Search PubMed⌕ Search

Biomedical subjects

D F Sahm

Publications and source records attributed to D F Sahm.

At least 55 records · Page 3Linked to original sources
PubMed1999

VanE, a new type of acquired glycopeptide resistance in Enterococcus faecalis BM4405.

Enterococcus faecalis BM4405 was resistant to low levels of vancomycin (MIC, 16 microg/ml) and was susceptible to teicoplanin (MIC, 0.5 microg/ml). No PCR product was obtained when the total DNA of this clinical isolate was used as a template with primers specific for glycopeptide resistance genes vanA, vanB, vanC, and vanD. However, a 604-bp PCR fragment was obtained when V1 and V2 degenerate primers were used and total DNA was digested with HindIII as a template. The product was cloned and sequenced. The deduced amino acid sequence had greater identity (55%) with VanC than with VanA (45%), VanB (43%), or VanD (44%). This was consistent with the fact that BM4405 synthesized peptidoglycan precursors that terminated in D-serine residues. After induction with vancomycin, weak D,D-dipeptidase and penicillin-insensitive D,D-carboxypeptidase activities were detected in cytoplasmic extracts of BM4405, whereas a serine racemase activity was found in the membrane preparation. This new type of acquired glycopeptide resistance was named VanE.

Amino Acid Sequence↗
PubMed1998

In vitro susceptibility and molecular analysis of gentamicin-resistant enterococci.

Enterococci with gentamicin MICs of 256 to 1,024 micrograms/mL were evaluated for susceptibility to ampicillin plus gentamicin synergism. Sixteen of eighteen enterococcal isolates were not susceptible to synergistic killing by ampicillin plus gentamicin; 11 possessed aac(6')-aph(2"), and 4 possessed aph(2")-Ic. A gentamicin MIC of 512 or 1,024 micrograms/mL predicted lack of ampicillin/gentamicin synergism, but a gentamicin MIC of 256 micrograms/mL did not. For six enterococcal strains possessing the gentamicin-resistance gene aph(2")-Ic, ampicillin plus dibekacin, ampicillin plus netilmicin, and ampicillin plus amikacin produced synergistic killing in five, three, and two strains, respectively.

Amikacin↗
PubMed1998

A new high-level gentamicin resistance gene, aph(2'')-Id, in Enterococcus spp.

Enterococcus casseliflavus UC73 is a clinical blood isolate with high-level resistance to gentamicin. DNA preparations from UC73 failed to hybridize with intragenic probes for aac(6')-Ie-aph(2'')-Ia and aph(2'')-Ic. A 4-kb fragment from UC73 was cloned and found to confer resistance to gentamicin in Escherichia coli DH5alpha transformants. Nucleotide sequence analysis revealed the presence of a 906-bp open reading frame whose deduced amino acid sequence had a region with homology to the aminoglycoside-modifying enzyme APH(2'')-Ic and to the C-terminal domain of the bifunctional enzyme AAC(6')-APH(2''). The gene is designated aph(2'')-Id, and its observed phosphotransferase activity is designated APH(2'')-Id. A PCR-generated intragenic probe hybridized to the genomic DNA from 17 of 118 enterococcal clinical isolates (108 with high-level gentamicin resistance) from five hospitals. All 17 were vancomycin-resistant Enterococcus faecium isolates, and pulsed-field typing revealed three distinct clones. The combination of ampicillin plus either amikacin or neomycin exhibited synergistic killing against E. casseliflavus UC73. Screening and interpretation of high-level aminoglycoside resistance in enterococci may need to be modified to include detection of APH(2'')-Id.

Anti-Bacterial Agents↗
PubMed1998

Ceftazidime-resistant Klebsiella pneumoniae and Escherichia coli isolates producing TEM-10 and TEM-43 beta-lactamases from St. Louis, Missouri.

Ceftazidime-resistant Escherichia coli and Klebsiella pneumoniae (49 and 102 isolates, respectively) were collected from Barnes-Jewish Hospital, St. Louis, Mo., from 1992 to 1996. They were uniformly resistant to ceftazidime, generally resistant to aztreonam, and variably susceptible to cefotaxime. Four representative E. coli strains and 15 Klebsiella strains were examined. From one to four beta-lactamases were produced per strain, with three possible enzymes related to ceftazidime resistance: enzymes with pI values of 5.6, 6.1, or 7.6. By pulsed-field gel electrophoresis there were at least 13 different Klebsiella strain types and 3 different E. coli strain types, indicating that the outbreak was not clonal. After cloning and sequencing of the beta-lactamase-encoding genes, the enzyme with a pI of 5.6 was identified as TEM-10. The enzyme with a pI of 6.1 was a novel TEM variant (TEM-43) with Lys at 104, His at 164, and Thr at 182. TEM-43 showed broad-spectrum hydrolytic activity against all penicillins, with the highest hydrolysis rate for ceftazidime compared to those for the other expanded-spectrum cephalosporins. Aztreonam was also a good substrate for TEM-43, with hydrolytic activity similar to that of ceftazidime and affinity higher than that of ceftazidime. The TEM-43 beta-lactamase was well inhibited by clavulanic acid and tazobactam at concentrations of < 10 nM. Sulbactam was less effective than the other inhibitors. The Thr182 mutation previously reported in an inhibitor-resistant beta-lactamase did not cause the TEM-43 enzyme to become resistant to any of the inhibitors.

Ceftazidime↗
PubMed1997

Surveillance for the emergence and dissemination of antimicrobial resistance in bacteria.

Effective surveillance of antimicrobial-resistant bacteria is important for developing rational empiric therapy guidelines and for guiding public health efforts to control and prevent the spread of infective agents. Surveillance must include a timely and thorough review of the test results generated in clinical microbiology laboratories because this data serves as the core of surveillance activities. Besides ensuring data accuracy and optimizing detection of emerging resistance, the role of clinical microbiology also includes supporting the production of informative surveillance reports, providing laboratory resources for outbreak investigations, and monitoring the performance of commonly used susceptibility testing methods. Once the accuracy of susceptibility results has been validated, the data are used by public health agencies and professional societies to monitor resistance trends on a local, state, national, and international level. This information is also used to develop policies for prudent antimicrobial use locally and nationally.

Anti-Bacterial Agents↗
PubMed1997

Heterogeneous expression of glycopeptide resistance in enterococci associated with transfer of vanB.

In mating experiments using a clinical strain that constitutively expresses vanB-encoded glycopeptide resistance, resistance transfer was detectable at a frequency of <10(-7) transconjugants/donor. Vancomycin MICs for transconjugants were 2- to 10-fold lower than those for the donor; both inducibly and constitutively resistant transconjugants were obtained. These findings demonstrate that the transfer of vanB among enterococci can be associated with substantial alterations in the level and control of glycopeptide resistance expression.

Anti-Bacterial Agents↗
PubMed1997

Rapid characterization schemes for surveillance isolates of vancomycin-resistant enterococci.

Surveillance cultures for vancomycin-resistant enterococci (VRE) and subsequent characterization of the isolates can be extremely burdensome and difficult. Therefore, efficient and reliable schemes for the characterization of surveillance isolates are needed. In this study, a commercial agar (bile esculin azide agar with 6 microg of vancomycin per ml [BEAA]; Remel, Lenexa, Kans.) was used in the initial screening step to establish relatively rapid (i.e., in < or = 24 h from the time of isolation) phenotype-based and PCR-based schemes for the detection and characterization of VRE. The phenotype-based scheme included Gram staining of growth on BEAA and subculture of cocci on sheep blood agar plates for vancomycin disk diffusion and pyrazinamidase (PYR) testing. For the PCR scheme, inocula for van gene detection were taken directly from the BEAA plates. The phenotypic approach was applied to 378 surveillance cultures that yielded growth on BEAA. Gram staining quickly eliminated gram-positive bacilli from further testing, and a negative PYR test classified 25 additional isolates as probable pediococci. A positive PYR test reliably identified 121 single-patient VRE isolates that included 83 Enterococcus faecium, 33 E. gallinarum, and 5 E. casseliflavus strains. The vancomycin inhibition zone size clearly distinguished VanA and VanB strains from VanC strains within 24 h of BEAA isolation. All VanA and VanB strains failed to produce zones of >6 mm, while only one VanC strain produced a zone of < 15 mm. Challenging this phenotypic scheme with 47 stock VRE isolates produced similar findings. In direct PCR analyses, false-negative vanA and vanB results occurred with 12% of the specimens. Many of the false-negative reactions also failed to produce an internal control product, which underscores the need for including control primers when a PCR scheme is used. In summary, the phenotype- and the PCR-based schemes provide efficient methods for characterizing VRE within 24 h of isolation.

Anti-Bacterial Agents↗
PubMed1996

Nosocomial outbreak of gastroenteritis due to Salmonella senftenberg.

We describe a prolonged nosocomial outbreak of Salmonella senftenberg, an uncommon human pathogen. We detected 22 cases of infection due to S. senftenberg that occurred from March 1993 through November 1994 and involved 18 patients and four healthy employees. All infected persons had consumed food prepared by the hospital kitchen. The estimated attack rate for the period of the outbreak was 0.19-0.23 cases per 100,000 meals served. Infection control interventions included observation of food preparation, disinfection of kitchen devices, and education of food handlers. The consumption of lettuce (11 of 15 patients who could recount extended dietary histories vs. 4 of 20 controls; P = .005), cauliflower (5 of 15 vs. 0/20; P = .02), cottage cheese (4 of 15 vs. 0/20; P = .03), and deli turkey (8 of 15 vs. 0/20; P < .001) was associated with S. senftenberg infection. The isolates had identical antibiograms and pulsed-field gel electrophoretic patterns. Cultures of stool samples from food handlers as well as food items, kitchen devices, and kitchen surroundings were negative for S. senftenberg. Interruption of the outbreak occurred coincidentally with the institution of infection control measures. This prolonged outbreak of salmonellosis was probably related to contamination in the kitchen from turkey, with cross-contamination via equipment.

Adult↗
PubMed1996

Factors influencing the vitek gram-positive susceptibility system's detection of vanB-encoded vancomycin resistance among enterococci.

Studies were conducted to identify factors contributing to the inability of the Vitek Gram-Positive Susceptibility system (GPS; bioMerieux, Vitek, Inc., Hazelwood, Mo.) to reliably detect vanB-mediated vancomycin resistance among enterococci. To some extent the accuracy of the GPS depended on a particular strain's level of resistance, as all isolates for which vancomycin MICs were > or = 128 mu g/ml were readily detected but detection of resistance expressed by several strains for which MICs were < or = 64 mu g/ml was sporadic. Factors besides the level of resistance were studied in two vanB strains. For one strain (Enterococcus faecium U8304), the ability of GPS to detect resistance was accurate and consistent, while for the other (Enterococcus faecalis V583), GPS results were inconsistent and unreliable. Using these isolates, we established that growth medium had the most notable effect on the detection of resistance. In the absence of vancomycin, Vitek GPS broth supported growth comparable to that obtained with brain heart infusion broth for both E. faecium U8304 and E. faecalis V583. However, in the presence of vancomycin the growth patterns changed dramatically so that neither VanB strain grew well in Vitek broth, and growth of V583 was barely detectable after 8 h of incubation. In contrast, good growth of both strains was observed in brain heart infusion broth supplemented with vancomycin. Additionally, the same medium effect was observed with other inducibly resistant VanB strains. In conclusion, although Vitek broth can support good enterococcal growth, this medium does not sufficiently support expression of vancomycin resistance by certain strains to allow them to be detected by the Vitek automated system. Furthermore, this observation establishes that the type of growth medium used can substantially influence the expression of vancomycin resistance and indicates that medium-based strategies should be explored for the enhancement of resistance detection among commercial systems.

Anti-Bacterial Agents↗
PubMed1995

In vitro activities of various beta-lactam antimicrobial agents against clinical isolates of Escherichia coli and Klebsiella spp. resistant to oxyimino cephalosporins.

Broth microdilution testing was used to study the activity of several beta-lactam antimicrobial agents, including piperacillin-tazobactam and cefepime, against 108 clinically derived Escherichia coli and Klebsiella sp. strains resistant to oxyimino cephalosporins (i.e., putative extended-spectrum beta-lactamase producers). On the basis of the percentage of susceptible strains, imipenem (100%), cefotetan (> or = 92%), and piperacillin-tazobactam (> or = 86%) were the most active agents. Cefepime activity (52 to 64% susceptible) was comparable to that of cefotaxime (40 to 63% susceptible) and aztreonam (20 to 63% susceptible). Among all beta-lactams tested, imipenem and cefotetan demonstrated the highest and most consistent level of activity and were the least affected by challenges with increased sizes of inocula of these resistant organisms.

Anti-Bacterial Agents↗
PubMed1995

Inducible and constitutive expression of vanC-1-encoded resistance to vancomycin in Enterococcus gallinarum.

Clinical isolates Enterococcus gallinarum AIB39 and E. gallinarum GS1 were studied to establish whether the expression of vanC-1-mediated resistance may be inducible or constitutive. By growth curve analysis, strain AIB39 exhibited the same lag period (i.e., 1 to 1.5 h) whether it was subcultured to unsupplemented brain heart infusion broth or broth containing 6 micrograms of vancomycin per ml, a growth pattern typical of constitutively expressed resistance. Use of high-performance liquid chromatography (HPLC) to separate peptidoglycan precursor extracts substantiated this finding because the serine-terminating pentapeptide precursor UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ser was produced in the presence and absence of vancomycin, whereas no UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala was detected. In contrast, results with strain GS1 were consistent with inducible expression. GS1 demonstrated a lag time that was 3 to 4 h longer when it was subcultured to vancomycin-containing broth than when it was subcultured in unsupplemented broth. HPLC analysis showed that in the absence of vancomycin only UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala was detected, but in the presence of drug only UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala was found. Inducible expression of vanC-1-mediated resistance in E. gallinarum is consistent with recent findings suggesting the presence of at least two ligases in this species. Although vanC-1 may be intrinsic to E. gallinarum, our findings raise doubt regarding the natural mechanism of this gene's expression.

Amino Acid Sequence↗
PubMed1995

Multilaboratory evaluation of screening methods for detection of high-level aminoglycoside resistance in enterococci. National Committee for Clinical Laboratory Standards Study Group on Enterococci.

Since the early 1970s, the synergistic activity of an aminoglycoside with a cell wall-active agent has been predicted by determining the ability of an enterococcus to grow in the presence of high levels of the aminoglycoside (usually > or = 2,000 micrograms/ml). However, a variety of media and concentrations of aminoglycosides has been used for this screening procedure. In the present study, we sought to optimize the agar dilution, broth microdilution, and disk diffusion tests used to detect high-level gentamicin and streptomycin resistance in enterococci. For dilution tests, brain heart infusion agar or broth gave the best growth and performance. For agar dilution, 500 micrograms of gentamicin per ml, 2,000 micrograms of streptomycin per ml, and an inoculum of 1 x 10(6) CFU/ml were optimal, while for broth microdilution, 500 micrograms of gentamicin per ml, 1,000 micrograms of streptomycin per ml, and an inoculum of 5 x 10(5) CFU/ml were best. Growth of more than one colony in the agar dilution test was determined to be the best indicator of high-level resistance. For disk diffusion, Mueller-Hinton agar, 120-micrograms gentamicin disks, and 300-micrograms streptomycin disks with breakpoints of no zone for resistance and > or = 10 mm for susceptibility gave the best sensitivity and specificity if results for strains with zones of 7 to 9 mm are considered inconclusive, indicating that a broth or agar test should be performed to determine susceptibility or resistance.

Anti-Bacterial Agents↗
PubMed1995

Molecular characterization and multilaboratory evaluation of Enterococcus faecalis ATCC 51299 for quality control of screening tests for vancomycin and high-level aminoglycoside resistance in enterococci.

Studies were conducted to validate the use of Enterococcus faecalis ATCC 51299 (which is vancomycin resistant and resistant to high levels of gentamicin and streptomycin) and E. faecalis ATCC 29212 (which is susceptible to vancomycin and against which gentamicin or streptomycin and cell wall-active agents have synergistic kill activity) as controls in an agar screening test for vancomycin resistance and high-level streptomycin and gentamicin resistance and a broth microdilution screening test for high-level streptomycin and gentamicin resistance. Both organisms performed as expected in these tests and will serve as appropriate controls. However, E. faecalis ATCC 29212 was occasionally noted to produce light growth on the vancomycin screening plate with certain lots of agar. Quality control ranges for disk diffusion tests with disks with large amounts of streptomycin (300 micrograms) and gentamicin (120 micrograms) were established for E. faecalis ATCC 29212; zone limits are 16 to 22 mm for gentamicin and 14 to 19 mm for streptomycin. No zones for inhibition were seen when E. faecalis ATCC 51299 was tested with these high-content disks.

Aminoglycosides↗
PubMed1995

Investigation of the reformulated Remel Synergy Quad plate for detection of high-level aminoglycoside and vancomycin resistance among enterococci.

We investigated the accuracy of the recently released Remel Synergy Quad plate, a commercially available agar screening method for detecting high-level aminoglycoside and vancomycin resistance among enterococci that is based on the National Committee for Clinical Laboratory Standards recommended guidelines (National Committee for Clinical Laboratory Standards, M7-A3, 1993). The Synergy Quad correctly determined the gentamicin and streptomycin resistance status for > or = 97% of 147 Enterococcus faecalis and Enterococcus faecium isolates tested. Detection of vancomycin resistance also was reliable, as no false susceptibility occurred with 36 vancomycin-resistant E. faecalis and E. faecium strains and false resistance occurred only once with the 47 susceptible strains tested. One strain each of Enterococcus gallinarum and Enterococcus casseliflavus failed to grow on the screen, but because the true nature and significance of resistance in such isolates is unknown the implication of their screen negativity is uncertain. In summary, the Remel Synergy Quad provides a highly accurate and convenient method for susceptibility testing of enterococci against gentamicin, streptomycin, and vancomycin.

Aminoglycosides↗
PubMed1995

High-level gentamicin resistance among enterococci.

The bi-functional enzyme 6'-aminoglycoside acetyltransferase (AAC-6')-2" aminoglycoside phosphotransferase (APH-2"), which is encoded by the 2 kb fused gene aacA-aphD, mediates gentamicin resistance in Enterococcus faecalis. This gene is associated with the composite transposon Tn5281 that shares extensive homology with transposons Tn4001 and Tn4031 found in staphylococci. Typically these transposons are characterized by the resistance gene being flanked by 1.35 kb insertion sequences (IS256). The resistance gene (aacA-aphD) appears identical among all E. faecalis studied, but restriction enzyme analysis indicates notable heterogeneity within the flanking regions. Various combinations of IS256-like and IS257-like elements, or portions thereof, have been described in different resistant isolates. As gentamicin resistance has been described in other enterococcal species (E. faecium, E. avium, E. gallinarum, E. raffinosus, E. casseliflavus) homology with the resistance gene of E. faecalis has largely been maintained, but more extensive variations in the flanking sequences have been noted. Although many similarities exist among enterococcal gentamicin resistance determinants, heterogeneity in the flanking regions indicates that measurable divergence exists among these determinants. This divergence may be of value in characterizing the mechanisms and natural course of gentamicin resistance dissemination which now includes staphylococci, enterococci and streptococci.

Anti-Bacterial Agents↗
PubMed1994

Detection and surveillance of antimicrobial resistance.

The clinical microbiology laboratory is strategically positioned to recognize changing patterns in bacterial resistance to antimicrobials. This requires the application of accurate testing methods and a methodological survey of drug-resistance patterns among clinically important bacteria. This information can be assembled into comprehensive international databases, using a common format to facilitate monitoring.

Drug Resistance, Microbial↗