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Phenotypic variants of Staphylococcus aureus that display small colonies, reduced pigmentation, and decreased hemolysis and/or coagulase activity are periodically isolated by the clinical laboratory. Antimicrobial susceptibility testing (AST) of these isolates is complicated, because many do not grow on routine AST media, including Mueller-Hinton agar (MHA) and cation-adjusted Mueller-Hinton broth. This multicenter study evaluated cefoxitin disk diffusion for 37 atypical S. aureus isolates (156 readings) with MHA supplemented with 5% sheep's blood (BMHA), using mecA PCR as the reference standard. The correlation of two commercial PBP2a assays with mecA PCR was also assessed. Ten isolates were negative and 27 positive for mecA No major errors for cefoxitin were observed, but 19.5% very major errors (VMEs) were observed at 24 h of incubation, and 17.2% VMEs were observed at 48 h. The proportions of VMEs ranged from 14.7 to 23.0% at 24 h, and from 13.3 to 17.6% at 48 h, across three testing laboratories. PBP2a tests were performed from growth on BMHA and blood agar plates (BAP), with and without cefoxitin disk induction. The Alere PBP2a SA culture colony test sensitivities for mecA were 90.0% with uninduced growth and 97.4% with induced growth from BMHA. On BAP, sensitivity was 96.0% with induced growth. The sensitivities of the Oxoid PBP2' latex agglutination test were 85.7% with uninduced growth and 93.9% with induced growth from BMHA and 95.9% with induced growth on BAP. On the basis of these data, we recommend that laboratories perform only mecA PCR and/or PBP2a tests when requested to perform AST on atypical isolates of S. aureus., (Copyright © 2017 American Society for Microbiology.)

This study evaluated the Etest for direct antimicrobial susceptibility testing (AST) of bacteria from equine synovial specimens, incubated in BACTEC enrichment bottles. Ninety-four culture-positive broths were inoculated onto agar to directly determine the minimum inhibitory concentrations (MICs) of 13 antimicrobials, using the Etest (direct Etest). Results were compared with those obtained with the agar dilution reference method, the standard Etest, and the disc diffusion method, after subculture and standardisation of the inoculum. For categorical comparison of AST results, MICs were translated into susceptibility categories, using clinical breakpoints. The direct Etest predicted categorical susceptibility/resistance of bacteria from equine synovial fluid with acceptable accuracy (overall categorical agreement, 91%) and was more reliable than the disc diffusion test. The direct Etest was less accurate than the standard Etest for generating MICs ± 1 log dilution relative to the reference method (overall essential agreement, 69% vs. 89%). As the Etest generated a high percentage of inaccuracies with trimethoprim and sulfadiazine, these were less suitable antimicrobial agents for inclusion. In conclusion, the direct Etest reliably predicted the susceptibility of isolates from equine synovial fluid for the tested antimicrobials, except for trimethoprim and sulfadiazine. Since it did not require subculture and preparation of a standardised inoculum, direct Etest results were available at least 24 h earlier than with other methods, which could facilitate the diagnosis of synovial infections. However, when accuracy is prioritised over speed for MIC determination, the standard Etest is preferred over the direct Etest., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: To investigate the performance of the meropenem and imipenem double-ended Etest ± EDTA and the tablet-based (meropenem and meropenem + dipicolinic acid) KPC/MBL Confirm Kit to detect cfiA metallo-β-lactamase (MBL) in Bacteroides fragilis., Methods: Well-characterized B. fragilis isolates, most from previously published studies, harbouring the cfiA gene and covering a wide range of meropenem MICs were included (n = 21)., Results: The imipenem double-ended Etest showed an indeterminate result in 95% of the included isolates with the cfiA gene (20 of 21), whereas the meropenem double-ended Etest gave an MIC ratio ≥8 (positive test) with all the isolates. All isolates that were meropenem intermediate or resistant had a zone diameter difference ≥6 mm with the KPC/MBL Confirm Kit., Conclusions: The meropenem double-ended Etest and not imipenem should be preferred for phenotypic detection of MBLs in B. fragilis. The KPC/MBL Confirm Kit could be an alternative with isolates that are meropenem intermediate or resistant (MIC >2 mg/L)., (© 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.)

The aim of this study was to evaluate the accuracy of commercial gradient test (Etest) in the detection of triazole resistant Aspergillus fumigatus isolates using reference microdilution methods and the analysis of sequences of the cyp 51A gene. The study was performed on twenty clinical isolates which were identified as Aspergillus fumigatus based on the DNA sequences of the ITS1-2 fragment of ribosomal DNA and the β-tubulin gene, out of them seventeen isolates showed wild-type cyp51A sequence and three were positive for the mutation TR34/L98H. All isolates were tested for the susceptibility to itraconazole (ITZ), voriconazole (VOR) and posaconasole (POS) using microdilution methods, according to EUCAST and CLSI protocols, as well as using Etest. The results of microdilution and Etests were analysed separately according to clinical breakpoints (CBP) defined by EUCAST version 7.0 and epidemiological cut off values (ECV). Etest as well as reference methods excellently recognised the WT isolates, which were susceptible to all tested triazoles, regardless of the method and CBP or ECV criteria used. The Etest recognized three non-WT isolates as resistant or intermediately sensitive to ITZ and POS and one as resistant to VOR. The categorical concordance between Etests and EUCAST and Etests and the CLSI method ranged from 90 to 100%. The interpretation of the results obtained from routine A. fumigatus Etests requires great caution. The use of the confirmative examinations with reference AST methods as well as with molecular tests is recommended.

Disk diffusion testing is widely used to detect methicillin resistance in staphylococci, and cefoxitin is currently considered the best marker for mecA-mediated methicillin resistance. In low-inoculum diffusion testing (colony suspension at 10 6 CFU/ml), the addition of moxalactam in combination with cefoxitin has been reported to improve on cefoxitin alone for the detection of methicillin-heteroresistant staphylococci. However, moxalactam is absent from EUCAST and CLSI guidelines, which use high-inoculum diffusion testing (colony suspension at 10 8 CFU/ml), calling into question the potential interest of including moxalactam in their recommendations. The inhibition zone diameters of cefoxitin and moxalactam, alone and in combination, were evaluated for concordance with mecA and mecC positivity in a large collection of clinical Staphylococcus isolates (611 Staphylococcus aureus, Staphylococcus lugdunensis, and Staphylococcus saprophyticus isolates and 307 coagulase-negative staphylococci other than S. lugdunensis and S. saprophyticus isolates, of which 22% and 53% were mecA-positive, respectively) and in 25 mecC-positive S. aureus isolates using high-inoculum diffusion testing. Receiver operating characteristic, sensitivity, and specificity analyses indicated that the detection of mecA- and mecC-positive and negative isolates did not improve with moxalactam, either alone or in combination with cefoxitin, compared to cefoxitin alone. These findings were similar in both the S. aureus/S. lugdunensis/S. saprophyticus group and in the coagulase-negative staphylococci group. Our results do not support the use of moxalactam as an additional marker of methicillin resistance when testing with high-inoculum disk diffusion., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: In clinical microbiology, some instruments are able to automatically read inhibition zone diameters for antibiotic susceptibility testing (AST) performed by the disc diffusion (DD) method. The actual resolution of commercial reader systems is low and high-resolution scanners have been developed for microbiology. Here, we evaluated and compared the reading and interpretation of AST by the DD method using the Scan ® 1200 instrument as compared with the Sirscan ® system on 211 clinical strains and the possibility to read AST after 6 or 8 h of incubation as compared with 24 h on 121 additional Gram-negative strains and 76 non-fermenter Gram-negative and Gram-positive strains., Methods: Validation of the technique was assessed on three reference strains as requested by EUCAST for analysis of the repeatability and reproducibility of the method., Results: Correlation between the two methods, assessed using 211 clinical isolates (n = 2439 zones of growth inhibition measured), was excellent with a correlation coefficient of 0.97. For the earlier reading experiments, preliminary results demonstrate the possibility of reading AST for drug-species combinations after 6 and 8 h for Gram-negative bacteria with rapid growth (correlation coefficient at 6 h = 0.96 and at 8 h = 0.98) and at 8 or 10 h for Gram-positive bacteria., Conclusions: The Scan ® 1200 has many advantages thanks to its small size, rapidity of use and high-resolution imaging allowing the possibility to improve AST results after only 6-8 h of incubation. This AST reader system represents a robust alternative tool for routine use in clinical microbiology 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.)

The normalized resistance interpretation (NRI) method was developed in response to a call for a method to calibrate disc diffusion test results making inter-laboratory comparisons possible. The main use of NRI so far has been in individual laboratories, in medical and veterinary medicine and in the field of marine microbiology. The applications of NRI for disc diffusion tests are reviewed and, in addition, a detailed description of the calculation procedure is presented. NRI provides a fully objective method for ECOFF calculations of disc diffusion antimicrobial susceptibility test results., (© 2016 APMIS. Published by John Wiley & Sons Ltd.)

Background: One of the most important complications of fixed orthodontic treatment is the formation of white spots which are initial carious lesions. Addition of antimicrobial agents into orthodontic adhesives might be a wise solution for prevention of white spot formation. The aim of this study was to evaluate the antibacterial properties of a conventional orthodontic adhesive containing three different concentrations of silver/hydroxyapatite nanoparticles., Methods: One hundred and sixty-two Transbond XT composite discs containing 0, 1, 5, and 10 % silver/hydroxyapatite nanoparticles were prepared and sterilized. Antibacterial properties of these composite groups against Streptococcus mutans, Lactobacillus acidophilus, and Streptococcus sanguinis were investigated using three different antimicrobial tests. Disk agar diffusion test was performed to assess the diffusion of antibacterial agent on brain heart infusion agar plate by measuring bacterial growth inhibition zones. Biofilm inhibition test showed the antibacterial capacity of composite discs against resistant bacterial biofilms. Antimicrobial activity of eluted components from composite discs was investigated by comparing the viable counts of bacteria after 3, 15, and 30 days., Results: Composite discs containing 5 and 10 % silver/hydroxyapatite nanoparticles were capable of producing growth inhibition zones for all bacterial types. Results of biofilm inhibition test showed that all of the study groups reduced viable bacterial count in comparison to the control group. Antimicrobial activity of eluted components from composite discs was immensely diverse based on the bacterial type and the concentration of nanoparticles., Conclusions: Transbond XT composite discs containing 5 and 10 % silver/hydroxyapatite nanoparticles produce bacterial growth inhibition zones and show antibacterial properties against biofilms.

Background: Accurate detection of Neisseria gonorrhoeae antimicrobial resistance is essential for appropriate management and prevention of spread of infection in the community. In this study Calibrated Dichotomous Sensitivity (CDS) and Clinical Laboratory Standards Institute (CLSI) disc diffusion methods were compared with minimum inhibitory concentration (MIC) by Etest in Neisseria gonorrhoeae isolates from Karachi, Pakistan. CDS and CLSI disc diffusion techniques, and Etest for ceftriaxone, penicillin G, spectinomycin and ciprofloxacin against 100 isolates from years 2012-2014 were performed. Due to lack of CLSI breakpoints for azithromycin, it was interpreted using cut-offs from British Society of Antimicrobial Chemotherapy (BSAC). Due to lack of low concentration tetracycline discs, tetracycline was tested with CLSI disc diffusion and Etest only. Comparisons were based on the identified susceptibility, intermediate susceptibility and resistance (SIR) categories using the different methods. Complete percent agreement was percentage agreement achieved when test and reference method had identical SIR-category. Essential percent agreement was percentage agreement when minor discrepancies were disregarded., Results: There was 100 % and 99 % overall essential agreement and 50 % versus 23 % overall complete agreement by CDS and CLSI methods, respectively, with MICs for all tested antibiotics. Using either method, there was 100 % complete agreement for ceftriaxone and spectinomycin. There was 90 % versus 86 % complete agreement for ciprofloxacin, and 60 % and 75 % for penicillin using CDS and CLSI method, respectively. Essential agreement of 99 % and complete agreement of 62 % was found for tetracycline with CLSI method. There was 100 % essential and complete agreement by CDS, BSAC and Etest for azithromycin., Conclusion: No major errors with regard to identified SIR-categories were found for penicillin, ciprofloxacin, ceftriaxone and spectinomycin using CLSI and CDS methods. All isolates were susceptible to ceftriaxone and spectinomycin, and 99 % to azithromycin. In low-resource settings, both the CLSI and CDS disc diffusion techniques might be used for susceptibility testing of gonococcal isolates. However, these methods require considerable standardization and quality controls for adequate levels of reproducibility and correct interpretation to reflect appropriately the MIC values of the different antimicrobials. New, emerging, or rare resistance should be confirmed by MIC determination., Competing Interests: The author(s) declare that they have no competing interests.

Background: Chlorhexidine (CHX) is used in oral care products to help control dental plaque. In this study dental plaque bacteria were grown on media containing 2 μg/ml chlorhexidine gluconate to screen for bacteria with reduced CHX susceptibility. The isolates were characterized by 16S rRNA gene sequencing and antibiotic resistance profiles were determined using the disc diffusion method., Results: The isolates were variably resistant to multiple drugs including ampicillin, kanamycin, gentamicin and tetracycline. Two species, Chryseobacterium culicis and Chryseobacterium indologenes were able to grow planktonically and form biofilms in the presence of 32 μg/ml CHX. In the CHX and multidrug resistant C. indologenes we demonstrated a 19-fold up-regulation of expression of the HlyD-like periplasmic adaptor protein of a tripartite efflux pump upon exposure to 16 μg/ml CHX suggesting that multidrug resistance may be mediated by this system. Exposure of biofilms of these resistant species to undiluted commercial CHX mouthwash for intervals from 5 to 60 s indicated that the mouthwash was unlikely to eliminate them from dental plaque in vivo., Conclusions: The study highlights the requirement for increased vigilance of the presence of multidrug resistant bacteria in dental plaque and raises a potential risk of long-term use of oral care products containing antimicrobial agents for the control of dental plaque.

Background:: Topical antimicrobial drugs are indicated for limited superficial pyodermitis treatment, although they are largely used as self-prescribed medication for a variety of inflammatory dermatoses, including atopic dermatitis. Monitoring bacterial susceptibility to these drugs is difficult, given the paucity of laboratory standardization., Objective:: To evaluate the prevalence of Staphylococcus aureus topical antimicrobial drug resistance in atopic dermatitis patients., Methods:: We conducted a cross-sectional study of children and adults diagnosed with atopic dermatitis and S. aureus colonization. We used miscellaneous literature reported breakpoints to define S. aureus resistance to mupirocin, fusidic acid, gentamicin, neomycin and bacitracin., Results:: A total of 91 patients were included and 100 S. aureus isolates were analyzed. All strains were methicillin-susceptible S. aureus. We found a low prevalence of mupirocin and fusidic acid resistance (1.1% and 5.9%, respectively), but high levels of neomycin and bacitracin resistance (42.6% and 100%, respectively). Fusidic acid resistance was associated with more severe atopic dermatitis, demonstrated by higher EASI scores (median 17.8 vs 5.7, p=.009). Our results also corroborate the literature on the absence of cross-resistance between the aminoglycosides neomycin and gentamicin., Conclusions:: Our data, in a southern Brazilian sample of AD patients, revealed a low prevalence of mupirocin and fusidic acid resistance of S. aureus atopic eczema colonizer strains. However, for neomycin and bacitracin, which are commonly used topical antimicrobial drugs in Brazil, high levels of resistance were identified. Further restrictions on the use of these antimicrobials seem necessary to keep resistance as low as possible., Competing Interests: None

Clostridium difficile is a leading cause of diarrhea in hospitalized patients worldwide. While metronidazole and vancomycin are the most prescribed antibiotics for the treatment of this infection, teicoplanin, tigecycline and nitazoxanide are alternatives drugs. Knowledge on the antibiotic susceptibility profiles is a basic step to differentiate recurrence from treatment failure due to antimicrobial resistance. Because C. difficile antimicrobial susceptibility is largely unknown in Brazil, we aimed to determine the profile of C. difficile strains cultivated from stool samples of inpatients with diarrhea and a positive toxin A/B test using both agar dilution and disk diffusion methods. All 50 strains tested were sensitive to metronidazole according to CLSI and EUCAST breakpoints with an MIC90 value of 2μg/mL. Nitazoxanide and tigecycline were highly active in vitro against these strains with an MIC90 value of 0.125μg/mL for both antimicrobials. The MIC90 were 4μg/mL and 2μg/mL for vancomycin and teicoplanin, respectively. A resistance rate of 8% was observed for moxifloxacin. Disk diffusion can be used as an alternative to screen for moxifloxacin resistance, nitazoxanide, tigecycline and metronidazole susceptibility, but it cannot be used for testing glycopeptides. Our results suggest that C. difficile strains from São Paulo city, Brazil, are susceptible to metronidazole and have low MIC90 values for most of the current therapeutic options available in Brazil., (Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.)

Objective To 1) describe the correlation between the zones of inhibition in 1-µg oxacillin disk diffusion (ODD) tests and penicillin and ceftriaxone minimum inhibitory concentrations (MICs) of meningeal and non-meningeal strains of Streptococcus pneumoniae and 2) evaluate the usefulness of the ODD test as a predictor of susceptibility to penicillin in S. pneumoniae and as a quick and cost-effective method easily implemented in a routine clinical laboratory setting. Methods S. pneumoniae isolates from healthy nasopharyngeal carriers less than 2 years old, obtained in a multicentric cross-sectional study conducted in various Peruvian hospitals and health centers from 2007 to 2009, were analyzed. Using Clinical and Laboratory Standards Institute (CLSI) breakpoints, the correlation between the zones of inhibition of the ODD test and the MICs of penicillin and ceftriaxone was determined. Results Of the 571 S. pneumoniae isolates, 314 (55%) showed resistance to penicillin (MIC ≥ 0.12 µg/mL) and 124 (21.7%) showed resistance to ceftriaxone (MIC ≥ 1 µg/mL). Comparison of the ODD test zones of inhibition and the penicillin MICs, using the CLSI meningeal breakpoints, showed good correlation (Cohen's kappa coefficient = 0.8239). Conclusions There was good correlation between ODD zones of inhibition and penicillin meningeal breakpoints but weak correlation between the ODD results and non-meningeal breakpoints for both penicillin and ceftriaxone. Therefore, the ODD test appears to be a useful tool for predicting penicillin resistance in cases of meningeal strains of S. pneumoniae, particularly in low- and middle- income countries, where MIC determination is not routinely available.

The Etest on chocolate Mueller-Hinton agar was compared to broth microdilution (BMD) for 125 isolates of nutritionally variant streptococci. Vancomycin Etests yielded 31.1% essential agreement (EA) and 20.0% categorical agreement (CA). Penicillin Etests yielded 86.0% EA and 85.6% CA, whereas ceftriaxone Etests yielded 73.6% EA and 68.0% CA., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: The Middle East is regarded as a secondary reservoir for OXA-48 and New Delhi metallo-β-lactamase (NDM) carbapenemases. One of the main challenges in clinical microbiology diagnostics is the detection of carbapenemases. For this reason simple screening methods have been sought to detect carbapenemase producers to determine appropriate therapeutic measures and implement infection control interventions. The present study aimed to evaluate the efficacy of the modified Hodge test (MHT) and a boronic acid-based combined disk test using carbapenems as substrates for the phenotypic determination of OXA-48 and NDM type carbapenemases in 45 epidemiologically unrelated carbapenem-resistant clinical isolates of Klebsiella pneumoniae (13 isolates), Acinetobacter baumanii (20 isolates), and Pseudomonas aeruginosa (12 isolates)., Results: Boronic acid disk test using meropenem as substrate and 600 µg of 3- aminophenylboronic acid (APB) was the most sensitive method (83.33 %) for detection of OXA-48, while the most specific method was MHT (100 %). As regards NDM carbapenemase, boronic acid disk tests using imipenem and 600 µg of APB per disk, and meropenem with 300 or 600 µg of APB were the most  sensitive methods (87.50 %), while the most specific method was the MHT (100 %)., Conclusions: The results of the present study indicate that phenotypic screening with the MHT and the boronic acid disk test may be used to detect OXA-48 and NDM carbapenemases in Gram-negative bacilli clinical isolates, and that these tests can be easily applied in tertiary care settings with minimal infrastructure.

Microbial pathogens represent an increasing threat to human health. Although many infections can be successfully treated and cleared, drug resistance is a widespread problem. The existence of subpopulations of 'tolerant' cells (where a fraction of the population is able to grow above the population resistance level) may increase the rate of treatment failure; yet, existing methods to measure subpopulation effects are cumbersome. Here we describe diskImageR, a computational pipeline that analyses photographs of disk diffusion assays to determine the degree of drug susceptibility [the radius of inhibition, (RAD)], and two aspects of subpopulation growth [the fraction of growth (FoG) within the zone of inhibition, (ZOI), and the rate of change in growth from no drug to inhibitory drug concentrations, (SLOPE)]. diskImageR was used to examine the response of the human fungal pathogen Candida albicans to the antifungal drug fluconazole across different strain backgrounds and growth conditions. Disk diffusion assays performed under Clinical and Laboratory Standards Institute (CLSI) conditions led to more susceptibility and less tolerance than assays performed using rich medium conditions. We also used diskImageR to quantify the effects of three drugs in combination with fluconazole, finding that all three combinations affected tolerance, with the effect of one drug (doxycycline) being very strain dependent. The three drugs had different effects on susceptibility, with doxycycline generally having no effect, chloroquine generally increasing susceptibility and pyrvinium pamoate generally reducing susceptibility. The ability to simultaneously quantitate different aspects of microbial drug responses will facilitate the study of mechanisms of subpopulation responses in the presence of antimicrobial drugs.

In this study, we aimed to identify the molecular carbapenemase types of the Enterobacteriaceae isolates and to evaluate the performance of manually prepared and commercially available combination disc methods and the modified Hodge test. One hundred and forty carbapenemase producing isolates and 45 isolates as control group were included in our study. The Xpert CARBA-R test was used as the molecular method. Antibiotic susceptibility tests were performed using combined discs, manually prepared with APBA (3-aminophenyl boronic acid), DPA (dipicolinic acid), EDTA (Ethylene diamine tetra acetic acid), cloxacillin supplements and Mastdiscs Combi-D70C that includes four antibiotic discs with specific inhibitors and temocillin discs. The modified Hodge test was performed on all isolates. OXA-48 gene was identified in 129 isolates , the NDM gene was identified in 10 isolates and VIM in one isolate. Thirty inaccurate results (30/185, 16%) were detected by using the manually prepared confirmation test. The sensitivity and specificity of this test were identified respectively 85% and 73%. Also, the sensitivity and specificity of the Mastdiscs Combi-D70C were identified as 100%. Negative results were detected in 3 NDM isolates with the use of a modified Hodge test. Sensitivity and specificity were calculated for the modified Hodge test respectively 97% and 100%. Finally, molecular methods provide results rapidly but they are not always easily accessible. The modified Hodge test can be used only for screening as a first step test and is not one of the tests that can identify the type of the carbapenemase. When carbapenem-resistant Enterobacteriaceae are detected, a commercial kit like Mastdiscs Combi-D70 may be preferred instead of the manually prepared phenotypic verification tests., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: To determine the in vitrodisk diffusion and MIC patterns of the therapeutic alternatives for Salmonella Typhi., Study Design: Across-sectional study., Place and Duration of Study: Armed Forces Institute of Pathology, Rawalpindi, from June 2011 to May 2013., Methodology: Clinical samples were collected from suspected cases of Salmonella infections. Culture was obtained on standard media. Suspected Salmonella colonies were tested by API 20E and confirmed by serology. The isolates were tested for resistance to various antibiotics by Kirby-Bauer disc diffusion method. MIC was done on MDR and ciprofloxacin intermediate or resistant cases by E-strips for selected antibiotics., Results: One hundred and twenty-eight isolates of Salmonella Typhi were recovered from 2230 specimens. Resistance by disk diffusion technique was 72% for ampicillin, 41.2% for cotrimoxazole, 38% for chloramphenicol, 8% for ciprofloxacin, 4.7% for cefpodoxime, 3.5% each for ertapenem aztreonam and moxifloxacin 2.4% for ceftriaxone and 2.3% for doripenem. No resistance was noted for imipenem, cefepime and gatifloxacin. Imipenem MIC90was 0.38 and MIC50 was 0.25. For cefpirome, MIC90was 0.64 and MIC50 was 0.09. For aztreonam, MIC90 was 0.12 and MIC50 was 0.09. For cefpodoxime MIC90 was 0.75 and MIC50 was 0.38. For azithromycin, these values were 16.0 and 7.0; and for tigecycline they were 0.25 and 0.09., Conclusion: Imipenem, azithromycin, tigecycline, aztreonam, cefpodoxime and cefpirome are potential therapeutic agents for resistant Salmonella Typhi infection.

Introduction: Carbapenemase-producing Klebsiella pneumoniae (KPC) outbreaks may cause a huge economical burden on developing countries. Furthermore, KPC can be challenging to detect. We describe the laboratory strategy for the detection of KPC from 2011 to 2013 in a tertiary care hospital in Central America with approximately 1,000 beds., Methodology: A retrospective analysis of a clinical laboratory database was done to determine the pragmatic application of the combined-disk boronic acid test during a KPC outbreak in Panama. A total of 1,026 Klebsiella pneumoniae isolates were found, of which 133 were positive for KPC. The strategy during two phases was described according to the test employed as a confirmatory test for KPC. After the K. pneumoniae isolates were detected by the VITEK 2 system, blaKPC polymerase chain reaction (PCR) and the combined-disk boronic acid test were employed as a confirmatory test during phase one. The combined-disk boronic acid test was employed as a confirmatory test for KPC during phase two., Results: The sensitivity, specificity, positive predictive value, and negative predictive value of the boronic acid test were 100%, 97%, 91%, and 100%, respectively, when blaKPC PCR was employed as a confirmatory test during the start of the outbreak. Afterwards, modified VITEK 2 system parameters resulted in 116 suspicious KPC samples and the boronic acid test confirmed 102 isolates., Conclusions: The use of an automated bacterial identification system and the boronic acid test for the detection of KPC was an effective and low-cost strategy for a clinical laboratory in Panama during an outbreak.

ChromID ESBL agar and Brilliance ESBL agar were compared for the isolation of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae from 298 stools. These had comparable sensitivity and selectivity for the 116 positive samples. Pre-enrichment with cefpodoxime and extending incubation to 48 hours after direct plating both significantly increased sensitivity but reduced selectivity of both agars., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Disc diffusion is still largely based on manual procedures. Technical variations originate from inoculum preparation, variations in materials, individual operator plate streaking and reading accuracy. Resulting measurement imprecision contributes to categorization errors. Biological variation resembles the natural fluctuation of a measured parameter such as antibiotic susceptibility around a mean value. It is deemed to originate from factors such as genetic background or metabolic state. This study analysed the relative contribution of different technical and biological factors to total disc diffusion variation., Methods: For calculation of relative error factor contribution to disc diffusion variability, five experiments were designed keeping different combinations of error factors constant. A mathematical model was developed to analyse the individual error factor contribution to disc diffusion variation for each of the tested drug-species combinations., Results: The contribution of biological variation to total diameter variance ranged from 10.4% to 98.8% for different drug-species combinations. Highest biological variation was found for Enterococcus faecalis WT and vancomycin (98.8%) and for penicillinase-producing Staphylococcus aureus and penicillin G (96.0%). Average imprecision of automated zone reading revealed that 1.4%-5.3% of total imprecision was due to technical variation, while materials, i.e. antibiotic discs and agar plates, contributed between 2.6% and 3.9%. Inoculum preparation and manual plate streaking contributed 6.8%-24.8% and 6.6%-24.3%, respectively, to total imprecision., Conclusions: This study illustrates the relative contributions of technical factors that account for a significant part of total variance in disc diffusion. The highest relative contribution originated from the operator, i.e. manual inoculum preparation and plate streaking. Further standardization of inoculum preparation and plate streaking by automation could potentially increase the precision of disc diffusion and improve the correlation of susceptibility reports with clinical outcome., (© 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.)

Objectives: The objective of this study was to assess the performance of an algorithm based on the disc diffusion method for the screening of carbapenemase-producing Enterobacteriaceae (CPE) referred to the French National Reference Centre for Antibiotic Resistance., Methods: From April to June 2014, all isolates of Enterobacteriaceae referred to the French National Reference Centre for Antibiotic Resistance were included. The inhibition zone diameters of imipenem, ticarcillin/clavulanate and temocillin using EUCAST disc diffusion methodology were recorded. All isolates were subjected to the algorithm proposed by the Antibiogram Committee of the French Society of Microbiology (CA-SFM) for the screening of carbapenemase producers. Phenotypic, biochemical and molecular detection of carbapenemases was performed for all isolates., Results: A total of 621 consecutive enterobacterial isolates with decreased susceptibility to carbapenems were tested. They included 213 CPE [OXA-48-like (n = 183), NDM (n = 22), VIM (n = 3), KPC (n = 3) and OXA-48-like + NDM (n = 2)] and 408 non-carbapenemase producers. The CA-SFM algorithm combining cut-off values of 15 mm, 15 mm and 22 mm for ticarcillin/clavulanate, temocillin and imipenem, respectively, perfectly detected 204 isolates (32.8%) as non-carbapenemase producers, leading to a negative predictive value of 100% for this algorithm., Conclusions: Implementation of the CA-SFM algorithm in clinical microbiology laboratories may avoid additional testing for CPE in one-third of the enterobacterial isolates with decreased susceptibility to carbapenems., (© 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.)

16S rRNA methyltransferases confer resistance to most aminoglycosides, but discriminating their activity from that of aminoglycoside-modifying enzymes (AMEs) is challenging using phenotypic methods. We demonstrate that arbekacin, an aminoglycoside refractory to most AMEs, can rapidly detect 16S methyltransferase activity in Enterobacteriaceae with high specificity using the standard disk susceptibility test., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We compared levofloxacin (1 μg/disk) disk diffusion method to E test against 212 Helicobacter pylori strains. Using diameter breakpoints for susceptibility (≥15 mm) and resistance (≤9 mm), very major error, major error rate, and categoric agreement were 0.0%, 0.6%, and 93.9%, respectively. The method may be useful in low-resource laboratories., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Haemophilus influenzae is a major pathogen, and beta-lactams are first-line drugs. Resistance due to altered penicillin-binding protein 3 (rPBP3) is frequent, and susceptibility testing of such strains is challenging. A collection of 154 beta-lactamase-negative isolates with a large proportion of rPBP3 (67.5%) was used to evaluate and compare Etest (Haemophilus test medium [HTM]) and disk diffusion (EUCAST method) for categorization of susceptibility to aminopenicillins and cefuroxime, using MICs generated with broth (HTM) microdilution and clinical breakpoints from CLSI and EUCAST as the gold standards. In addition, the proficiency of nine disks in screening for the rPBP3 genotype (N526K positive) was evaluated. By Etest, both essential and categorical agreement were generally poor (<70%), with high very major errors (VME) (CLSI, 13.0%; EUCAST, 34.3%) and falsely susceptible rates (FSR) (CLSI, 87.0%; EUCAST, 88.3%) for ampicillin. Ampicillin (2 μg) with adjusted (+2 mm) zone breakpoints was superior to Etest for categorization of susceptibility to ampicillin (agreement, 74.0%; VME, 11.0%; FSR, 28.3%). Conversely, Etest was superior to 30 μg cefuroxime for categorization of susceptibility to cefuroxime (agreement, 57.1% versus 60.4%; VME, 2.6% versus 9.7%; FSR, 7.1% versus 26.8%). Benzylpenicillin (1 unit) (EUCAST screening disk) and cefuroxime (5 μg) identified rPBP3 isolates with highest accuracies (95.5% and 92.2%, respectively). In conclusion, disk screening reliably detects rPBP3 H. influenzae, but false ampicillin susceptibility is frequent with routine methods. We suggest adding a comment recommending high-dose aminopenicillin therapy or the use of other agents for severe infections with screening-positive isolates that are susceptible to aminopenicillins by gradient or disk diffusion., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Fluoroquinolone resistance is a serious and increasingly common problem in Salmonella. Two companion studies in this issue of the Journal of Clinical Microbiology (E. Deak, R. Skov, J. A. Hindler, and R. M. Humphries, J Clin Microbiol 53:3405-3410, 2015, http://dx.doi.org/10.1128/JCM.01393-15; R. Skov, E. Matuschek, M. Sjölund-Karlsson, J. Åhman, A. Petersen, M. Stegger, M. Torpdahl, and G. Kahlmeter, J Clin Microbiol 53:3411-3417, 2015, http://dx.doi.org/10.1128/JCM.01287-15) provide data to support the use of pefloxacin disk diffusion as a convenient and inexpensive surrogate laboratory method to detect fluoroquinolone resistance in Salmonella when the direct measurement of fluoroquinolone MICs is not feasible [corrected]. Recently updated CLSI and EUCAST susceptibility breakpoints will help to optimize clinical outcomes and reduce the likelihood of emergent resistance., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Determination of polymyxin susceptibility profile is important to monitor resistance rates and for implementing control measures for polymyxin-resistant carbapenem-resistant Enterobacteriaceae. Some laboratorial methods have been used to determine the polymyxin susceptibility profile. However, the performance of MicroScan WalkAway has been poorly reported for KPC-producing Klebsiella pneumoniae, so far. To evaluate two different methods, Etest and the MicroScan automated system, in determining minimal inhibitory concentration (MIC) of polymyxin among KPC-producing K. pneumoniae isolated from patients in two care units (ICUs) of a tertiary hospital in Porto Alegre, Southern Brazil. A total of 101 KPC-Kb isolates were obtained from rectal swabs and clinical specimens (urine, blood, and endotracheal aspirate). Colistin and polymyxin B MICs were determined using MicroScan WalkAway automated system and Etest, respectively. Discrepant results were resolved by broth microdilution (BMD). MicroScan showed 88.1% of sensitivity for predicting polymyxin B resistance in KPC-producing K. pneumoniae compared to the results obtained by Etest. All discrepant results were tested by BMD and these were concordant with results obtained by Etest. The MicroScan automated system does not seem to be very efficient for the screening of polymyxin-resistant isolates once an inappropriate sensitivity is achieved. The results presented here show the need for confirmation of the susceptibility profile by use of a dilution method (Etest or BMD)., (© 2015 APMIS. Published by John Wiley & Sons Ltd.)

Detection of fluoroquinolone resistance in Salmonella enterica has become increasingly difficult due to evolving resistance mechanisms to this antimicrobial class in this organism. We evaluated two quinolone disks and five fluoroquinolone disks for their ability to act as a surrogate agent for the detection of fluoroquinolone resistance in a collection of 136 S. enterica isolates, including 111 with intermediate or resistant ciprofloxacin MICs mediated by a variety of resistance mechanisms. Ciprofloxacin, ofloxacin, and pefloxacin disks detected all isolates resistant to ciprofloxacin (0% very major error) and yielded false resistance (major error) in 8, 4, and 12% of susceptible isolates, respectively. Ciprofloxacin and pefloxacin provided clearer differentiation of susceptible and resistant isolates., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Fluoroquinolones (FQs) are among the drugs of choice for treatment of Salmonella infections. However, fluoroquinolone resistance is increasing in Salmonella due to chromosomal mutations in the quinolone resistance-determining regions (QRDRs) of the topoisomerase genes gyrA, gyrB, parC, and parE and/or plasmid-mediated quinolone resistance (PMQR) mechanisms including qnr variants, aac(6')-Ib-cr, qepA, and oqxAB. Some of these mutations cause only subtle increases in the MIC, i.e., MICs ranging from 0.12 to 0.25 mg/liter for ciprofloxacin (just above the wild-type MIC of ≤0.06 mg/liter). These isolates are difficult to detect with standard ciprofloxacin disk diffusion, and plasmid-mediated resistance, such as qnr, is often not detected by the nalidixic acid screen test. We evaluated 16 quinolone/fluoroquinolone disks for their ability to detect low-level-resistant Salmonella enterica isolates that are not serotype Typhi. A total of 153 Salmonella isolates characterized for the presence (n = 104) or absence (n = 49) of gyrA and/or parC topoisomerase mutations, qnrA, qnrB, qnrD, qnrS, aac(6')-Ib-cr, or qepA genes were investigated. All isolates were MIC tested by broth microdilution against ciprofloxacin, levofloxacin, and ofloxacin and by disk diffusion using EUCAST or CLSI methodology. MIC determination correctly categorized all isolates as either wild-type isolates (MIC of ≤0.06 mg/liter and absence of resistance genes) or non-wild-type isolates (MIC of >0.06 mg/liter and presence of a resistance gene). Disk diffusion using these antibiotics and nalidixic acid failed to detect some low-level-resistant isolates, whereas the 5-μg pefloxacin disk correctly identified all resistant isolates. However, pefloxacin will not detect isolates having aac(6')-Ib-cr as the only resistance determinant. The pefloxacin disk assay was approved and implemented by EUCAST (in 2014) and CLSI (in 2015)., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Aim of this study was to verify whether Mueller-Hinton agar supplemented with 2 % glucose and methylene blue (MH-GM), which is used for disk diffusion susceptibility testing of Candida species by the Clinical and Laboratory Standards Institute, is suitable for testing Malassezia pachydermatis. A variant of the disk diffusion procedure utilizing a 9-mm tablet was used to test 31 isolates against clotrimazole and miconazole using MH-GM as test medium. The MH-GM agar optimally supported the growth of all M. pachydermatis isolates, provided that the yeast inoculum was prepared with a lipid source (Tween 40 and 80). Zone edges were frequently definite and clear, facilitating the measurement of zone size and minimizing subjectivity. The inhibition zones correlated with MIC values obtained in a broth dilution assay. The agar diffusion method with MH-GM as the test medium appears as a suitable procedure for testing the susceptibility of M. pachydermatis to CTZ and MCZ in clinical laboratories. This test format may allow processing a large number of isolates in epidemiological studies. This may in turn facilitate clarifying to what extent the problem "drug resistance" accounts for cases of treatment failure in dogs with Malassezia otitis and dermatitis.

Introduction: Propolis is a natural product derived from beekeeping. It has anesthetic, anti-inflammatory, immune-stimulant and antibacterial properties on grampositive and gramnegative bacteria. However, little is known regarding its activity on Helicobacter pylori. This bacteria colonizes about half of the world's population and is associated with chronic gastritis, peptic ulcer and gastric cancer., Objective: The aim of this study was to evaluate the inhibitory activity of 22 propolis extracts from nine of the 11 beekeeping Chilean regions on 10 strains of H. pylori isolated from gastric mucosa., Methods: The antibacterial activity of the extracts was determined using the well diffusion method and diffusion disks., Results: 100% of the extracts were active on the tested strains, showing inhibition halos equal to or greater than 15 mm by both methods., Conclusions: Our results show an effective anti H. pylori activity of propolis. However, additional microbiological studies are needed before a potential clinical utility of these natural products is warranted.

The disk diffusion (DD) method remains the most popular manual technique for antibiotic susceptibility testing (AST) in clinical microbiology laboratories. This is because of its simplicity, reproducibility, and limited cost compared to (automated) microdilution systems, which are usually less sensitive at detecting certain important mechanisms of resistance. Here, we evaluate the PREVI® Isola automated seeder system using a new protocol for spreading bacterial suspensions (eight deposits of calibrated inocula of bacteria, followed by two rounds of rotation) in comparison with manual DD reference testing on a large series of clinical and reference strains. The average time required for seeding one agar plate for DD with this new protocol was 51 s per plate, i.e., 70 agar plates/h. Reproducibility and repeatability was assessed on three reference and three randomly chosen clinical strains, as usually requested by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and was excellent compared to the manual method. The standard deviations of zones of growth inhibition showed no statistical discrimination. The correlation between the two methods, assessed using 294 clinical isolates and a panel of six antibiotics (n = 3,528 zones of growth inhibition measured), was excellent, with a correlation coefficient of 0.977. The new PREVI® Isola protocol adapted for DD had a sensitivity of 99 % and a specificity of 100 % compared to the manual technique for interpreting DD as recommended by the EUCAST.

In this study, we introduce a new approach for determination of epidemiologic cutoffs (ECOFFs) and resistant-population cutoffs (RCOFFs) based on receiver operating characteristic (ROC) curves. As an example, the method was applied for determination of ECOFFs for seven different beta-lactam antibiotics and wild-type populations of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. In addition, RCOFFs were determined for bacterial populations with defined resistance mechanisms ("resistotypes"), i.e., extended-spectrum beta-lactamase (ESBL)-positive E. coli, ESBL-positive K. pneumoniae, and ESBL-positive E. cloacae; AmpC cephalosporinase-positive E. coli and AmpC-positive K. pneumoniae; and broad-spectrum beta-lactamase (BSBL)-positive E. coli. RCOFFs and ECOFFs are instrumental for a systematic characterization of associations between resistotypes and wild-type populations., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The current phenotypic methods for detecting carbapenemase-producing Enterobacteriaceae (CPE) allow differentiation between class A and B carbapenemases, but they cannot confirm in a single test class D OXA-48 carbapenemase producers. In this study, we evaluated a new phenotypic test, the OXA-48 disk test, which is based on an imipenem disk and two blank disks adjacent to the imipenem disk, loaded with the tested strain and impregnated with EDTA and EDTA plus phenyl boronic acid (PBA), respectively. The evaluation of the OXA-48 disk test was performed with 81 genotypically confirmed OXA-48-type-producing Enterobacteriaceae isolates (41 extended-spectrum β-lactamase [ESBL] producers, 3 AmpC producers, and 37 non-ESBL, non-AmpC producers). To measure the specificity of the test, 173 genotypically confirmed OXA-48-negative Enterobacteriaceae isolates (57 Klebsiella pneumoniae carbapenemase [KPC] producers, 34 VIM producers, 23 KPC/VIM producers, 22 NDM producers, and 37 AmpC or ESBL producers and porin deficient) that were nonsusceptible to at least one carbapenem were chosen for testing. Using the imipenem disk and the distortion of the inhibition halo around both blank disks containing EDTA and EDTA/PBA, the test differentiated all but 3 of the 81 OXA-48 producers (sensitivity of 96.3%). The test was negative for OXA-48 production in all but 4 of the 173 carbapenem-nonsusceptible isolates producing other carbapenemases, AmpCs, or ESBLs (specificity of 97.7%). This evaluation shows that the OXA-48 disk test is an accurate phenotypic method for the direct differentiation of OXA-48-producing Enterobacteriaceae. Its use along with combined disk tests employing inhibitor-supplemented carbapenem disks might allow the differentiation of the currently known carbapenemase types in Enterobacteriaceae species and provide important infection control information., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

This study aimed to evaluate the in vitro antifungal susceptibility of Candida species of head-and-neck-irradiated patients (Group 1), non-institutionalized (Group 2) and institutionalized elders (Group 3) using Etest® methodology. Candida was isolated from saliva and presumptively identified by CHROMagar Candida(r), confirmed by morphological criteria, carbohydrate assimilation (API 20C AUX®) and genetic typing (OPE 18). The collection was made from 29, 34 and 29 individuals (Groups 1, 2 and 3, respectively) with 67 isolates. Etest® strips (ketoconazole, itraconazole, fluconazole, amphotericin B and flucytosine) on RPMI (Roswell Park Memorial Institute) agar, on duplicate, were used to evaluate susceptibility. ATTC (American Type Culture Collection) 10231 (Candida albicans) was used as quality control. Among the 67 isolates of Candida species, most were susceptible to azoles, flucytosine and amphotericin B. None of the isolates showed resistance and dose-dependent susceptibility to amphotericin B. There were nine strains resistant to itraconazole, six to fluconazole and two to ketoconazole and ten dose-dependent, mainly to flucytocine. The highest MIC (minimum inhibitory concentration) to C. albicans, C. tropicalis, C. parapsilosis was 2.671 μg.mL-1, 8.104 μg.mL-1, 4.429 μg.mL-1, all for flucytosine. C. krusei and C. glabrata were associated with higher MIC for azoles and C. glabrata with higher MIC to flucytosine. In summary, susceptibility to all tested antifungal agents was evident. The isolates were more resistant to itraconazole and dose-dependent to flucytosine. A comparison of C. albicans in the three groups showed no outliers. Higher MIC was associated with C. krusei and C. glabrata.

Background: We evaluated the combined use of the modified Hodge test (MHT) and carbapenemase inhibition test (CIT) using phenylboronic acid (PBA) and EDTA to detect carbapenemase-producing Enterobacteriaceae (CPE) and metallo-β-lactamase (MBL)-producing Pseudomonas spp., Methods: A total of 49 isolates of CPE (15 Klebsiella pneumoniae carbapenemase [KPC], 5 Guiana extended-spectrum β-lactamase [GES]-5, 9 New Delhi metallo-β-lactamase [NDM]-1, 5 Verona integron-encoded metallo-β-lactamase [VIM]-2, 3 imipenem-hydrolyzing β-lactamase [IMP], and 12 oxacillinase [OXA]-48-like), 25 isolates of MBL-producing Pseudomonas spp. (14 VIM-2 and 11 IMP), and 35 carbapenemase-negative controls were included. The MHT was performed for all isolates as recommended by the Clinical and Laboratory Standards Institute. Enhanced growth of the indicator strain was measured in mm with a ruler. The CIT was performed by directly dripping PBA and EDTA solutions onto carbapenem disks that were placed on Mueller-Hinton agar plates seeded with the test strain., Results: Considering the results of the MHT with the ertapenem disk in Enterobacteriaceae and Pseudomonas spp., the CIT with the meropenem disk in Enterobacteriaceae, and the imipenem disk in Pseudomonas spp., three combined disk tests, namely MHT-positive plus PBA-positive, EDTA-positive, and MHT-positive plus PBA-negative plus EDTA-negative, had excellent sensitivity and specificity for the detection of KPC- (100% sensitivity and 100% specificity), MBL- (94% sensitivity and 100% specificity), and OXA-48-like-producing isolates (100% sensitivity and 100% specificity), respectively., Conclusions: Combined use of the MHT and CIT with PBA and EDTA, for the detection of CPE and MBL-producing Pseudomonas spp., is effective in detecting and characterizing carbapenemases in routine laboratories.

We determined the susceptibility of 102 clinical isolates Cryptococcus neoformans from Durban, South Africa, to amphotericin B, fluconazole, flucytosine, and voriconazole using broth microdilution (BMD) according to the Clinical and Laboratory Standards Institute M27-A3 document and compared these results with Etest and Vitek 2(®). Essential agreement (EA) of Etest and Vitek 2(®) compared to BMD was determined. Low MICs that were below the epidemiological cutoff values of the 4 antifungal agents tested were demonstrated by all isolates. The EA of Etests for fluconazole, amphotericin, and voriconazole was 95.1%, 83.3%, and 91.2%, respectively, and for Vitek 2(®) EA for fluconazole, amphotericin, and flucytosine was 97.1%, 95.1%, and 97.1%, respectively. The Vitek 2(®) showed good agreement with BMD and is a suitable alternative. Etests demonstrated good EA for azoles only. Clinical cryptococcal isolates from Durban remain susceptible to current recommended antifungal therapy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The study objective was to use Bayesian latent class analysis to evaluate the accuracy of susceptibility test results obtained from disk diffusion and broth microdilution using bacteria recovered from beef feedlot cattle. Isolates of Escherichia coli and Mannheimia haemolytica were tested for susceptibility to ampicillin, ceftiofur, streptomycin, sulfisoxazole, tetracycline, and trimethoprim-sulfamethoxazole. Results showed that neither testing method was always or even generally superior to the other. Specificity (ability to correctly classify non-resistant isolates) was extremely high for both testing methods, but sensitivity (ability to correctly classify resistant isolates) was lower, variable in the drugs evaluated, and variable between the two bacterial species. Predictive values estimated using Bayesian Markov chain Monte Carlo models showed that the ability to predict true susceptibility status was equivalent for test results obtained with the two testing methods for some drugs, but for others there were marked differences between results obtained from disk diffusion and broth microdilution tests.

In dairy farms, antimicrobial drugs are frequently used for treatment of (sub)clinical mastitis. Determining the antimicrobial susceptibility of mastitis pathogens is needed to come to a correct use of antimicrobials. Strains of Staphylococcus aureus (n=768), Streptococcus uberis (n=939), Streptococcus dysgalactiae (n=444), Escherichia coli (n=563), and Klebsiella species (n=59) originating from routine milk samples from (sub)clinical mastitis were subjected to the disk diffusion method. Disks contained representatives of frequently used antibiotics in dairy. A limited number of clinical breakpoints were available through CLSI, and showed that susceptibility of Staph. aureus, E. coli, and Klebsiella was moderate to high. For streptococcal species however, a large variation between the tested species and the different antimicrobials was observed. In a next step, wild type populations were described based on epidemiological cut off values (EUCAST). Because of the limited number of official cut off values, the data were observed as a mastitis subpopulation and self-generated cut off values were created and a putative wild type population was suggested. The need for accurate clinical breakpoints for veterinary pathogens is high. Despite the lack of these breakpoints, however, a population study can be performed based on the distribution of inhibition zone diameters on the condition that a large number of strains is tested., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Different antimicrobial susceptibility testing methods to detect low-level vancomycin resistance in enterococci were evaluated in a Scandinavian multicenter study (n=28). A phenotypically and genotypically well-characterized diverse collection of Enterococcus faecalis (n=12) and Enterococcus faecium (n=18) strains with and without nonsusceptibility to vancomycin was examined blindly in Danish (n=5), Norwegian (n=13), and Swedish (n=10) laboratories using the EUCAST disk diffusion method (n=28) and the CLSI agar screen (n=18) or the Vitek 2 system (bioMérieux) (n=5). The EUCAST disk diffusion method (very major error [VME] rate, 7.0%; sensitivity, 0.93; major error [ME] rate, 2.4%; specificity, 0.98) and CLSI agar screen (VME rate, 6.6%; sensitivity, 0.93; ME rate, 5.6%; specificity, 0.94) performed significantly better (P=0.02) than the Vitek 2 system (VME rate, 13%; sensitivity, 0.87; ME rate, 0%; specificity, 1). The performance of the EUCAST disk diffusion method was challenged by differences in vancomycin inhibition zone sizes as well as the experience of the personnel in interpreting fuzzy zone edges as an indication of vancomycin resistance. Laboratories using Oxoid agar (P<0.0001) or Merck Mueller-Hinton (MH) agar (P=0.027) for the disk diffusion assay performed significantly better than did laboratories using BBL MH II medium. Laboratories using Difco brain heart infusion (BHI) agar for the CLSI agar screen performed significantly better (P=0.017) than did those using Oxoid BHI agar. In conclusion, both the EUCAST disk diffusion and CLSI agar screening methods performed acceptably (sensitivity, 0.93; specificity, 0.94 to 0.98) in the detection of VanB-type vancomycin-resistant enterococci with low-level resistance. Importantly, use of the CLSI agar screen requires careful monitoring of the vancomycin concentration in the plates. Moreover, disk diffusion methodology requires that personnel be trained in interpreting zone edges.

We studied whether the Etest can be used as an alternative to agar dilution to determine antimicrobial susceptibilities of ceftriaxone, cefixime, and cefpodoxime in Neisseria gonorrhoeae surveillance. One hundred fifteen clinical and laboratory isolates of N. gonorrhoeae were tested following the Clinical Laboratory Improvement Amendments (CLIA)-approved CLSI standard agar dilution method and, separately, by the Etest according to the manufacturer's recommendations. The MICs were determined and compared. Ten laboratory-generated mutants were used to simulate substantially nonsusceptible specimens. The Etest and agar dilution methods were well correlated. Statistical tests produced regression R2 values of 88%, 82%, and 85% and Pearson correlation coefficients of 92%, 91%, and 92% for ceftriaxone, cefixime, and cefpodoxime, respectively. When paired comparisons were made, the two tests were 88.7%, 80%, and 87% within 1 log2 dilution from each other for ceftriaxone, cefixime, and cefpodoxime, respectively. The within-2-log2 agreements were 99.1%, 98.3%, and 94.8% for ceftriaxone, cefixime, and cefpodoxime, respectively. Notwithstanding the good correlations and the within-2-log2 general agreement, the Etest results produced slightly lower MICs than the agar dilution results. In conclusion, we found that the Etest can be effectively used as an alternative to agar dilution testing to determine the susceptibility of N. gonorrhoeae to ceftriaxone, cefixime, and cefpodoxime, although we recommend further research into extremely resistant isolates. For isolates within the typical range of clinical MICs, reexamination of the Etest interpretation of susceptible and nonsusceptible categories would likely allow for successful transition from agar dilution to the Etest.

We evaluated the utility of 2 methods for detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from signal-positive blood culture bottles: loop-mediated isothermal amplification (LAMP) assay, and direct cefoxitin disk diffusion (DCDD) test using a 30 μg cefoxitin disk. In parallel, standard microbiological identification and oxacillin susceptibility testing with MecA PCR was performed. Of 60 blood cultures positive for Gram-positive cocci in clusters, LAMP (via detection of the FemA and MecA genes) showed 100% sensitivity and specificity for identification of MRSA/MSSA. When coagulase-negative staphylococci were tested, sensitivity for detection of methicillin resistance was 91.7% and specificity was 100%. DCDD along with direct tube coagulase assay detected only 80.6% of MRSA/MSSA. LAMP showed higher diagnostic accuracy although DCDD was more cost-effective and did not require additional reagents or supplies.

To detect antibiotic-resistant bacteria in areas remote from microbiology laboratories, we designed portable culture devices performing an analogue of the Kirby-Bauer disk diffusion test inside patterned papers embedded in tape. We quantified the antibiotic susceptibility of several strains of Escherichia coli and Salmonella typhimurium by measuring blue-colored zones of inhibited growth.

Background: The purpose of this study was to objectively compare methodological approaches that might be utilized in designing an antimicrobial resistance (AMR) surveillance program in beef feedlot cattle. Specifically, four separate comparisons were made to investigate their potential impact on estimates for prevalence of AMR. These included investigating potential differences between 2 different susceptibility testing methods (broth microdilution and disc diffusion), between 2 different target bacteria (non-type-specific E. coli [NTSEC] and Mannheimia haemolytica), between 2 strategies for sampling feces (individual samples collected per rectum and pooled samples collected from the pen floor), and between 2 strategies for determining which cattle to sample (cattle that were culture-positive for Mannheimia haemolytica and those that were culture-negative)., Results: Comparing two susceptibility testing methods demonstrated differences in the likelihood of detecting resistance between automated disk diffusion (BioMIC®) and broth microdilution (Sensititre®) for both E. coli and M. haemolytica. Differences were also detected when comparing resistance between two bacterial organisms within the same cattle; there was a higher likelihood of detecting resistance in E. coli than in M. haemolytica. Differences in resistance prevalence were not detected when using individual animal or composite pen sampling strategies. No differences in resistance prevalences were detected in E. coli recovered from cattle that were culture-positive for M. haemolytica compared to those that were culture-negative, suggesting that sampling strategies which targeted recovery of E. coli from M. haemolytica-positive cattle would not provide biased results., Conclusions: We found that for general purposes, the susceptibility test selected for AMR surveillance must be carefully chosen considering the purpose of the surveillance since the ability to detect resistance appears to vary between these tests depending upon the population where they are applied. Continued surveillance of AMR in M. haemolytica recovered by nasopharyngeal swab is recommended if monitoring an animal health pathogen is an objective of the surveillance program as results of surveillance using fecal E. coli cannot be extrapolated to this important respiratory pathogen. If surveillance of E. coli was pursued in the same population, study populations could target animals that were culture-positive for M. haemolytica without biasing estimates for AMR in E. coli. Composite pen-floor sampling or sampling of individuals per-rectum could possibly be used interchangeably for monitoring resistance in E. coli.

Objective: To compare the sensitivity and specificity of different phenotypic methods for detection of Amp C betalactamase producing bacteria., Study Design: Analytical study., Place and Duration of Study: Department of Microbiology, Army Medical College / National University of Sciences and Technology (NUST), Islamabad, Pakistan, from June 2010 to December 2010., Methodology: A total of 150 clinical isolates were screened for presence of Amp C beta-lactamase by using the cefoxitin disc. The confirmatory methods evaluated were inhibitor based assay (boronic acid), Amp C disc test and Amp C Etest. Three dimensional enzyme extract assay was used as the reference method for determining the sensitivity and specificity., Results: Among the total isolates tested, 62.8% bacteria showed the presence of Amp C beta-lactamase by standard three dimensional enzyme extract assay. Among the three methods compared, boronic acid disk test found out to be highly sensitive (88%) and specific (92%) for the detection of Amp C beta-lactamase producing bacteria., Conclusion: Detection of Amp C production is crucial in order to establish the antibiotic therapy and to attain the favourable clinical outcomes. Implementation of simple tests like boronic acid disk tests in the laboratories will help to alleviate the spread of Amp C beta-lactamase harboring organisms.

Purpose: To evaluate the discordance between Vitek 2 and Etest vancomycin minimum inhibitory concentration (MIC) testing methods in methicillin-resistant Staphylococcus aureus (MRSA) isolates., Methods: Inclusion criteria consisted of MRSA isolates with blood, respiratory, or wound origin of culture, inpatient or emergency department location at time of culture and isolates with a Vitek 2 MIC reported. Isolates were subjected to Etest and the resulting MICs were compared to the corresponding Vitek 2 MICs, and both the rate and degree of discordance were evaluated., Results: Seventy-five MRSA isolates met the inclusion criteria. Etest resulted in an MIC value greater than that of Vitek 2 in 64 isolates (85.3%). Furthermore, of the 35 isolates with an MIC of ≤ 0.5 mcg/mL via Vitek 2, Etest reported 27 isolates with an MIC >1 mcg/mL. Of the 39 isolates with a Vitek 2 MIC of 1 mcg/mL, Etest reported 30 isolates with an MIC >1 mcg/mL., Conclusion: We have demonstrated that Etest commonly reports MICs greater than 1 mcg/mL, even among isolates with MICs as low as ≤ 0.5 mcg/mL, according to Vitek 2. The clinical significance of these findings is unknown and would be an area of further study.