MIC Results Research Articles

Epidemiological MIC cut-off values for tigecycline calculated from Etest MIC values using normalized resistance interpretation

To apply the normalized resistance interpretation (NRI) method to Etest MIC results which have higher precision than conventional log2 dilution MIC tests due to the inclusion of intermediate values. If successful, NRI might provide an objective tool for the definition of epidemiological MIC cut-off values. MICs of tigecycline and other antimicrobial agents were determined for 4771 clinical isolates comprising five Gram-positive and 13 Gram-negative species or species groups using the Etest. Histograms of MIC values were constructed for each species and NRI calculations were applied to them. An upper MIC limit of 2.5 SD above the theoretical mean of the normalized distribution was used for setting the epidemiological cut-off values. Calculated cut-off values for wild-type strains were between 0.11 and 0.96 mg/L for Gram-positive species, and between 0.44 and 8.3 mg/L for Gram-negative species, except for Pseudomonas aeruginosa, which had a cut-off value of 450 mg/L, consistent with earlier reports on the lack of activity of tigecycline against this species. NRI offers an objective method for the analysis of MICs produced using Etests and the determination of epidemiological MIC cut-off values.

Oxazolidinone susceptibility patterns in 2004: report from the Zyvox® Annual Appraisal of Potency and Spectrum (ZAAPS) Program assessing isolates from 16 nations

To investigate the activity of linezolid (an oxazolidinone), a potent choice for community- and hospital-acquired infections, via a worldwide surveillance network called the Zyvox Annual Appraisal of Potency and Spectrum (ZAAPS) Program. A total of 4098 Gram-positive strains were collected from 42 laboratories located in North America (five sites in Canada), South America (10 sites), Europe (16 sites) and the Far East (11 sites). Each country or site submitted 200 isolates (Canada submitted 200 isolates for each of five sites; total 1000) for confirmation of organism identification and reference MIC processing. Nearly 25 comparator agents were tested along with quality control strains, and interpretative criteria from the CLSI, formerly the NCCLS, M100-S15 were applied. No linezolid resistance was detected in strains from 16 monitored countries in 2004. Linezolid remained highly active against Streptococcus pneumoniae, viridans group and beta-haemolytic streptococci (MIC90, 1 mg/L). Against Staphylococcus aureus, linezolid showed 99.5% of results at 0.5-2 mg/L with only one isolate at 4 mg/L. Oxacillin-resistant S. aureus rates varied between nations and ranged from 1.4% in Sweden to 29.5% in the UK to 65.2% in Mexico. Linezolid MIC values were generally one log2 dilution step lower for coagulase-negative staphylococci (CoNS) when compared with S. aureus. No CoNS strains produced a linezolid result at 4 mg/L. Compared with ZAAPS 2002 and 2003 results for enterococci where seven resistant strains were identified, the 2004 data revealed no resistance and 98.1% of linezolid MIC results were at 1 or 2 mg/L. Vancomycin-resistant enterococci (5.3% overall) varied markedly by country including a high of 47.2% in Korea. Linezolid continues to be effective in vitro against Gram-positive pathogens from five continents and no oxazolidinone-resistant strains were identified among the 4098 systemically collected strains (2004) or among 20 158 non-United States isolates for the entire ZAAPS Program (2002-04).

Assessment of Accuracy of Disk Diffusion Tests for the Determination of Antimicrobial Susceptibility of Common Bovine Mastitis Pathogens: A Novel Approach

A novel approach was used to assess disk diffusion accuracy for determination of antibiotic susceptibility of various bovine mastitis pathogens (Escherichia coli, Staphylococcus aureus, Staphylococcus chromogenes, and Streptococcus dysgalactiae). MIC and disk diffusion diameters were compared for 587 bovine mastitis bacterial isolates collected in Israel and 3,186 drug-organism combinations. Results were analyzed by ROC curves, Bayesian statistics, and standard descriptive methods. Low correlation was observed between results of disk diffusion and MIC for S. dysgalactiae and all antimicrobial agents, S. aureus and erythromycin and neomycin, and E. coli and gentamicin, neomycin, and polymyxin B. On a few occasions in which correlation was satisfactory, accepted susceptibility breakpoints to some of the antimicrobial agents resulted in high discrepancies with MIC results and new breakpoints were suggested-e.g., 21 mm for S. aureus susceptibility to penicillin G instead of 29 mm recommended by the National Committee for Clinical Laboratory Standards (NCCLS) and <21 mm, resistant, 21-25 mm, intermediate, and >25 mm, susceptible for susceptibility of E. coli to trimethoprim/sulfamethoxazole. Thus, this approach enabled determination of the most accurate breakpoints that best fitted the specific prevalence of susceptibility in Israel. Thus, we suggest its adoption by microbiology diagnostic laboratories for the provision of accurate antimicrobial susceptibility results when using the disk diffusion test.

Antibiotic Susceptibility Testing of Mycoplasma genitalium by TaqMan 5′ Nuclease Real-Time PCR

Mycoplasma genitalium is an important pathogen in male nongonococcal urethritis (NGU). Isolation of M. genitalium from clinical specimens by axenic culture is very difficult and time-consuming, and very few strains are available for antibiotic susceptibility testing. Primary isolation of M. genitalium by coculture with Vero cells improves the isolation rate significantly. However, some strains cannot be adapted to axenic culture. In this study, we determined the antibiotic susceptibility of M. genitalium strains grown in Vero cell culture with dilutions of antibiotics. Growth of M. genitalium was monitored by a quantitative PCR assay detecting a single-copy region of the mgpB adhesin gene. Growth inhibition in the presence of antibiotics was expressed as a percentage of the DNA load of controls grown in the absence of antibiotics. Eighteen strains were examined, including 6 new strains isolated from urethral swab specimens and 4 new strains isolated from urine specimens collected from Japanese men. Eight strains adapted to axenic culture were also tested by the conventional broth dilution method. The two methods had an acceptable correlation. Azithromycin was the most active drug against M. genitalium. Among the fluoroquinolones, moxifloxacin had the highest activity, with MICs ranging from 0.03 to 0.5 mg/liter, whereas ciprofloxacin and levofloxacin were considerably less active, with MICs ranging from 0.5 to 16 mg/liter and 0.25 to 4 mg/liter, respectively. MICs for tetracycline ranged from 0.125 to 4 mg/liter. This new method could increase the number of M. genitalium strains available for antibiotic susceptibility testing and significantly shorten the time from sampling to MIC results.

Evaluation of PPI-0903M (T91825), a novel cephalosporin: bactericidal activity, effects of modifying in vitro testing parameters and optimization of disc diffusion tests

To evaluate bactericidal activity of PPI-0903M and in vitro testing parameters for this compound. A total of 110 strains were tested to determine MIC and MBC values of PPI-0903M. MIC values were determined by reference broth microdilution whereas MBC was assessed by plating the broth from the MIC well and from those wells above the MIC for each organism onto appropriate drug-free growth media. Seventeen strains were tested by the kill-curve methodology to again evaluate the bactericidal activity of PPI-0903M. Broth microdilution results for 15 strains (in triplicate) were compared with the standard, reference method after modifying several testing parameters. Optimal disc content was assessed by testing seven reference strains with five PPI-0903M disc concentrations (5, 10, 30, 50 and 100 microg). PPI-0903MMBC/MIC ratios were < or = 4 in 90% of strains tested. Kill-curve studies showed >99.9% killing at concentrations < or = 4x MIC within 8 to 24 h against staphylococci and Enterobacteriaceae tested. PPI-0903M MIC results were rarely influenced by the test or medium changes evaluated [high inoculum, low calcium or reduced pH (5.0)]. Human serum had no effect on in vitro antimicrobial activity of PPI-0903M. The 10 microg disc content would be recommended for optimal correlation of MIC breakpoints of < or = 1-4 mg/L. PPI-0903M was generally bactericidal against tested species, and the MIC results were stable across numerous susceptibility test condition changes.

Quality Control and Reference Guidelines for CLSI Broth Microdilution Susceptibility Method (M38-A Document) for Amphotericin B, Itraconazole, Posaconazole, and Voriconazole

Although standard conditions are available for testing the susceptibilities of filamentous fungi to antifungal agents by the Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) broth microdilution assay, quality control (QC) MIC limits have not been established for any mold-agent combination. This multicenter (eight-center) study documented the reproducibility of tests for one isolate of Paecilomyces variotii ATCC MYA-3630 and 11 other mold isolates (three isolates of Aspergillus fumigatus; two isolates of A. terreus; one isolate each of A. flavus, A. nidulans, Fusarium moniliforme, and F. solani; and two isolates of Scedosporium apiospermum) by the CLSI reference broth microdilution method (M 38-A document). Control limits (amphotericin B, 1 to 4 microg/ml; itraconazole, 0.06 to 0.5 microg/ml; posaconazole, 0.03 to 0.25 microg/ml; voriconazole, 0.015 to 0.12 microg/ml) for the selected QC P. variotii ATCC MYA-3630 were established by the analysis of replicate MIC results. Reference isolates and corresponding MIC ranges were also established for 6 of the 12 molds evaluated. MIC limits were not proposed for the other five molds tested due to low testing reproducibility for these isolates.

Dalbavancin activity against selected populations of antimicrobial-resistant Gram-positive pathogens

Dalbavancin, a dimethylaminepropyl amide derivative of the lipoglycopeptide A40926, was tested against 375 antimicrobial-resistant Gram-positive pathogens collected worldwide during 2001–2003. The isolates were tested by reference and Clinical Laboratory Standards Institute broth microdilution susceptibility methods, and dalbavancin was compared with over 20 other antimicrobials. Vancomycin resistance determinants among enterococci were identified using PCR primer sets for vanA and vanB. Dalbavancin was generally more potent than vancomycin or teicoplanin. Dalbavancin was highly active against penicillin- and ceftriaxone-resistant Streptococcus pneumoniae strains (MIC 90, ≤ 0.016 μg/mL). Dalbavancin was also very active against teicoplanin-nonsusceptible coagulase-negative staphylococci (CoNS; MIC range, 0.03–0.25 μg/mL), but dalbavancin MIC results were slightly elevated compared with wild type strains. Dalbavancin inhibited vanB enterococci (MIC range, 0.03–0.12 μg/mL) and was active against other resistant, non- vanA enterococcal species. However, vanA enterococcal strains were not as susceptible to dalbavancin (MIC 50, 16 μg/mL). In summary, dalbavancin was very active against a wide spectrum of resistant Gram-positive isolates and demonstrated greater potency than vancomycin or teicoplanin.

Reevaluation of Enterobacteriaceae MIC/disk diffusion zone diameter regression scattergrams for 9 β-lactams: adjustments of breakpoints for strains producing extended spectrum β-lactamases

Validity of the current susceptibility breakpoint criteria for 9 β-lactam antimicrobials and performance of proposed alternative breakpoints to improve prediction of clinical outcomes were analyzed by testing a contemporary collection of 350 Enterobacteriaceae, enriched for an overrepresentative collection of 70 (20.0%) strains producing extended-spectrum β-lactamases (ESBLs). The majority of the strains were isolated from bloodstream infections (83.7% of the entire collection and 85.7% of the ESBL subset). The 9 β-lactam antimicrobials analyzed were aztreonam, cefepime, cefotaxime, cefotetan, cefoxitin, ceftazidime, ceftriaxone, ceftizoxime, and cefuroxime. Reference broth microdilution MIC results were compared with those zone diameters obtained by the standardized disk diffusion test. The correlation coefficient ( r) was acceptable for all antimicrobials, ranging from 0.87 (cefotetan) to 0.97 (aztreonam, cefotaxime, and ceftazidime). Using the current susceptible breakpoint criteria for Enterobacteriaceae, the intermethod categorical agreement ranged from 90.6% (cefotaxime) to 98.0% (ceftazidime). Very major (false-susceptible by disk test) and major errors (false-resistant) were nil (0.0%) for 6 of the 9 β-lactams. Minor error rates ranged from only 0.9% (cefotetan) to 9.4% (cefotaxime). The proposed MIC breakpoint criteria (generally lower) adjusted to levels to accurately detect ESBL-producing strains and better predict clinical outcomes, also had acceptable intermethod concordance ranging from 90.6% (cefotetan) to 100.0% (ceftazidime). Remarkably, an improvement in the intermethod categorical agreement ranging from +1.7% to +8.3% was observed for 7 of the 9 antimicrobials, including the ESBL index or screening compounds (aztreonam, ceftazidime, cefotaxime, and ceftriaxone). No change in the breakpoint criteria, with removal of the intermediate category was tentatively proposed for cefoxitin and cefuroxime, resulting in an increase of the serious intermethod errors (very major and major), but the absolute intermethod agreement remained highly acceptable at 90.6% to 93.4%. Although the current breakpoint criteria remain acceptable in minimizing intermethod discords, the alternative susceptible breakpoint criteria proposed by combining pharmacokinetic/pharmacodynamic (PK/PD), microbiology MIC population analyses, and clinical success parameters possess improved intermethod agreement for the ESBL screening drugs and 5 other broad-spectrum β-lactam compounds. The Clinical Laboratory Standards Institute (formerly, the National Committee for Clinical Laboratory Standards) should consider these changes to facilitate the detection of all Enterobacteriaceae with low PK/PD target attainment rates, therefore having the potential for suboptimal responses with usual therapeutic dosing.

Comparative Antimicrobial Characterization of LBM415 (NVP PDF-713), a New Peptide Deformylase Inhibitor of Clinical Importance

LBM415 (NVP PDF-713) is the first member of the peptide deformylase (PDF) inhibitor class being developed for clinical trials as a parenteral and oral agent for treatment of community-acquired respiratory tract disease and serious infections caused by antimicrobial-resistant gram-positive cocci. In this study susceptibility testing results from 1,306 recent clinical isolates selected to over-represent resistance trends among the species were summarized. All staphylococci (153 strains; MIC at which 90% of isolates were inhibited [MIC90], 2 microg/ml), Streptococcus pneumoniae (170 strains; MIC90, 1 microg/ml), other streptococci (150 strains; MIC90, 1 microg/ml), enterococci (104 strains; MIC90, 4 microg/ml), Moraxella catarrhalis (103 strains; MIC90, 0.5 microg/ml), and Legionella pneumophila (50 strains; MIC90, 0.12 microg/ml) were inhibited at < or = 8 microg of LBM415/ml, as were 97% of Haemophilus influenzae isolates (300 strains; MIC90, 4 to 8 microg/ml). Among other bacterial groups, 100% of gram-positive and -negative anaerobes, including 22 Bacteroides spp. strains (31 strains total; MIC90, 1 microg/ml), were inhibited by < or = 4 microg/ml, whereas Enterobacteriaceae (112 strains) and most nonfermentative bacilli (107 strains) were not inhibited at readily achievable concentrations. The compound was found to have a dominantly bacteriostatic action, and spontaneous single-step mutational rates occurred at low levels (10(-6) to <10(-8)). Drug interaction studies failed to identify any class-specific synergistic interactions, nor were antagonistic interactions observed. Variations in broth and agar MIC test conditions demonstrated that, whereas the agar-based method trended towards a 1-log2 dilution-higher MIC than the broth method and was inoculum dependent, other variations in incubation environment, medium supplements, pH, or calcium concentration had little influence on LBM415 MIC results. Use of the efflux inhibitor phe-arg-beta-naphthylamide showed an average of 1 log2 dilution decrease in H. influenzae MICs, demonstrating the contribution of efflux pumps in influencing susceptibility to PDF inhibitors. The in vitro activity of LBM415 against targeted bacterial species, including resistant subsets, and other laboratory characteristics of this novel compound demonstrate the potential of PDF inhibitors as a new class of antimicrobial agents.

Reproducibility assessment of tigecycline MIC results by broth microdilution methods using commercially prepared dry-form panels

We assessed the reproducibility of tigecycline and piperacillin/tazobactam MIC results tested by broth microdilution methods on commercially prepared dry-format panels. Fifteen bacterial isolates were tested 3 times daily for 3 days for a total of 9 replicate results per strain. The within-day and between-days reproducibility was 100% for tigecycline at ±1 log 2 dilution step and >98% for piperacillin/tazobactam at ±1 log 2 dilution step. Identical MIC results were observed for 84.4% and 78.7% of tests for same-day replicates for tigecycline and piperacillin/tazobactam, respectively. These results demonstrate the excellent reproducibility of in vitro susceptibility tests for both tigecycline and piperacillin/tazobactam using a commercially prepared dry-form broth microdilution MIC panel.

First Extended-Spectrum-β-Lactamase (CTX-M-15)-Producing Salmonella enterica Serotype Typhimurium Isolate Identified in Lebanon

In Salmonella, most extended-spectrum β-lactamases (ESBLs) are derivatives of TEM and SHV β-lactamase families, although other groups, including PER and CTX-M, have been described recently (1, 2). CTX-M-type ESBLs display levels of resistance to cefotaxime and ceftriaxone significantly higher than those to ceftazidime (2). Since 1995, CTX-M-type ESBLs have disseminated dramatically in several parts of the world (1). In January 2004, a Salmonella enterica serotype Typhimurium isolate, CAM18, was recovered from a stool specimen collected from a 6-year-old child upon admission to a hospital in Northern Lebanon. The child had no history of travel. The isolate had a high resistance level to cefotaxime and ceftazidime and was also resistant to several aminoglycosides, sulfamethoxazole-trimethoprim, and tetracycline. It was susceptible to cefoxitin, imipenem, quinolones, and chloramphenicol. ESBL production was detected by the double-disk synergy test (6). The ESBL phenotype was transferred to Escherichia coli K-12 resistant to nalidixic acid with an efficiency of 10−2 per donors. Certain transconjugants also acquired resistance to tetracycline and kanamycin (TC-pCAM18-1), while others acquired resistance to tetracycline alone (TC-pCAM18-2). The addition of clavulanic acid reduced the MICs of all β-lactams tested (Table ​(Table11). TABLE 1. Characteristics of S. enterica serotype Typhimurium CAM18, E. coli K-12, and two transconjugants Results of isoelectric focusing (8) and of amplifications using TEM, SHV, CTX-M, and OXA-1 primers (3, 10, 11) are shown in Table ​Table1.1. The β-lactamase with a pI of 5.4 was most probably TEM-1. Sequencing of blaOXA-1 amplicons revealed the presence of OXA-30, a β-lactamase with a pI of 7.3 differing from OXA-1 by one amino acid and recently reported in E. coli (4) and S. enterica serotype Typhimurium (5). Sequencing of blaCTX-M amplicons revealed that coding regions were 100% identical to the coding region of the blaCTX-M-15 gene (GenBank accession number {type:entrez-nucleotide,attrs:{text:AY044436,term_id:15636728,term_text:AY044436}}AY044436). Amplification and sequencing of the entire coding sequence of blaCTX-M-15 (14) confirmed that the β-lactamase with a pI of 8.6 was CTX-M-15, an enzyme that has been previously identified by several researchers (for a recent review, see reference 1). Unlike most CTX-M enzymes, CTX-M-15, an Asp-240-Gly variant of CTX-M-3, increased the catalytic efficiency against ceftazidime (12), as observed with MIC results (Table ​(Table11). The presence of ISEcp1, a mobile sequence located upstream of several CTX-M genes, was investigated by a PCR assay as previously described (13). Sequencing of PCR products obtained with isolate CAM18 and transconjugants revealed that the ISEcp1 element was located in the same position as that found in Indian and Turkish isolates (7, 9). Moreover, the 160-bp sequence upstream of blaCTX-M-15 was 100% identical to the corresponding sequences of Indian (GenBank accession number {type:entrez-nucleotide,attrs:{text:AY044436,term_id:15636728,term_text:AY044436}}AY044436) and Canadian (accession number {type:entrez-nucleotide,attrs:{text:AY458016,term_id:38606064,term_text:AY458016}}AY458016) isolates. Kanamycin resistance was associated only with strain TC-pCAM18-1, while tetracycline resistance was exhibited by both transconjugants. This result in addition to plasmid purification revealed that blaTEM and kanamycin resistance genes reside on a plasmid of 20 kb and blaCTX-M-15, blaOXA-30, and tetracycline resistance genes reside on a larger plasmid of 60 kb. We report for the first time the isolation of a CTX-M-type ESBL in Lebanon. In addition, this is also the first report of an ESBL-producing Salmonella in Lebanon. A study done in 2003 at St. George hospital (Beirut, Lebanon) on 49 Salmonella strains showed 100% susceptibility to ceftazidime and cefotaxime (Z. Daoud, personal communication). The appearance of the CTX-M-15-producing S. enterica serotype Typhimurium in the community in 2004 is a serious threat to public health.

Determination of Selectivity and Efficacy of Fatty Acid Synthesis Inhibitors

Type II fatty acid synthesis (FASII) is essential to bacterial cell viability and is a promising target for the development of novel antibiotics. In the past decade, a few inhibitors have been identified for this pathway, but none of them lend themselves to drug development. To find better inhibitors that are potential drug candidates, we developed a high throughput assay that identifies inhibitors simultaneously against multiple targets within the FASII pathway of most bacterial pathogens. We demonstrated that the inverse t(1/2) value of the FASII enzyme-catalyzed reaction gives a measure of FASII activity. The Km values of octanoyl-CoA and lauroyl-CoA were determined to be 1.1 +/- 0.3 and 10 +/- 2.7 microM in Staphylococcus aureus and Bacillus subtilis, respectively. The effects of free metals and reducing agents on enzyme activity showed an inhibition hierarchy of Zn2+ > Ca2+ > Mn2+ > Mg2+; no inhibition was found with beta-mercaptoethanol or dithiothreitol. We used this assay to screen the natural product libraries and isolated an inhibitor, bischloroanthrabenzoxocinone (BABX) with a new structure. BABX showed IC50 values of 11.4 and 35.3 microg/ml in the S. aureus and Escherichia coli FASII assays, respectively, and good antibacterial activities against S. aureus and permeable E. coli strains with minimum inhibitory concentrations ranging from 0.2 to 0.4 microg/ml. Furthermore, the effectiveness, selectivity, and the in vitro and in vivo correlations of BABX as well as other fatty acid inhibitors were elucidated, which will aid in future drug discovery.

A7.10 Therapeutic efficacy of parenteral or oral DX-619, a novel des-fluoro(6)-quinolone, in MRSA infection models

The Tigecycline Evaluation and Surveillance Trial (TEST Program) determined the in vitro activity of tigecycline over a large population of organisms from geographically diverse sites. Tigecycline was compared to amikacin, ampicillin, amoxicillin/clavulanic acid, imipenem, cefepime, ceftazidime, ceftriaxone, levofloxacin, minocycline, piperacillin/tazobactam, linezolid, penicillin, and vancomycin against 3989 commonly encountered clinical Gram-negative and Gram-positive pathogens collected from sites in the United States during 2004. The tigecycline activity was equivalent to imipenem against Enterobacteriaceae. Tigecycline inhibited extended-spectrum β-lactamase and AmpC phenotypes at MIC90 values (minimum inhibitory concentration) of ≤2 μg/mL. In vitro results for tigecycline were similar to other broad-spectrum antimicrobial agents against nonfermenters with MIC90 results of 2 μg/mL against Acinetobacter spp. and >16 μg/mL against Pseudomonas aeruginosa. Tigecycline demonstrated potent activity against Staphylococcus aureus (MIC90, 0.25 μg/mL) and enterococci (MIC90, 0.12 μg/mL) regardless of methicillin or vancomycin susceptibility. Tigecycline MIC values were unaffected by penicillin nonsusceptibility and β-lactamase production among fastidious respiratory pathogens (Streptococcus pneumoniae [MIC90, 0.5 μg/mL] and Haemophilus influenzae [MIC90, 0.25 μg/mL]). Tigecycline offers excellent activity against most of the commonly encountered nosocomial and community-acquired bacterial pathogens.

Evaluation of the BD PHOENIX Automated Microbiology System for Detection of Methicillin Resistance in Coagulase-Negative Staphylococci

The new BD PHOENIX automated microbiology system (Becton Dickinson Diagnostic Systems, Sparks, Md.) is designed for automated rapid antimicrobial susceptibility testing and identification of clinically relevant bacteria. In our study, the accuracy and speed of the BD PHOENIX oxacillin MIC determination for detecting methicillin resistance was evaluated for 200 clinical isolates of coagulase-negative staphylococci (CoNS). Compared to mecA PCR, the BD PHOENIX system detected methicillin resistance with a sensitivity of 99.2%. According to the actual NCCLS oxacillin MIC breakpoint of > or =0.5 microg/ml, the specificity was only 64.9%, attributable to false-positive results in 26 mecA-negative strains, including 16 non-Staphylococcus epidermidis strains. Alternative oxacillin breakpoints of > or =1, > or =2, and > or =4 microg/ml resulted in increased specificities of 83.8, 94.6, and 100% and high sensitivities of 99.2, 99.2, and 96.7%, respectively. Similarly, NCCLS broth microdilution oxacillin MICs exhibited a sensitivity of 100% but a low degree of specificity. However, the previous oxacillin MIC breakpoint of > or =4 microg/ml performed with a sensitivity of 98.4% and a specificity of 98.7%. BD PHOENIX oxacillin MIC results were available after 9 h for 40.5% of the examined CoNS strains and were completed after 17 h. Our results revealed the high reliability of the BD PHOENIX system as a phenotypic method for detection of resistance to oxacillin in mecA-positive CoNS. However, for the improvement of specificity, reevaluation of the optimal oxacillin MIC breakpoint for CoNS appears to be necessary.

Hétérogénéité des milieux gélosés cœur–cervelle (BHI) : effets sur la détermination des concentrations minimales inhibitrices (CMI) à la vancomycine et à la teicoplanine des souches de Staphylococcus aureus

The influence of BHI media commercially available on the results of glycopeptides MIC measured by E-test method was studied on 36 S. aureus isolates (21 MRSA and 15 MSSA). The MIC obtained with the vancomycin and the teicoplanin determined by the E-test method, on the ready prepared BHI plates (AES) and the plate prepared at the laboratory among the four dehydrated bases (AES, Biorad, Oxoïd, and Becton Dickinson), were compared. The mean of the MIC showed variations from 3,14 (Biorad) to 5,25 mg/L (Oxoïd) and from 3,33 (Biorad) to 9,75 mg/L (ready prepared AES) respectively for the vancomycin and for the teicoplanin. A variance analysis (Test de Friedman) showed a significant difference between the five media (p <0,001) with the two antibiotics. The comparison of media 2 by 2 allowed that all combinations excepted one (Biorad vs Becton with the vancomycin) were statistically different (p <0,001). The variation of the MIC observed in relation to the origin of the product of BHI media requires the inclusion of glycopeptide-intermediate S. aureus reference strains to control the prepared culture media.

Commercial broth microdilution panel validation and reproducibility trials for NVP PDF-713 (LBM 415), a novel inhibitor of bacterial peptide deformylase

NVP PDF-713 (LBM 415) is a peptide deformylase inhibitor being progressed into clinical trials. Dry-form broth microdilution panels of NVP PDF-713 were compared to reference MIC panels of 552 recent clinical isolates. Most (99.2%) dry-form MIC results were within +/- 1 log(2) dilution of the reference panel MICs. Of the bacteria tested, Streptococcus pneumoniae and Haemophilus influenzae showed a bias towards higher and lower MICs, respectively. Same-day and between-day reproducibility tests showed that 98.9% and 96.7% of MIC values, respectively, were within +/- 1 log(2) dilution step, thereby demonstrating a high degree of reliability of the dry-form MIC product for clinical studies.

Antibiograms of resistant Gram-negative bacteria from Scottish CF patients

Background: Over a 19-month pilot phase, 93 multiply resistant Gram-negative isolates from Scottish cystic fibrosis patients were sent to a referral laboratory for further investigation. Methods: In common with the referring diagnostic laboratories, disc diffusion testing was carried out. Antibiotic susceptibility testing was also established by MIC methodology. NCCLS methods were used throughout. Twenty antibiotics were tested. Results: Comparing disc diffusion results against MIC results, there were 167 (14%) major errors. By MIC, Pseudomonas aeruginosa ( n=59), Stenotrophomonas maltophilia ( n=16), Burkholderia cepacia ( n=10) and Alcaligenes xylosoxidans ( n=7) were susceptible to 18%, 11%, 4% and 35% of the antibiotics tested, respectively. Colistin and tobramycin were the most active agents against P. aeruginosa with 60% and 49%, respectively, testing susceptible. Minocycline and gentamicin were most active against S. maltophilia with 58% and 18%, respectively, testing susceptible. B. cepacia were most susceptible to co-trimoxazole (10%) and ciprofloxacin (10%). Five and six of the seven A. xylosoxidans isolates were susceptible to piperacillin and imipenem, respectively. Conclusions: Improved methods for susceptibility testing of such clinical isolates need to be employed in routine diagnostic laboratories. Levels of resistance in referred isolates were very high and similar to those described in the USA.

Potency and spectrum reevaluation of cefdinir tested against pathogens causing skin and soft tissue infections: A sample of North American isolates

Cefdinir is a widely used orally administered cephalosporin for community-acquired respiratory tract infections and skin and soft tissue infections (SSTI). A total of 415 nonduplicate isolates of community-acquired SSTI (CA-SSTI) were collected from medical centers in North America and susceptibility tested against cefdinir and various compounds indicated for the treatment of CA-SSTI. The cefdinir MIC 50/90 in μg/mL/% susceptible for strains of the 7 principal CA-SSTI pathogens were: oxacillin-susceptible Staphylococcus aureus (0.5/0.5/100%), oxacillin-susceptible coagulase-negative staphylococci (0.06/0.12/100%), group A streptococci (≤0.03/≤0.03/100%), group B streptococci (≤0.03/0.06/100%), viridans group streptococci (0.25/2/88%), Klebsiella spp. (0.12/1/95%), and Escherichia coli (0.25/0.5/95%). Cefdinir was the most potent oral cephalosporin tested against staphylococci and the Enterobacteriaceae species, and 8-fold to 64-fold more potent than cephalexin against these pathogens. β-Hemolytic streptococci was highly susceptible to cefdinir (MIC 90, ≤ 0.03–0.06 μg/mL), while viridans group streptococci showed slightly elevated MIC results. Cephalexin MIC values for streptococcal strains (MIC 90, 1–32 μg/mL) were 32-fold to 64-fold higher than those of cefdinir or other oral cephalosporins evaluated. Only 0.5% of all 415 recent CA-SSTI pathogens were resistant to cefdinir (MIC, ≥ 4 mg/L). Cefdinir showed a spectrum and potency comparable or superior to other orally administered β-lactams (cephalexin).

Rapid detection of quinolone-resistant Salmonella by real time SNP genotyping

A total of 63 isolates were screened for the gyrA mutation ( 87Asp–Tyr) in Salmonella enterica serovars using real time PCR. All of the isolates were successfully identified as resistant or susceptible, consistent with the MIC result of the agar dilution method and gyrA sequencing.

Broth microdilution susceptibility testing for Leptospira spp.

Leptospirosis in humans has traditionally been treated with penicillin or doxycycline. The choice of therapy offered at the time of initial patient presentation is often empirical, as definitive diagnosis can take weeks. Determining the activity of numerous antimicrobial agents against a wide range of Leptospira serovars may broaden empirical therapeutic options. Various antimicrobials have been shown to be active against a limited number of serovars in in vitro studies, chiefly by the use of broth macrodilution techniques. We developed a broth microdilution technique using the commercially available growth indicator alamarBlue. MICs produced by this technique were compared to MICs and minimal bactericidal concentrations produced by the traditional broth macrodilution technique. The internal validity of our methods was assessed with 11 runs over numerous days with a single isolate of Leptospira interrogans serovar Icterohaemorrhagiae. By either method, the MICs for these internal-validity runs fell within 2 dilutions of each other for more than 90% of antimicrobials. A broader application of these two techniques included 12 serovars (including seven species) of Leptospira and six antimicrobials (penicillin G, doxycycline, chloramphenicol, erythromycin, cefotaxime, and ciprofloxacin). Observed reproducibility fell within 2 dilutions for 99% of the duplicate result sets for the MIC microdilution method, compared to 89% for the MIC macrodilution method. The macrodilution method tended to have a higher MIC at which 90% of the isolates were inhibited (MIC(90)) than did the microdilution method, but the MIC(90)s of both methods were within 2 dilutions of each other for all six drugs. The macrodilution and microdilution techniques produced similar results, with microdilution allowing a faster, more streamlined method of producing MIC results.