Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content

Top–down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content

Mechanisms of Decreased Susceptibility to Cefpodoxime in Escherichia coli

Cefpodoxime is one of five antimicrobial agents recommended by the National Committee for Clinical Laboratory Standards for screening isolates of Klebsiella spp. and Escherichia coli for extended-spectrum β-lactamase (ESBL) production. In a prior study, we noted that among 131 E. coli isolates for which the MIC of at least one extended-spectrum cephalosporin (ESC) or aztreonam was ≥2 μg/ml (suggesting the presence of ESBL production), there were 59 isolates (45.0%) for which the MIC of cefpodoxime was 2 to 4 μg/ml (i.e., a positive ESBL screening test), but the MICs of ceftazidime, cefotaxime, and ceftriaxone were ≤1 μg/ml (below the ESBL screening breakpoint). Thus, the results appeared to be false-positive ESBL screening tests. These 59 isolates were divided into five phenotypic groups based on the susceptibility patterns of the organisms to a variety of β-lactam agents and further characterized. The first group (32 isolates) all produced a TEM-1 β-lactamase, and changes in the major outer membrane proteins were detected in representative strains. The second group (18 isolates) lacked bla(TEM) but showed a number of porin changes; some also showed a modest elevation in production of the AmpC chromosomal β-lactamase. In the third phenotypic group (seven isolates) all expressed an OXA-30 β-lactamase. Some also harbored altered porins. The two remaining phenotypes each had a distinct pattern of porin changes with or without β-lactamase production. These data indicate that several factors are associated with decreased susceptibility to cefpodoxime in E. coli, but none of the mechanisms are related to ESBL production. Current screening methods produced false-positive ESBL results for these isolates. Such isolates should not be classified as containing ESBLs, nor should interpretations of ESCs or aztreonam susceptibility be changed to resistant on test reports for these isolates

2003. In vitro activities of daptomycin, linezolid, and quinupristin-dalfopristin against a challenge panel of staphylococci and enterococci, including vancomycin-intermediate S. aureus and vancomycin-resistant E. faecium. Microb. Drug Resist

ABSTRACT We assessed the in vitro activities of daptomycin, linezolid, and quinupristin-dalfopristin (QD) against a contemporary challenge panel of 88 staphylococcal and 90 enterococcal isolates. The staphylococci selected included vancomycin-intermediate Staphylococcus aureus (VISA), methicillin-resistant S. aureus, and coagulasenegative staphylococci. Enterococcal isolates included vancomycin-resistant Enterococcus faecium (VREF) containing either vanA, vanB1, or vanD. The MICs of daptomycin, linezolid, and QD were determined using commercial broth microdilution panels. All three VISA isolates were susceptible to daptomycin, linezolid, and QD. QD was the most active agent against staphylococcal isolates (MIC 50 # 0.5 mg/ml and MIC 90 5 1 mg/ml), including those with decreased susceptibility to vancomycin. QD was also the most active agent against VREF (MIC 90 # 0.5 mg/ml). No differences were seen for susceptibility of vanA, vanB1, and vanD VREF strains for daptomycin, linezolid, or QD. Daptomycin was the most effective against E. faecalis. On the basis of manufacturer-suggested interpretive criteria, 92% of isolates were susceptible (MIC 90 5 4 mg/ml). All isolates tested were susceptible to at least one antimicrobial agent for which interpretive criteria have been defined. Population analysis of three S. aureus isolates for which the daptomycin MICs were 8 mg/ml showed a pattern of homogeneous resistance. 38

Utility of NCCLS Guidelines for Identifying Extended-Spectrum β-Lactamases in Non-Escherichia coli and Non-Klebsiella spp. of Enterobacteriaceae

NCCLS screening and confirmation methods for detecting extended-spectrum β-lactamases (ESBLs) apply only to Escherichia coli and Klebsiella spp., yet ESBLs have been found in other members of the family Enterobacteriaceae. We evaluated the effectiveness of NCCLS methods for detecting ESBLs in 690 gram-negative isolates of Enterobacteriaceae that excluded E. coli, Klebsiella pneumoniae, and Klebsiella oxytoca. Isolates were collected between January 1996 and June 1999 from 53 U.S. hospitals participating in Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology). The antimicrobial susceptibility patterns of the isolates were determined by using the NCCLS broth microdilution method (BMD), and those isolates for which the MIC of ceftazidime, cefotaxime, ceftriaxone, or aztreonam was ≥2 μg/ml or the MIC of cefpodoxime was ≥8 μg/ml (positive ESBL screen test) were further tested for a clavulanic acid (CA) effect by BMD and the disk diffusion method (confirmation tests). Although 355 (51.4%) of the isolates were ESBL screen test positive, only 15 (2.2%) showed a CA effect. Since 3 of the 15 isolates were already highly resistant to the five NCCLS indicator drugs, ESBL detection would have an impact on the reporting of only 1.7% of the isolates in the study. Only 6 of the 15 isolates that showed a CA effect contained a bla(TEM), bla(SHV), bla(CTX-M), or bla(OXA) β-lactamase gene as determined by PCR (with a corresponding isoelectric focusing pattern). Extension of the NCCLS guidelines for ESBL detection to Enterobacteriaceae other than E. coli and Klebsiella spp. does not appear to be warranted in the United States at present, since the test has poor specificity for this population and would result in changes in categorical interpretations for only 1.7% of Enterobacteriaceae tested

Testing for Induction of Clindamycin Resistance in Erythromycin-Resistant Isolates of Staphylococcus aureus

Disk diffusion and broth microdilution (BMD) were used to perform clindamycin (CLI) induction testing on 128 selected nonduplicate isolates of Staphylococcus aureus. Disk diffusion testing involved placing CLI and erythromycin (ERY) disks approximately 12 mm apart (measured edge to edge) on a Mueller-Hinton agar plate that had been inoculated with an S. aureus isolate; the plate was then incubated for 16 to 18 h. Two distinct induction phenotypes (labeled D and D(+)) and four noninduction phenotypes (designated as negative [Neg], hazy D zone [HD], resistant [R], and susceptible [S]) were observed in disk diffusion results. A clear, D-shaped zone of inhibition around the CLI disk was designated as the D phenotype and was observed for 21 isolates while a D-shaped zone containing inner colonies growing up to the CLI disk was designated as D(+) (17 isolates). In addition, 10 isolates were CLI susceptible and ERY resistant but were not inducible and showed no blunting of the CLI zone (Neg phenotype). Isolates that were CLI and ERY resistant (constitutive macrolide-lincosamide-streptogramin B resistance) demonstrated either a double zone of inhibition with an inner ring of reduced growth up to the edge of the disks (HD phenotype; 33 isolates) or solid growth around the CLI and ERY disks (R phenotype; 16 isolates). Finally, 31 isolates were susceptible by disk testing to both CLI and ERY (S phenotype). PCR results showed that isolates with a D phenotype harbored ermA, isolates with a D(+) phenotype contained either ermC (16 isolates) or ermA and ermC (one isolate), and all 10 isolates with a Neg phenotype contained msrA. All isolates with an HD or R phenotype harbored at least one erm gene. Isolates showing the D(+) phenotype by disk diffusion were also detected by BMD using a variety of CLI and ERY concentrations; however, isolates with the D phenotype were more difficult to detect by BMD and will likely require optimization of ERY and CLI concentrations in multilaboratory studies to ensure adequate sensitivity. Thus, at present, disk diffusion is the preferred method for testing S. aureus isolates for inducible CLI resistance

Evaluation of the NCCLS Extended-Spectrum β-Lactamase Confirmation Methods for Escherichia coli with Isolates Collected during Project ICARE

To determine whether confirmatory tests for extended-spectrum β-lactamase (ESBL) production in Escherichia coli are necessary, we selected 131 E. coli isolates that met the National Committee for Clinical Laboratory Standards (NCCLS) screening criteria for potential ESBL production from the Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) strain collection. For all 131 isolates, the broth microdilution (BMD) MIC of at least one extended-spectrum cephalosporin was ≥2 μg/ml. For 21 of 131 (16%) isolates, the ESBL confirmatory test was positive; i.e., the BMD MICs of ceftazidime or cefotaxime decreased by ≥3 doubling dilutions in the presence of clavulanic acid (CA) or the disk diffusion zone diameters increased by ≥5 mm around ceftazidime or cefotaxime disks in the presence of CA. All 21 isolates were shown by PCR to contain at least one of the genes bla(TEM), bla(SHV), and bla(OXA), and in isoelectric focusing (IEF) tests, all isolates demonstrated at least one β-lactamase band consistent with a TEM, SHV, or OXA enzyme. Of the 21 isolates, 3 showed a CA effect for cefotaxime by BMD but not by disk diffusion testing. A total of 59 (45%) of the 131 isolates demonstrated decreased susceptibility to cefpodoxime alone (MIC = 2 to 4 μg/ml), and none had a positive ESBL confirmatory test result. These were classified as false positives according to ESBL screen test results. For the remaining 51 (39%) isolates, the cefpodoxime MICs ranged from 16 to >128 μg/ml and the MICs for the other extended-spectrum cephalosporins were highly variable. All 51 isolates gave negative ESBL confirmatory test results. Most showed IEF profiles consistent with production of both a TEM and an AmpC β-lactamase, and representative isolates of several phenotypic groups showed changes in porin profiles; these 51 isolates were considered true negatives. In all, only 16% of 131 E. coli isolates identified as potential ESBL producers by the current NCCLS screening criteria were confirmed as ESBL producers. Thus, changing the interpretation of extended-spectrum cephalosporins and aztreonam results from the susceptible to the resistant category without confirming the presence of an ESBL phenotype would lead to a large percentage of false resistance results and is not recommended. However, by increasing the cefpodoxime MIC screening breakpoint to ≥8 μg/ml, 45% of the false-positive results could be eliminated. NCCLS has incorporated this change in the cefpodoxime screening breakpoint in its recent documents

Identification of Some Charcoal-Black-Pigmented CDC Fermentative Coryneform Group 4 Isolates as Rothia dentocariosa and Some as Corynebacterium aurimucosum: Proposal of Rothia dentocariosa emend. Georg and Brown 1967, Corynebacterium aurimucosum emend. Yassin et al. 2002, and Corynebacterium nigricans Shukla et al. 2003 pro synon. Corynebacterium aurimucosum

Sixty-three clinical isolates of charcoal-black-pigmented, gram-positive coryneform rods were received for identification by the Centers for Disease Control and Prevention (CDC) and were provisionally designated CDC fermentative coryneform group 4 (FCG4). Forty-five of these were characterized by morphological, physiologic, antimicrobial susceptibility, cellular fatty acids, 16S rRNA gene sequencing, and DNA-DNA hybridization analyses. Nitrate reduction, cellular fatty acid analysis, 16S rRNA gene sequencing, and DNA-DNA hybridization studies segregated these strains into two groups: FCG4a (8 strains) and FCG4b (37 strains). The FCG4a strains, only one of which was from a female genitourinary source, produced cellular fatty acid and biochemical profiles similar to those observed with reference strains of Rothia dentocariosa and Rothia mucilaginosa, while the FCG4b strains were similar to Corynebacterium species. DNA-DNA hybridization analysis demonstrated species-level relatedness among six FCG4a tested strains and showed that they were a charcoal-black-pigmented variant of R. dentocariosa. Sixteen isolates of the FCG4b group, mainly from female genitourinary tract specimens, as well as the type strains of two recently named species, Corynebacterium aurimucosum and Corynebacterium nigricans, were shown by DNA-DNA hybridization analysis and the sequencing of the 16S rRNA gene to be related at the species level and unrelated to the type strain of R. dentocariosa; therefore, the Corynebacterium-like strains were classified as a charcoal-black-pigmented variant of C. aurimucosum, because this name has nomenclatural priority over C. nigricans. These findings indicate that FCG4 represents a heterogeneous group that contains pigmented variants of both R. dentocariosa and C. aurimucosum; hence, the descriptions of both R. dentocariosa and C. aurimucosum have been amended to include charcoal-black-pigmented variants, and C. nigricans is a pro synonym of C. aurimucosum