Independent risk factors for the co-colonization of vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus in the region most endemic for vancomycin-resistant Staphylococcus aureus isolation.

In the majority of cases of vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant Enterococcus faecalis (VR E. faecalis) served as the vanA donor to S. aureus. Previous studies that evaluated the risk factors for co-colonization with VRE and MRSA did not differentiate between VR E. faecalis and VR E. faecium. This study aimed to identify variables associated with VR E. faecalis and MRSA co-colonization. A retrospective case-control study from January 2008 to December 2009 was conducted at the Detroit Medical Center. Data were extracted from charts and pharmacy records. Unique patients co-colonized with VR E. faecalis and MRSA (defined as isolation of MRSA within 7 days of VR E. faecalis isolation) were compared with patients with VR E. faecalis who were not co-colonized with MRSA. A total of 546 patients with VR E. faecalis isolation were identified. 85 (15.6 %) VR E. faecalis patients were co-colonized with MRSA and 461 (84.4 %) VR E. faecalis patients were not co-colonized with MRSA. The mean age of the study cohort was 65.9 ± 16.4 years, 424 (77.7 %) were African-American, and 270 (49.5 %) were residing in long-term care institutions. Independent predictors of co-colonization of VR E. faecalis and MRSA were male gender, impaired consciousness, ICU stay prior to VR E. faecalis isolation, indwelling devices, and isolation of VR E. faecalis from wounds. MRSA was frequently isolated from the same culture specimen as VR E. faecalis (n = 39, 45.9 %), most commonly from wounds. This large study of patients with VR E. faecalis identified the severity of illness, indwelling devices, and chronic wounds as independent predictors of co-colonization with VR E. faecalis and MRSA.

Characterization of Streptomyces MITKK-103, a newly isolated actinomycin X2-producer.

A new actinomycete strain designated MITKK-103 was isolated from the soil of a flowerpot using a humic acid agar medium. The newly isolated strain was able to produce a large amount of actinomycin X2 even under nonoptimized growing conditions and serves as a promising source of this antibiotic. Actinomycin X2 has higher cytotoxicity toward cultured human leukemia (HL-60) cells than does actinomycin D, and it induces cell death via apoptosis. A nearly complete 16S ribosomal DNA (rDNA) sequence from the isolate was determined and found to have high identity (98.5-100%) with Streptomyces galbus, Streptomyces griseofuscus, and Streptomyces padanus, indicating that MITKK-103 belongs to the genus Streptomyces. The isolate clustered with species belonging to the S. padanus clade in a 16S-rDNA-based phylogenetic tree and showed 75% overall homology to S. padanus ATCC 25646 in DNA-DNA relatedness analysis. Although the growth of the isolate was somewhat different from the three species mentioned, the strain MITKK-103 most closely resembles S. padanus on the basis of the morphological and phenotypic characteristics, phylogenetic analysis, and genotypic data. As such, this is the first report of a strain of S. padanus capable of producing actinomycins.

Disparity between changes in mRNA abundance and enzyme activity in Corynebacterium glutamicum: implications for DNA microarray analysis.

The relationship between changes in mRNA abundance and enzyme activity was determined for three genes over a span of nearly 3 h during amino acid production in Corynebacterium glutamicum. Gene expression changes during C. glutamicum fermentations were examined by complementary DNA (cDNA) microarrays and by a second method for quantitating RNA levels, competitive reverse transcriptase-PCR (RT-PCR). The results obtained independently by both methods were compared and found to be in agreement, thus validating the quantitative potential of DNA microarrays for gene expression profiling. Evidence of a disparity between mRNA abundance and enzyme activity is presented and supports our belief that it is difficult to generally predict protein activity from quantitative transcriptome data. Homoserine dehydrogenase, threonine dehydratase, and homoserine kinase are enzymes involved in the biosynthesis of l-isoleucine and other aspartate-derived amino acids in C. glutamicum. Our data suggest that different underlying regulatory mechanisms may be connected with the expression of the genes encoding each of these three enzymes. Indeed, whereas in one case the increases in enzyme activity exceeded those in the corresponding mRNA abundance, in another case large increases in the levels of gene expression were not congruent with changes in enzyme activity.

Development and validation of corynebacterium DNA microarrays.

We have developed DNA microarray techniques for studying Corynebacterium glutamicum. A set of 52 C. glutamicum genes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of a C. glutamicum fermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To establish a set of benchmark metrics for this technique, we compared the variability between replicate spots on the same slide, between slides hybridized with cDNAs from the same labeling reaction, and between slides hybridized with cDNAs prepared in separate labeling reactions. We found that the results were both robust and statistically reproducible. Spot-to-spot variability was 3.8% between replicate spots on a given slide, 5.0% between spots on separate slides (though hybridized with identical, labeled cDNA), and 8.1% between spots from separate slides hybridized with samples from separate reverse transcription reactions yielding an average spot to spot variability of 7.1% across all conditions. Furthermore, when we examined the changes in gene expression that occurred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods. These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement of C. glutamicum amino acid-producing strains.

A novel Sinorhizobium meliloti operon encodes an alpha-glucosidase and a periplasmic-binding-protein-dependent transport system for alpha-glucosides.

The most abundant carbon source transported into legume root nodules is photosynthetically produced sucrose, yet the importance of its metabolism by rhizobia in planta is not yet known. To identify genes involved in sucrose uptake and hydrolysis, we screened a Sinorhizobium meliloti genomic library and discovered a segment of S. meliloti DNA which allows Ralstonia eutropha to grow on the alpha-glucosides sucrose, maltose, and trehalose. Tn5 mutagenesis localized the required genes to a 6.8-kb region containing five open reading frames which were named agl, for alpha-glucoside utilization. Four of these (aglE, aglF, aglG, and aglK) appear to encode a periplasmic-binding-protein-dependent sugar transport system, and one (aglA) appears to encode an alpha-glucosidase with homology to family 13 of glycosyl hydrolases. Cosmid-borne agl genes permit uptake of radiolabeled sucrose into R. eutropha cells. Analysis of the properties of agl mutants suggests that S. meliloti possesses at least one additional alpha-glucosidase as well as a lower-affinity transport system for alpha-glucosides. It is possible that the Fix+ phenotype of agl mutants on alfalfa is due to these additional functions. Loci found by DNA sequencing to be adjacent to aglEFGAK include a probable regulatory gene (aglR), zwf and edd, which encode the first two enzymes of the Entner-Doudoroff pathway, pgl, which shows homology to a gene encoding a putative phosphogluconolactonase, and a novel Rhizobium-specific repeat element.

A novel screening method for isolating exopolysaccharide-deficient mutants.

A screening method based on differential staining of the wild type and exopolysaccharide-deficient mutants of Rhizobium (Sinorhizobium) meliloti by the lipophilic dye Sudan Black B is described. Mutants defective in the production of either succinoglycan or EPS II (galactoglucan) were isolated by using this method, which might also prove useful for isolating exopolysaccharide-defective derivatives of other bacteria.

The phbC (poly-beta-hydroxybutyrate synthase) gene of Rhizobium (Sinorhizobium) meliloti and characterization of phbC mutants.

Defined insertion mutations have been constructed in the Rhizobium (Sinorhizobium) meliloti phbC gene, which encodes poly-beta-hydroxybutyrate (PHB) synthase. The locus was isolated and subcloned from a genomic library of R. meliloti Rm1021 by complementation of phbC mutation of Alcaligenes eutrophus. PHB production was detected in wild-type R. meliloti under nutrient-limited conditions but not in rich medium. No PHB production was detected in the R. meliloti phbC mutants. The DNA sequence of the R. meliloti phbC gene was determined. The deduced polypeptide sequence is homologous to previously identified PhbCs from other bacteria. The R. meliloti phbC locus maps to pRmeSU47a, the smaller of the two megaplasmids in this strain.

Identification of the Rhizobium meliloti alcohol dehydrogenase gene (adhA) and heterologous expression in Alcaligenes eutrophus.

A screen for Rhizobium meliloti genes which improve the growth of Alcaligenes eutrophus on sucrose identified the first alcohol dehydrogenase gene (adhA) isolated from the Rhizobiaceae. R. meliloti adhA is constitutively expressed in A. eutrophus and has alcohol dehydrogenase activity. R. meliloti adhA mutants retain some alcohol dehydrogenase activity.