Biomedical subjects

Dietrich Mack

Publications and source records attributed to Dietrich Mack.

30 records · Page 2Linked to original sources

PubMed2003

Extended-spectrum beta-lactamases: implications for the clinical microbiology laboratory, therapy, and infection control.

Extended-spectrum beta-lactamase (ESBL) producing gram-negative bacilli are a growing concern in human medicine today. When producing these enzymes, organisms (mostly K. pneumoniae and E. coli) become highly efficient at inactivating the newer third-generation cephaloporins (such as cefotaxime, ceftazidime, and ceftriaxone). In addition, ESBL-producing bacteria are frequently resistant to many classes of non-beta-lactam antibiotics, resulting in difficult-to-treat infections. This review gives an introduction into the topic and is focused on various aspects of ESBLs; it covers the current epidemiology, the problems of ESBL detection and the clinical relevance of infections caused by ESBL-producing organisms. Therapeutic options and potential strategies for dealing with this growing problem are also discussed in this article.

Gram-Negative Bacteria↗

PubMed2003

Comparison of BDPhoenix and VITEK2 automated antimicrobial susceptibility test systems for extended-spectrum beta-lactamase detection in Escherichia coli and Klebsiella species clinical isolates.

The present study compares the ability to detect extended-spectrum beta-lactamases (ESBL) among a collection of 34 ESBL producing clinical isolates belonging to Escherichia coli and Klebsiella species with two new rapid susceptibility and identification instruments-VITEK2 (bioMérieux, Marcy l'Etoile, France) vs. BDPhoenix (BD Biosciences, Sparks, MD). ESBL content in these isolates was previously characterized on the basis of PCR amplification and sequencing results which were used as the reference method in our evaluation. BDPhoenix correctly determined the ESBL outcome for all strains tested (100% detection rate), whereas VITEK2 was not able to detect the ESBL status in 5 isolates (85% detection rate). Detailed analysis revealed that the discrepancies were mainly observed with 'difficult-to-detect' strains. Misidentification was either due to low oximino cephalosporin MIC in these strains or was associated with pronounced 'cefotaximase' or 'ceftazidimase' phenotypes. Klebsiella oxytoca chromosomal beta-lactamase (K1) is phenotypically quite similar to ESBL enzymes. In order to evaluate whether the K1 and ESBL enzymes could be discriminated, we expanded our analysis by 8 clinical K. oxytoca strains with K1 phenotypes. VITEK2 gave excellent identification of these strains whereas 7 out of 8 were falsely labeled ESBL-positive by the BDPhoenix system.

Anti-Bacterial Agents↗

PubMed2003

Establishment of an arbitrary PCR for rapid identification of Tn917 insertion sites in Staphylococcus epidermidis: characterization of biofilm-negative and nonmucoid mutants.

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.

Bacterial Adhesion↗

PubMed2003

Glucose-related dissociation between icaADBC transcription and biofilm expression by Staphylococcus epidermidis: evidence for an additional factor required for polysaccharide intercellular adhesin synthesis.

Biofilm formation in Staphylococcus epidermidis depends, in the majority of the strains, on the activity of the icaADBC locus. The expression of the operon that encodes the synthetic enzymes of the intercellular polysaccharide adhesin (PIA) depends on a variety of exogenic environmental conditions and is, at least in part, regulated by the alternative sigma factor sigma(B). We investigated the transcriptional regulation of the ica operon and the respective phenotypes expressed under growth conditions differing in the content of glucose in the growth medium. In the presence of glucose, S. epidermidis exhibited a PIA- and biofilm-positive phenotype whereas ica transcription was down-regulated in the postexponential and stationary phases of growth. Surprisingly, maximum transcription of ica was detectable in the stationary phase of growth in the absence of glucose despite the expression of a PIA- and biofilm-negative phenotype. In vitro enzymatic assays and phenotypic characterization showed that the abundant amount of ica mRNA was functionally active because induction of stationary-phase cells with glucose led to immediate PIA synthesis. Induction of biofilm formation could be completely inhibited by chloramphenicol, which, given at a later stage of biofilm accumulation, also inhibited further development of preformed biofilm, indicating that continuous translation of an additional, icaADBC-independent factor is required for the expression of a biofilm-positive phenotype.

Adhesins, Bacterial↗

PubMed2002

Evaluation of different detection methods of biofilm formation in Staphylococcus aureus.

The icaADBC gene locus of Staphylococcus aureus and its polysaccharide intercellular adhesin (PIA/PNSG) were recently identified, but biofilm formation has rarely been detected in vitro. In this study we evaluated a tissue culture plate (TCP) assay and a tube test, as well as Congo red agar, using the two basic media trypticase soy broth (TSB) and brain heart infusion (BHI) broth with different sugar supplements for detection of biofilm formation in 128 ica-positive S. aureus isolates. Of the S. aureus strains, 57.1% displayed a biofilm-positive phenotype under optimized conditions in the TCP test. The tube test correlated well with the TCP test for strongly biofilm-producing strains, whereas weak producers were not safely discriminated from biofilm-negative strains. Screening on Congo red agar displayed a strong correlation with the TCP and the tube test for only 3.8%, and is therefore not recommended for investigation of biofilm formation in S. aureus.

Agar↗

PubMed2002

Minimal attachment killing (MAK): a versatile method for susceptibility testing of attached biofilm-positive and -negative Staphylococcus epidermidis.

Due to its ability to attach to polymeric surfaces Staphylococcus epidermidis is a common pathogen in chronic, medical device-associated infections. Attached S. epidermidis displays reduced susceptibility against a variety of antimicrobial substances, and little correlation between standard susceptibility test results and clinical outcome of antibiotic treatment is observed. In this study we established a new, versatile, and easy method of antimicrobial susceptibility testing for attached Staphylococcus epidermidis, suitable for both biofilm-negative and biofilm-positive attached bacteria using readily available equipment. For three biofilm-positive wild-type strains and their biofilm-negative mutants minimal attachment killing concentrations (MAK) of penicillin, oxacillin, vancomycin, and gentamicin were determined. Depending on strain and investigated antibiotics, a heterogeneous MAK (MAK(hetero)) could be differentiated from a homogeneous resistance (MAK(homo)), favoring a model of few persisters within attached cells under antibiotic treatment. For the biofilm-negative mutants, a lower MAK(homo) was detected than for the corresponding wild types for some of the tested antibiotics, which probably resulted from higher bacterial inocula of wild-type strains, whereas the MAK(hetero) were comparable for mutants and wild types for most of the tested antibiotics and strains. These data indicate that biofilm formation is not a necessary prerequisite for persistence of attached S. epidermidis cells under antibiotic treatment, which could explain therapeutic failure in foreign body-associated infections due to biofilm-negative S. epidermidis isolates. The highly individual resistance phenotypes of the investigated strains with different antibiotics suggests that MAK determination could help to predict the therapeutic outcome of foreign body-associated infections with both biofilm-positive and biofilm-negative S. epidermidis.

Anti-Bacterial Agents↗

PubMed2002

Influence of Tn917 insertion on transcription of the icaADBC operon in six biofilm-negative transposon mutants of Staphylococcus epidermidis.

Insertion of Tn917 into the icaADBC operon determines a biofilm-negative phenotype in biofilm-producing Staphylococcus epidermidis due to the inactivation of the genes responsible for the synthesis of the polysaccharide intercellular adhesin. We previously characterized six isogenic biofilm-negative transposon mutants of S. epidermidis 1457 with Tn917 insertions in either icaA or icaC. Northern blot analysis using ica- and Tn917-specific probes revealed that ica sequences located upstream and downstream of the transposon insertion site were still transcribed in five mutants in which Tn917 was inserted in the same transcriptional orientation. Outward-directed transcription initiating from within the transposon resulted in the complete expression of individual ica genes. Our results indicate that not only the inactivation of the entire operon but the isolated interruption of individual icaA and icaC genes led to a biofilm-negative phenotype in S. epidermidis. Tn917 mutagenesis may also result in the activation of genes located downstream of the insertion site.

Amino Acid Sequence↗

PubMed2002

Cloning and sequencing of Enterobacter aerogenes OmpC-type osmoporin linked to carbapenem resistance.

Using outbreak-related strains of Enterobacter aerogenes, we cloned and sequenced ompK39, the structural gene coding for outer membrane protein OmpK39. Its lack of expression was closely associated with a phenotype exhibiting low-level carbapenem resistance. Detailed alignment of the predicted amino acid sequence revealed that OmpK39 is a member of the OmpC subclass of enterobacterial porins, with the highest degree of homology to Klebsiella pneumoniae OmpK36. Based on a computerized alignment including Escherichia coli PhoE and OmpF, the 3D structures of which are known from X-ray studies, OmpK39 can be assumed to form the typical beta-barrel structure which is common to all enterobacterial porins. Since no inhibitory DNA sequences could be detected in ompk39 in the resistant strains, porin deficiency leading to carbapenem resistance seems to involve alterations in key regulatory genes and/or the promotor sequence rather than a direct mutation in the structural gene.

Amino Acid Sequence↗

PubMed2002

Alcoholic ingredients in skin disinfectants increase biofilm expression of Staphylococcus epidermidis.

The pathogenesis of Staphylococcus epidermidis is correlated with biofilm formation. We investigated the effect of three common alcoholic skin disinfectants, ethanol, n-propanol and isopropanol, on the biofilm formation of 37 clinical, icaADBC-positive S. epidermidis isolates. In alcohol-supplemented media 18 strains displayed increased biofilm expression. Sixteen of 19 strains were generally incapable of biofilm formation. In three representative isolates, the increase in biofilm formation was paralleled by increased polysaccharide intercellular adhesin synthesis. Regarding the widespread use of alcoholic skin disinfectants, it is possible that the alcohol-inducible biofilm phenotype of S. epidermidis could add to the development of foreign body-related infections.

Biofilms↗

PubMed2002

Differential expression of methicillin resistance by different biofilm-negative Staphylococcus epidermidis transposon mutant classes.

Biofilm formation mediated by polysaccharide intercellular adhesin (PIA) is the major virulence factor of Staphylococcus epidermidis and is often associated with methicillin resistance. Transposon Tn917 insertions leading to a biofilm-negative phenotype in the biofilm-producing S. epidermidis strain 1457 (mecA-negative) were transferred into the methicillin-resistant, biofilm-producing S. epidermidis 1057 (mecA-positive) by transduction. According to their phenotypes and genotypes, the mutants could be separated into genetic classes I to IV (D. Mack, H. Rohde, S. Dobinsky, J. Riedewald, M. Nedelmann, J. K. M. Knobloch, H.-A. Elsner, and H. H. Feucht, Infect. Immun. 68:3799-3807, 2000). All transductants of S. epidermidis 1057 had phenotypes for biofilm formation similar to those of the corresponding mutants of S. epidermidis 1457. With a mecA-specific probe, identical hybridization patterns were observed for wild-type S. epidermidis 1057 and all the transductants. There were minor changes in oxacillin MICs for Class II and III transductants compared to those for wild-type S. epidermidis 1057. On population analysis, S. epidermidis 1057 displayed a heterogeneous expression type of resistance with an oxacillin MIC of > or =6 microg/ml for more than 90% of the cells. An almost identical profile was observed with biofilm-negative class I mutants, where the transposon insertions inactivate the icaADBC gene locus essential for PIA synthesis. In contrast, class III mutants were more sensitive to oxacillin with a MIC of < or =1 microg/ml for more than 90% of the cells. The class IV mutant displayed homogeneous resistance with a MIC of > or =50 microg/ml for more than 90% of the cells. On oxacillin gradient plates, the class II mutant displayed decreased resistance. Apparently, different independent mutations leading to a biofilm-negative phenotype of S. epidermidis by influencing expression of icaADBC on the level of transcription significantly influence the expression of methicillin resistance. However, transcription of mecA was not significantly altered in the different transductants compared to the wild type, independent of mecA induction with oxacillin, indicating that other mechanisms influencing phenotypic expression of methicillin resistance are involved.

Bacterial Proteins↗

PubMed2002

In vitro activity of moxifloxacin against bacteria isolated from odontogenic abscesses.

We evaluated the antimicrobial susceptibility of 87 pathogens isolated from 37 patients with odontogenic abscesses. The most prevalent bacteria were viridans group streptococci and Prevotella species. Considering all bacterial isolates, 100% were susceptible to amoxicillin-clavulanic acid, 98% were susceptible to moxifloxacin and to levofloxacin, 76% were susceptible to doxycycline, 75% were susceptible to clindamycin, and 69% were susceptible to penicillin.

Adolescent↗

PubMed2002

Rapid detection of methicillin resistance in coagulase-negative Staphylococci with the VITEK 2 system.

The aim of the present study was to evaluate the accuracy of the new VITEK 2 system (bioMérieux, Marcy l' Etoile, France) for the detection of methicillin resistance in coagulase-negative staphylococci (CoNS) by using AST-P515 and AST-P523 test cards. Analyses of the VITEK 2 oxacillin MIC determination evaluated according to the actual breakpoint (>/=0.5 micro g/ml) of the National Committee for Clinical Laboratory Standards resulted in a high sensitivity of 99.2% but a moderate specificity of 80%. The newly included oxacillin resistance (OR) test of the VITEK 2 system displayed a high sensitivity and a high specificity of 97.5 and 98.7%, respectively. Concordance between the results of the mecA PCR and the VITEK 2 oxacillin MIC was observed for almost all Staphylococcus epidermidis strains, but the reduced specificity was attributable to higher oxacillin MICs for mecA-negative non-S. epidermidis strains, especially S. saprophyticus, S. lugdunensis, and S. cohnii. Evaluation of alternative oxacillin MIC breakpoints of 1, 2, or 4 micro g/ml resulted in improved degrees of specificity of 84, 90.7, and 97.3%, respectively. Only minor changes occurred in the corresponding sensitivity values, which were 98.4, 97.5, and 97.5%, respectively. Methicillin resistance in CoNS was detected after 7 and 8 h in 91.1 and 93.5% of the mecA-positive strains, respectively, by the VITEK 2 OR test and in 86.3 and 89.5% of the mecA-positive strains, respectively, by VITEK 2 oxacillin MIC determination. After 7 and 8 h the VITEK 2 OR test classified 59.2 and 78.9% of the mecA-negative strains, respectively, as susceptible to oxacillin, whereas comparable values were obtained 2 h later by VITEK 2 oxacillin MIC determination. The results of our study encourage the use of the VITEK 2 system, which proved to be a highly reliable and rapid phenotypic method for the detection of methicillin resistance in CoNS.

Bacterial Proteins↗