathogens according to infection type should be used., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Background: The intestinal microbiota functions as a reservoir of antibiotic resistance., Objectives: To evaluate penicillin V (phenoxymethylpenicillin) effects on the faecal microbiota with focus on beta-lactam resistance., Methods: We included 31 primary care patients with group A streptococcal pharyngotonsillitis treated with penicillin V for 5 (800 mg × 4) or 10 days (1000 mg × 3). Twenty-nine patients contributed with three faecal swab samples each. The faecal specimens were collected at the start of penicillin V treatment, after the last dose and at follow-up 7-9 days after completed treatment. Samples were inoculated semiquantitatively on selective screening agar plates to study beta-lactam resistance, species shifts among Enterobacterales and enterococci, and colonization with Candida spp. and Clostridioides difficile . Representative colonies were identified using MALDI-TOF. Results were analysed by non-parametric statistical methods., Results: An increase in the proportion of patients colonized with ampicillin-resistant Enterobacterales, from 52% to 86% ( P  = 0.007), and Enterobacterales with decreased susceptibility to third-generation cephalosporins, from 32% to 52% ( P  = 0.034), was observed between the first and second samples. This increase was no longer significant at follow-up. New colonization with ampicillin-resistant Enterobacterales species and non-Enterobacterales Gram-negative species was observed, and persisted at follow-up., Conclusions: Following treatment with penicillin V, we observed decreased susceptibility to ampicillin and third-generation cephalosporins, and prolonged colonization with non- Escherichia coli Gram-negative species. These findings challenge the perception that penicillin V has limited ecological effect on the intestinal microbiota, and emphasizes the importance of avoiding even narrow-spectrum antimicrobials when possible., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Objectives: Colistin is a last-resort antibiotic, but there has been a rapid increase in colistin resistance, threatening its use in the treatment of infections with carbapenem-resistant Enterobacterales (CRE). Plasmid-mediated colistin resistance, in particular the mcr-1 gene, has been identified and WGS is the go-to method in identifying plasmids carrying mcr-1 genes. The goal of this study is to demonstrate the use of optical DNA mapping (ODM), a fast, efficient and amplification-free technique, to characterize plasmids carrying mcr-1 ., Methods: ODM is a single-molecule technique, which we have demonstrated can be used for identifying plasmids harbouring antibiotic resistance genes. We here applied the technique to plasmids isolated from 12 clinical Enterobacterales isolates from patients at a major hospital in Thailand and verified our results using Nanopore long-read sequencing., Results: We successfully identified plasmids encoding the mcr-1 gene and, for the first time, demonstrated the ability of ODM to identify resistance gene sites in small (∼30 kb) plasmids. We further identified bla CTX-M genes in different plasmids than the ones encoding mcr-1 in three of the isolates studied. Finally, we propose a cut-and-stretch assay, based on similar principles, but performed using surface-functionalized cover slips for DNA immobilization and an inexpensive microscope with basic functionalities, to identify the mcr-1 gene in a plasmid sample., Conclusions: Both ODM and the cut-and-stretch assay developed could be very useful in identifying plasmids encoding antibiotic resistance in hospitals and healthcare facilities. The cut-and-stretch assay is particularly useful in low- and middle-income countries, where existing techniques are limited., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Background: Pseudomonas aeruginosa bacteraemia is a common and serious infection. No consensus exists regarding whether definitive combination therapy is superior to monotherapy. We aimed to evaluate the impact of combination therapy on mortality., Methods: This was a multicentre retrospective study (nine countries, 25 centres), including 1277 patients with P. aeruginosa bacteraemia during 2009-15. We evaluated the association between β-lactam plus aminoglycoside or quinolone combination therapy versus β-lactam monotherapy and mortality. The primary outcome was 30 day all-cause mortality. Univariate and multivariate Cox regression analyses were conducted, introducing combination as a time-dependent variable. Propensity score was conducted to adjust for confounding for choosing combination therapy over monotherapy., Results: Of 1119 patients included, 843 received definitive monotherapy and 276 received combination therapy (59% aminoglycoside and 41% quinolone). Mortality at 30 days was 16.9% (189/1119) and was similar between combination (45/276; 16.3%) and monotherapy (144/843; 17.1%) groups (P = 0.765). In multivariate Cox regression, combination therapy was not associated with reduced mortality (HR 0.98, 95% CI 0.64-1.53). No advantage in terms of clinical failure, microbiological failure or recurrent/persistent bacteraemia was demonstrated using combination therapy. Likewise, adverse events and resistance development were similar for the two regimens., Conclusions: In this retrospective cohort, no mortality advantage was demonstrated using combination therapy over monotherapy for P. aeruginosa bacteraemia. Combination therapy did not improve clinical or microbiological failure rates, nor affect adverse events or resistance development. Our finding of no benefit with combination therapy needs confirmation in well-designed randomized controlled trials., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: The worldwide emergence of antibiotic resistance calls for effective exploitation of existing antibiotics. Antibiotic combinations with different modes of action can synergize for successful treatment. In the present study, we used microcalorimetry screening to identify synergistic combination treatments against clinical MDR isolates. The synergistic effects were validated in a murine infection model., Methods: The synergy of meropenem combined with colistin, rifampicin or amikacin was tested on 12 isolates (1 Escherichia coli, 5 Klebsiella pneumoniae, 3 Pseudomonas aeruginosa and 3 Acinetobacter baumannii) in an isothermal microcalorimeter measuring metabolic activity. One A. baumannii strain was tested with two individual pairings of antibiotic combinations. The microcalorimetric data were used to predict in vivo efficacy in a murine peritonitis/sepsis model. NMRI mice were inoculated intraperitoneally and after 1 h treated with saline, drug X, drug Y or X+Y. Bacterial load was determined by cfu in peritoneal fluid and blood after 4 h., Results: In vitro, of the 13 combinations tested on the 12 strains, 3 of them exhibited a synergistic reduction in MIC (23% n = 3/13), 5 showed an additive effect (38.5% n = 5/13) and 5 had indifferent or antagonistic effects (38.5% n = 5/13). There was a significant correlation (P = 0.024) between microcalorimetry-screening FIC index values and the log reduction in peritoneal fluid from mice that underwent combination treatment compared with the most effective mono treatment. No such correlation could be found between chequerboard and in vivo results (P = 0.16)., Conclusions: These data support microcalorimetic metabolic readout to predict additive or synergistic effects of combination treatment of MDR infections within hours., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Background: Genomic epidemiology of antibiotic resistance is not sufficiently studied in low-income countries., Objectives: To determine prevalence of ESBL production, and resistome and virulome profiles, of Klebsiella pneumoniae isolated at Jimma Medical Center, Ethiopia., Methods: Strains isolated from patients with suspected infections between June and November 2016 were characterized by MALDI-TOF for species identification and disc diffusion for antimicrobial susceptibility testing. All K. pneumoniae isolates were characterized by double disc diffusion for ESBL production and all ESBL-producing strains (ESBL-KP) were subjected to WGS on the Illumina (HiSeq 2500) platform. DNA was extracted by automated systems (MagNA Pure 96). Genome assembly was performed using SPAdes (v. 3.9) and draft genomes were used for analysing molecular features of the strains. Maximum likelihood trees were generated using FastTree/2.1.8 based on SNPs in shared genomic regions to identify transmission clusters., Results: Of the 146 K. pneumoniae strains isolated, 76% were ESBL-KP; 93% of the ESBL-KP strains showed resistance to multiple antimicrobial classes. bla CTX-M-15 (84.4%) was the most prevalent ESBL gene. Resistance genes for aminoglycosides and/or fluoroquinolones [ aac(6')-Ib-cr (65.1%)], phenicols [ catB3 (28.4%)], sulphonamides [ sul1 (61.2%) and sul2 (60.5%)], trimethoprim [ dfrA27 (32.1%)], macrolides [ mph(A) (12.8%)] and rifampicin [ arr2 / arr3 (39.4%)] were prevalent. Plasmids of the IncF and IncR families were prevalent among ST218, ST147, ST15 and ST39. KL64 and KL57 capsular types and O1 and O2 LPSs were prevalent. A high-risk clone, ST218-KL57 encoding rmpA1/rmpA2 and iutA , was detected. Phylogenetic analysis showed a cluster of clonally related strains from different units of the hospital., Conclusions: Prevalence of ESBL-KP was high and bla CTX-M-15 was the predominant ESBL gene. ESBL genes had spread through both clonal and polyclonal expansion of high-risk and hypervirulent clones. Nosocomial transmission of MDR strains between different units of the hospital was observed., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Recent data on polymyxin pharmacokinetics, pharmacodynamics, toxicity, and clinical outcomes suggest these agents have limited clinical utility. Pharmacokinetics-pharmacodynamics data show a steady-state concentration of 2 μg/mL is required for killing bacteria with colistin minimum inhibitory concentrations of 2 μg/mL. Less than 50% of patients with normal renal function achieve this exposure, and it is associated with high risk of nephrotoxicity. This exposure does not achieve bacterial stasis in pneumonia models. Randomized and observational studies consistently demonstrate increased mortality for polymyxins compared with alternative agents. The Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) are 2 global organizations that establish interpretive criteria for in vitro susceptibility data. CLSI has recently taken the step to eliminate the "susceptible" interpretive category for the polymyxins, whereas EUCAST maintains this interpretive category. This viewpoint describes the opinions of these organizations and the data that were used to inform their perspectives., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Objectives: MDR bacteria have become a prevailing health threat worldwide. We here aimed to use optical DNA mapping (ODM) as a rapid method to trace nosocomial spread of bacterial clones and gene elements. We believe that this method has the potential to be a tool of pivotal importance for MDR control., Methods: Twenty-four Escherichia coli samples of ST410 from three different wards were collected at an Ethiopian hospital and their plasmids were analysed by ODM. Plasmids were specifically digested with Cas9 targeting the antibiotic resistance genes, stained by competitive binding and confined in nanochannels for imaging. The resulting intensity profiles (barcodes) for each plasmid were compared to identify potential clonal spread of resistant bacteria., Results: ODM demonstrated that a large fraction of the patients carried bacteria with a plasmid of the same origin, carrying the ESBL gene blaCTX-M-15, suggesting clonal spread. The results correlate perfectly with core genome (cg)MLST data, where bacteria with the same plasmid also had very similar cgMLST profiles., Conclusions: ODM is a rapid discriminatory method for identifying plasmids and antibiotic resistance genes. Long-range deletions/insertions, which are challenging for short-read next-generation sequencing, can be easily identified and used to trace bacterial clonal spread. We propose that plasmid typing can be a useful tool to identify clonal spread of MDR bacteria. Furthermore, the simplicity of the method enables possible future application in low- and middle-income countries., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Background: The optimal antibiotic regimen for Pseudomonas aeruginosa bacteremia is controversial. Although β-lactam monotherapy is common, data to guide the choice between antibiotics are scarce. We aimed to compare ceftazidime, carbapenems, and piperacillin-tazobactam as definitive monotherapy., Methods: A multinational retrospective study (9 countries, 25 centers) including 767 hospitalized patients with P. aeruginosa bacteremia treated with β-lactam monotherapy during 2009-2015. The primary outcome was 30-day all-cause mortality. Univariate and multivariate, including propensity-adjusted, analyses were conducted introducing monotherapy type as an independent variable., Results: Thirty-day mortality was 37/213 (17.4%), 42/210 (20%), and 55/344 (16%) in the ceftazidime, carbapenem, and piperacillin-tazobactam groups, respectively. Type of monotherapy was not significantly associated with mortality in either univariate, multivariate, or propensity-adjusted analyses (odds ratio [OR], 1.14; 95% confidence interval [CI], 0.52-2.46, for ceftazidime; OR, 1.3; 95% CI, 0.67-2.51, for piperacillin-tazobactam, with carbapenems as reference in propensity adjusted multivariate analysis; 542 patients). No significant difference between antibiotics was demonstrated for clinical failure, microbiological failure, or adverse events. Isolation of P. aeruginosa with new resistance to antipseudomonal drugs was significantly more frequent with carbapenems (36/206 [17.5%]) versus ceftazidime (25/201 [12.4%]) and piperacillin-tazobactam (28/332 [8.4%] (P = .007)., Conclusions: No significant difference in mortality, clinical, and microbiological outcomes or adverse events was demonstrated between ceftazidime, carbapenems, and piperacillin-tazobactam as definitive treatment of P. aeruginosa bacteremia. Higher rates of resistant P. aeruginosa after patients were treated with carbapenems, along with the general preference for carbapenem-sparing regimens, suggests using ceftazidime or piperacillin-tazobactam for treating susceptible infection., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Minimum inhibitory concentration (MIC) testing, unlike routine drug susceptibility testing (DST) at a single critical concentration, quantifies drug resistance. The association of MICs and treatment outcome in multidrug-resistant (MDR)-tuberculosis patients is unclear. Therefore, we correlated MICs of first- and second-line tuberculosis drugs with time to sputum culture conversion (tSCC) and treatment outcome in MDR-tuberculosis patients., Methods: Clinical and demographic data of MDR-tuberculosis patients in Sweden, including DST results, were retrieved from medical records from 1992 to 2014. MIC determinations were performed retrospectively for the stored individual Mycobacterium tuberculosis (Mtb) isolates using broth microdilution in Middlebrook 7H9. We fitted Cox proportional hazard models correlating MICs, DST results, and clinical variables to tSCC and treatment outcome., Results: Successful treatment outcome was observed in 83.5% (132/158) of MDR-tuberculosis patients. Increasing MICs of fluoroquinolones, diabetes, and age >40 years were significantly associated with unsuccessful treatment outcome. Patients treated with pyrazinamide (PZA) had a significantly shorter tSCC compared to patients who were not (median difference, 27 days)., Conclusions: Increasing MICs of fluoroquinolones were correlated with unsuccessful treatment outcome in MDR-tuberculosis patients. Further studies, including MIC testing and clinical outcome data to define clinical Mtb breakpoints, are warranted. PZA treatment was associated with shorter tSCC, highlighting the importance of PZA DST., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Background: Little is known of the long-term risks of bloodstream infection (BSI) with extended spectrum β-lactamase-producing Enterobacteriaceae (EPE) in previously-colonized individuals. We investigated EPE-BSI risks and associated risk factors during 6 years following EPE colonization., Methods: We performed a population-based cohort study in Sweden using national health registers. Subjects were followed from their first EPE finding in feces (n = 5513) or urine (n = 17189). The effects of co-morbidity, sociodemography, and outpatient antibiotic dispensation on EPE-BSI risks were assessed. The EPE-BSI risks were compared to those of 45161 matched population-based reference subjects., Results: The cumulative 6-year EPE-BSI incidences were 3.8%, 1.6%, and 0.02% in the urine, feces, and reference cohorts, respectively. The incidences decreased exponentially during the first 6-12 months. Among EPE-exposed subjects, urological disorders were associated with the highest adjusted cause-specific hazard ratio (aCSHR) for subsequent EPE-BSIs (3.40, 95% confidence interval 2.47-4.69). The aCSHRs were between 1.62-2.20 for male sex, immunosuppression, diabetes, malignancy, lung disease, baseline urine source, and Klebsiella pneumoniae, compared to the Escherichia coli baseline sample. Antibiotics with selective activity against gram-negative bacilli-but mostly not EPE (trimethoprim-sulfamethoxazole, fluoroquinolones, oral cephalosporins, and penicillins with extended spectrums)-and pivmecillinam were associated with doubled EPE BSI risk during the 3 months after antibiotic dispensation in EPE-colonized subjects., Conclusions: EPE in urine or feces is a substantial risk factor for subsequent EPE-BSIs, but the risk declines rapidly during the first year after detection. In EPE-colonized individuals, specific risk factors can be used to identify subgroups for targeted interventions, such as eradication therapy.

Objectives: To examine performance of EUCAST disc diffusion and supplementary MIC methods for detection of Enterobacteriaceae with reduced susceptibility to meropenem using EUCAST screening recommendations., Methods: Sixty-one Nordic laboratories delivered data on EUCAST disc diffusion (n = 61), semi-automated meropenem MIC (n = 23; VITEK2, n = 20 and Phoenix, n = 3) and gradient meropenem MIC (n = 58) methods. The strains (n = 27) included the major carbapenemase classes (A, n = 4; B, n = 9; D, n = 6) involved in the global spread of carbapenemase-producing Enterobacteriaceae (CPE) and non-CPE strains (n = 8) covering a range of broth microdilution (BMD) meropenem MICs., Results: A triplicate Klebsiella variicola (meropenem MIC 0.5 mg/L) harbouring OXA-48 and Escherichia coli ATCC 25922 showed an overall good precision. Meropenem zone diameters below the EUCAST screening cut-off (<27 mm) were reported for strains with MIC ≥1 mg/L (n = 21), irrespective of resistance mechanism. For three strains (MIC = 0.5 mg/L) with OXA-48/-181, eight laboratories provided meropenem zone diameters above the screening cut-off. Very major errors (VMEs) were not observed. The overall distributions of major errors (MEs) and minor errors (mEs) were 9% and 36% (disc diffusion), 26% and 18% (VITEK2) and 7% and 20% (gradient MIC), respectively. Differences in ME and mE distributions between disc diffusion and MIC gradient tests compared with semi-automated methods were significant (P < 0.0001), using BMD MICs as a reference for categorization., Conclusions: The EUCAST disc diffusion method is a robust method to screen for CPE but isolates with meropenem MICs <1 mg/L pose challenges. The high ME rate in semi-automated methods might deter appropriate use of carbapenems in CPE infections with limited therapeutic options.

Objectives: Despite the fact that the blaOXA-48 and blaNDM-1 genes have successfully disseminated among Klebsiella pneumoniae isolates worldwide, outbreaks remain unidentified in Iran. Here we examined the molecular epidemiology of 96 carbapenem-resistant K. pneumoniae recovered from an Iranian hospital., Methods: A total of 96 non-replicate carbapenem-resistant K. pneumoniae were recovered from clinical specimens in a university hospital. Detection of ESBLs and carbapenemases produced by studied strains was performed using PCR and DNA sequencing. The bacterial isolates were assigned to clonal lineages by PFGE and MLST. In addition, plasmids were analysed by PCR-based replicon typing and conjugation assays., Results: All isolates harboured blaOXA-48 and blaNDM-1 genes together or alone. Almost all strains also carried ESBL genes. Eighty-seven isolates of K. pneumoniae were categorized into seven pulsotypes. The predominant strain clusters/pulsotypes associated with the outbreak corresponded to ST11 (48/96) and ST893 (31/96). Plasmids carrying blaOXA-48 and blaNDM-1 were successfully transferred to Escherichia coli K12 as the recipient strain. blaOXA-48 was located on IncL/M plasmids of ∼39 kb, while blaNDM-1 was carried by either an IncFII plasmid of ∼50 kb or an untypeable plasmid of ∼4 or 10 kb., Conclusions: We describe two separate outbreaks of blaOXA-48- and blaNDM-1-carrying K. pneumoniae strains associated with dissemination of the ST11 and ST893 clones, with the ICU acting as the epicentre. The spread of plasmids carrying carbapenemase genes resulting in fulminant antimicrobial resistance is a severe concern.

Over recent decades, several publications have described optimization procedures for antibiotic therapy in the individual patient based on antimicrobial MIC values. Most methods include therapeutic drug monitoring and use a single MIC determination plus the relevant pharmacokinetics/pharmacodynamics to adjust the dose to optimize antimicrobial drug exposure and antibacterial effects. However, the use of an MIC obtained by a single MIC determination is inappropriate. First, routine clinical laboratories cannot determine MICs with sufficient accuracy to guide dosage owing to the inherent assay variation in the MIC test. Second, the variation in any MIC determination, whatever method is used, must be accounted for. If dose adjustments are made based on therapeutic drug monitoring and include MIC determinations, MIC variation must be considered to prevent potential underdosing of patients. We present the problems and some approaches that could be used in clinical practice.

Objectives: The need for rapid antibiotic susceptibility testing increases with escalating levels of antimicrobial resistance in Enterobacteriaceae. Our objective was to evaluate the accuracy of reading EUCAST disc diffusion, ROSCO ESBL and carbapenemase detection kits and the Mast Carbapenemase Activity Test (CAT-ID) disc, after 6 h of incubation., Methods: We used a collection of 128 isolates of Escherichia coli and Klebsiella pneumoniae with a wide variety of resistance mechanisms. Inhibition zones read from digital photo images with the BD Kiestra™ Total Lab Automation System after 6 h of incubation were compared with standard reading, after 18 h, of the same Mueller-Hinton agar plates., Results: For WT isolates, zones were generally smaller at 6 h than at 18 h. Cefotaxime had excellent categorical agreement of 99%, despite the high number of challenge isolates. However, for some other antimicrobials, hetero-resistant subpopulations were commonly invisible at 6 h, which resulted in an unacceptable number of errors when using standard EUCAST breakpoints. Accurate ESBL detection was possible at 6 h for isolates lacking other β-lactamases. Carbapenemase detection was not reliable after 6 h., Conclusions: Inhibition zone reading at 6 h is an accurate method for susceptibility testing of extended-spectrum cephalosporins for Enterobacteriaceae. For other antimicrobials, 6 h reading can be used for preliminary reports of clearly resistant or susceptible isolates, preferably with application of adjusted breakpoints including an area of uncertainty between susceptible and resistant values., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objectives: Community carriage of ESBL-producing Escherichia coli (EPE) is common worldwide and there is a need to understand the connection between carriage and infection. We compared the molecular characteristics of EPE among Swedish community carriers with those of EPE causing invasive infections., Methods: We collected 2134 faecal samples from randomly selected Swedish inhabitants and examined them for the presence of EPE. All participating volunteers answered a questionnaire about putative risk factors for EPE carriage. Suspected EPE isolates (n = 418) from patients with bloodstream infection (BSI) were collected from Swedish laboratories. Isolates were genotypically and phenotypically characterized., Results: Our results show that the EPE population found in carriers generally had lower pathogenicity compared with the isolates from BSIs, since carriers had a lower proportion of E. coli belonging to phylogroup B2, ST131 and ST131 subclone H30-Rx. Isolates from carriers also had lower levels of multiresistance. The Swedish carriage rate of EPE was 4.7% (101/2134) among healthy volunteers. Risk factors associated with carriage were travel to countries in Asia (OR = 3.6, 95% CI = 1.4-9.2) and Africa (OR = 3.6, 95% CI = 1.7-7.7) and a diet without pork (OR = 0.5, 95% CI = 0.3-0.8 for pork eaters)., Conclusions: E. coli host factors previously associated with higher pathogenicity were all more common in BSIs compared with carriers. This indicates that the risk of invasive infection with EPE may be relatively modest in many community carriers and that EPE carriage of high-risk strains should be the focus of attention for prevention., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objectives: We evaluated the RAPIDEC(®) CARBA NP assay (bioMérieux SA, Marcy-l'Étoile, France), a colorimetric test for rapid detection of carbapenemases, at two sites: Karolinska University Laboratory and PHE's national reference laboratory., Methods: A total of 138 bacterial isolates previously characterized as positive for class A, B and/or D carbapenemase genes and 138 supposed non-carbapenemase producers were tested with RAPIDEC(®) CARBA NP according to the manufacturer's protocol. Two carbapenemase-producing isolates carried both NDM and OXA-48-like genes. Molecular detection of the expected carbapenemase gene(s) was used as the gold standard, and was performed by conventional and real-time PCR in-house assays., Results: The RAPIDEC(®) CARBA NP assay detected 135 of 138 carbapenemase producers; one OXA-48-producing Klebsiella pneumoniae and two Acinetobacter baumannii producing OXA-23 or OXA-24 were not detected. Among 'negative' controls, 135 of 138 isolates were negative by RAPIDEC(®) CARBA NP. The exceptions were one Klebsiella oxytoca, which was later found to produce GES-5 carbapenemase, one Pseudomonas aeruginosa with OprD loss and increased efflux, and one Enterobacter cloacae with impermeability. When numbers were adjusted for the GES-5 producer, the overall sensitivity of the RAPIDEC(®) CARBA NP test was 97.8% and its specificity was 98.5%., Conclusions: The assay took less than 2.5 h to carry out, was user-friendly and had a high overall performance, making it an attractive option for clinical laboratories., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Fluoroquinolones (FQs) are important in the treatment of MDR-TB and in the definition of XDR-TB. Our objective was to investigate how discrepancies in the phenotypic and genotypic methods for antimicrobial susceptibility testing could affect the interpretation of antimicrobial susceptibility test results., Methods: We analysed MICs of ofloxacin and levofloxacin in Middlebrook 7H10 broth (7H10) as well as sequencing of the quinolone resistance-determining region of the gyrA gene and the MTBDRsl assay in 75 resistant isolates, including MDR and XDR strains of Mycobacterium tuberculosis., Results: Among 75 resistant isolates, 27 had mutations associated with FQ resistance. Among isolates with resistance mutations in gyrA, 26% (seven of 27) were susceptible to levofloxacin and ofloxacin by phenotypic testing at 1 mg/L and 2 mg/L. The most common mutation was in codon 94 and these isolates had significantly increased MICs of levofloxacin (2-8 mg/L) compared with isolates with mutations in codon 90 (0.25-2 mg/L, P < 0.05). The sensitivity and specificity for the MTBDRsl assay compared with gyrA sequencing were 96% and 98%, respectively., Conclusion: Current critical concentrations may classify up to 26% of isolates with gyrA mutations as susceptible to FQs due to a close relationship between susceptible and resistant populations. These results should be considered while improving clinical breakpoints for M. tuberculosis and may have an impact on the definition of XDR-TB., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

In the face of increasing antimicrobial resistance and the paucity of new antimicrobial agents it has become clear that new antimicrobial strategies are urgently needed. One of these is to revisit old antibiotics to ensure that they are used correctly and to their full potential, as well as to determine whether one or several of them can help alleviate the pressure on more recent agents. Strategies are urgently needed to 're-develop' these drugs using modern standards, integrating new knowledge into regulatory frameworks and communicating the knowledge from the research bench to the bedside. Without a systematic approach to re-developing these old drugs and rigorously testing them according to today's standards, there is a significant risk of doing harm to patients and further increasing multidrug resistance. This paper describes factors to be considered and outlines steps and actions needed to re-develop old antibiotics so that they can be used effectively for the treatment of infections., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: The microbial etiology of community-acquired pneumonia (CAP) is still not well characterized. During the past few years, polymerase chain reaction (PCR)-based methods have been developed for many pathogens causing respiratory tract infections. The aim of this study was to determine the etiology of CAP among adults-especially the occurrence of mixed infections among patients with CAP-by implementing a new diagnostic PCR platform combined with conventional methods., Methods: Adults admitted to Karolinska University Hospital were studied prospectively during a 12-month period. Microbiological testing methods included culture from blood, sputum, and nasopharyngeal secretion samples; sputum samples analyzed by real-time quantitative PCR for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis; nasopharyngeal specimens analyzed by use of PCR; serological testing for Mycoplasma pneumoniae, Chlamydophila pneumoniae, and viruses common in the respiratory tract; and urine antigen assays for detection of pneumococcal and Legionella pneumophila antigens., Results: A microbial etiology could be identified for 67% of the patients (n = 124). For patients with complete sampling, a microbiological agent was identified for 89% of the cases. The most frequently detected pathogens were S. pneumoniae (70 patients [38%]) and respiratory virus (53 patients [29%]). Two or more pathogens were present in 43 (35%) of 124 cases with a determined etiology., Conclusions: By supplementing traditional diagnostic methods with new PCR-based methods, a high microbial yield was achieved. This was especially evident for patients with complete sampling. Mixed infections were frequent (most commonly S. pneumoniae together with a respiratory virus).

Objectives: The aim of this study was to establish wild-type MIC distributions of first-line drugs for Mycobacterium tuberculosis, as well as to explore the usefulness of such distributions when setting clinical breakpoints., Methods: We determined the MICs of rifampicin, isoniazid and ethambutol for M. tuberculosis using a Middlebrook 7H10 dilution method for 90 consecutive clinical isolates, 8 resistant strains and 16 isolates from the WHO proficiency test panel. M. tuberculosis H37Rv was used for quality control and susceptibility results using 7H10 were compared with the results obtained with BACTEC460., Results: The agreement with BACTEC460 was very high for isoniazid (99.1%) and rifampicin (99.1%) but lower for ethambutol (94.7%). Intra- and inter-assay variation was below one MIC dilution. The MIC distributions for isoniazid and rifampicin provided a clear separation between susceptible and resistant strains. Regarding ethambutol, the current breakpoint for 7H10 (5 mg/L) is close to the wild-type and all strains (n = 6) showing a disagreement between BACTEC460 and 7H10 were distributed very close to the breakpoint (MIC 4-8 mg/L). This problematic relation was confirmed by investigating isolates from the WHO panel with an agreement <95% (64%-88% among 26 laboratories, n = 4) for which the MICs were 4-8 mg/L., Conclusions: Utilizing the wild-type MIC distribution was found to be as useful in M. tuberculosis as in other bacteria when setting clinical breakpoints. We suggest that the present clinical breakpoints should be re-evaluated, taking into account wild-type MIC distributions and available pharmacokinetic data.

Background: The class A carbapenemase KPC has disseminated rapidly worldwide, challenging the treatment of Gram-negative infections. This report describes the first KPC-producing Klebsiella pneumoniae isolates identified in Norway (n=6) and the second isolate from Sweden., Methods: Antimicrobial susceptibility profiles were determined using Etest. PCR and sequencing were used to determine the bla(KPC) variant, the surrounding genetic structure and the presence of AmpC and extended-spectrum beta-lactamase genes. PFGE and multilocus sequence typing (MLST) were used for epidemiological comparisons. Localization of bla(KPC) was investigated by S1 nuclease digestion, followed by PFGE and Southern blot hybridization., Results: All isolates expressed a multidrug-resistant phenotype with some variability in the carbapenem susceptibility profile. The Norwegian isolates carried bla(KPC-2), while the Swedish isolate carried bla(KPC-3). All isolates carried TEM-1, but were negative for bla(CTX-M) and bla(AmpC) genes. SHV-11 and SHV-12 were detected in the Norwegian isolates, while the Swedish isolate carried only SHV-11. Isolates from four patients were associated with import from Greece (n=3) and Israel. The other isolates were probably associated with local transmissions. PFGE and MLST showed that the isolates were clonally related, with three isolates displaying ST258, a single locus variant of ST11 previously associated with the clonal spread of CTX-M-15-producing K. pneumoniae in Hungary. In all isolates, bla(KPC) was located on plasmids as part of isoform a of Tn4401., Conclusions: The emergence of KPC-producing isolates of K. pneumoniae in Norway and Sweden is associated with multiple import events and probable local transmission of a successful multiresistant ST258 clone, closely related to the CTX-M-15-producing ST11 clone previously described in Hungary.

The current beta-lactamase classifications have reached a high level of complexity, making them less accessible to clinicians, infection control professionals, hospital management and politicians. From the clinical perspective, a revised comprehensible nomenclature scheme is therefore needed. The term extended-spectrum beta-lactamases (ESBLs) has reached a broader audience over time, but is currently restricted to functional class 2be/molecular class A, clavulanic acid inhibited enzymes with activity against extended-spectrum cephalosporins. The proposed new classification expands the definition of ESBL to other clinically important acquired beta-lactamases with activity against extended-spectrum cephalosporins and/or carbapenems. The classical class A ESBLs have been designated ESBLA in this classification, whereas plasmid-mediated AmpC and OXA-ESBLs are classed as miscellaneous ESBLs (ESBLM). Lastly, the carbapenemases have been designated ESBLCARBA, ESBLs with hydrolytic activity against carbapenems. We believe that this simplified classification may encourage new groups of healthcare professionals to engage in the effort to prevent the spread of acquired beta-lactamases.

Objectives: The aims were to study if efflux and down-regulation of porins contribute to cefuroxime resistance in Klebsiella pneumoniae and to co-resistance to unrelated antibiotics., Methods: Ten cefuroxime-non-susceptible but cefotaxime-susceptible blood culture isolates of K. pneumoniae and one multiply antibiotic-resistant (MAR) laboratory strain (selected by chloramphenicol) were examined. Transcription of the genes acrA, ompK35, ramA, marA and soxS was determined with quantitative RT-PCR., Results: All clinical isolates and the MAR laboratory strain had similar antibiograms with non-susceptibility to cefuroxime, tigecycline, chloramphenicol and nalidixic acid. Phenylalanine arginine beta-naphthylamide (PAbetaN) increased susceptibility to tigecycline, chloramphenicol and nalidixic acid, but not to cefuroxime. Increased acrA transcription and decreased ompK35 transcription was seen in all strains. Increased ramA transcription was seen in all strains except one clinical isolate., Conclusions: This multidrug-resistant phenotype of K. pneumoniae is associated with increased acrA and ramA transcription and decreased ompK35 transcription. Since the cefuroxime resistance was not reversed by PAbetaN, it was probably attributable to decreased levels of OmpK35, rather than to efflux.

The lack of oral treatment alternatives for enterococcal urinary tract infections (UTIs) has led to a renewed interest in trimethoprim. Enterococci can incorporate exogenously produced folates and thereby reverse the effect of trimethoprim. Although a large proportion of enterococci appear susceptible to trimethoprim in vitro using standard media devoid of folates, a 360-fold increase in the MIC can be seen when susceptibility testing is performed in media containing fresh urine. Even if trimethoprim has a favourable pharmacokinetic profile, with high serum and very high urine concentrations, pharmacodynamic (PD) estimates show that a large proportion of the apparent wild-type isolates (as categorized by standard susceptibility testing) have unfavourable PD indices. The clinical efficacy of trimethoprim in enterococcal UTI is debated. We could identify not more than 38 evaluable cases of enterococcal UTI in the literature. The eradication rate was 82%. Case reports where patients on co-trimoxazole for UTI have developed bacteraemia with enterococci susceptible to trimethoprim seem to support experimental findings that standard antimicrobial susceptibility testing poorly predicts the clinical outcome of trimethoprim therapy. The European Committee on Antimicrobial Susceptibility Testing and the national breakpoint committees in Europe have recently debated the role of trimethoprim in the treatment of enterococcal UTI and agreed to categorize wild-type enterococci as intermediate to trimethoprim and trimethoprim/sulfamethoxazole. This allows the distinction between enterococci with and without acquired resistance mechanisms to trimethoprim. This review discusses the microbiological, experimental, clinical and PD aspects of the usage of trimethoprim for enterococcal UTI.

Objectives: To evaluate four phenotypic tests for the detection of metallo-beta-lactamase (MBL) production in Pseudomonas aeruginosa in a low MBL prevalence setting., Methods: Sixty clinical isolates of P. aeruginosa resistant to imipenem and/or meropenem and seven MBL-positive control strains were examined by: (i) MBL Etest; (ii) combined imipenem discs supplemented with EDTA (IPM-EDTA); (iii) beta-lactam discs on dipicolinic acid plates (DF-DIPI); and (iv) the Cica-beta test. Spectrophotometric analysis of crude cell extracts for imipenem hydrolysis along with consensus PCRs for bla(VIM) and bla(IMP) was used as reference methods., Results: Two clinical isolates (3%) were MBL-positive. The MBL Etest and IPM-EDTA test scored positive for all MBL-positive isolates, but showed specificities of 86% and 91%, and positive predictive values (PPVs) of only 20% and 29%, respectively. Adding resistance to ceftazidime (MIC >8 mg/L) as a criterion for MBL testing would reduce the number of isolates to be screened by 50% and increase the PPVs of the MBL Etest and IMP-EDTA test to 29% and 40%, respectively. The Cica-beta test correctly identified all MBL-negative isolates, but misidentified one MBL-positive clinical isolate as an extended-spectrum beta-lactamase (ESBL)-producer and one as inconclusive (producing multiple beta-lactamases). No reliable breakpoints could be defined for the DF-DIPI test due to overlapping inhibition zone diameters for MBL-positive and -negative isolates., Conclusions: None of the phenotypic tests were optimal due to low sensitivity or specificity, resulting in low PPVs. Including ceftazidime resistance to the MBL-screening criteria would significantly improve the performance of the MBL Etest and IPM-EDTA disc test.