Permeabilizing effects of sub-inhibitory concentrations of microcystin on the growth of Escherichia coli.

Microcystin, a cyanotoxin produced by Microcystis aeruginosa, lacks potent antibacterial activity. When tested in combination, in vitro, inhibitory values for a range of hydrophobic antibiotics were significantly reduced in the presence of at least 1/20 x the minimum inhibitory concentration of microcystin. The degree of inhibition was equivalent to that of a well-characterised permeabilizing agent, polymyxin B nonapeptide. The permeabilizing ability of sub-inhibitory concentrations of microcystin to affect the envelope of Escherichia coli was demonstrated by a rapid and sustained reduction in absorbance values of lysozyme-treated cells and by enhanced uptake of crystal violet in microcystin-treated cultures. Direct effects of appropriate concentrations of microcystin on the integrity of bacterial outer and inner membranes were measured by release of specific enzyme markers. Although the exact mechanism for permeabilizing E. coli with microcystin has not been elucidated, the effects were consistent with permeability changes to the enterobacterial outer membrane caused by polymyxin B nonapeptide.

Genomic and phenomic differentiation of Rhodococcus equi and related strains.

16S rDNA sequence and pyrolysis mass spectrometric analyses were carried out on representatives of Rhodococcus equi and marker strains of genera that encompass mycolic acid containing actinomycetes. The R. equi strains formed a monophyletic clade within the evolutionary radiation occupied by members of the genera Nocardia and Rhodococcus. The 16S rDNA sequence data also showed R. equi to be an heterogeneous taxon. This heterogeneity was underscored by the pyrolysis mass spectrometric data. These observations are in line with those of previous studies where similar profiles of relatedness were found between pyrolysis mass spectral data and the results of DNA:DNA pairing and numerical phenetic studies.

Parentage and kinship studies in an obligate brood parasitic bird, the brown-headed cowbird (Molothrus ater), using microsatellite DNA markers.

Recent studies suggest that single-locus microsatellite DNA markers have the potential to unambiguously resolve parentage among individuals in natural populations where maternity is known. However, their power for determining parentage when neither parent is known is unclear. Here we investigate the usefulness of microsatellite DNA markers to determine parentage in a brood parasitic bird, the brown-headed cowbird (Molothrus ater), where, for a given offspring, no a priori knowledge of either parent is available. Seven polymorphic microsatellite DNA markers isolated from brown-headed cowbirds and yellow warblers (Dendroica petechia) were used to genetically characterize an individually marked breeding population of male and female cowbirds at Delta Marsh, Manitoba. Forty-four males, 21 females, and 61 cowbird chicks were genotyped at seven loci using DNA amplified from blood and tissue samples. The mean exclusion probabilities pooled across all seven loci were 0.9964 for males and 0.9948 for females. Two null (non-amplifying) alleles at one locus were discovered and accounted for by constructing alternate nonoverlapping primer sets. Exclusion analyses performed using all individuals determined both paternity and maternity for 43 chicks and paternity only for 4 chicks. Another microsatellite locus was then used to determine paternity for three additional chicks. Relatedness analyses placed 12 of the 18 remaining chicks not assigned both maternity and paternity into four unique full sibling groups. Overall, 90.16% (55 of 61) of all offspring examined were placed into distinct parent/sibling groups, demonstrating that this marker set is extremely useful for parentage studies in this species.

Rhodococcal systematics: problems and developments.

Various approaches that have been used in the development of a system of classification for the genus Rhodococcus are discussed. The application of chemotaxonomic, molecular systematic and numerical phenetic methods have greatly contributed to improvements in the systematics of rhodococci and related mycolic-acid containing actinomycetes. The genus currently encompasses twelve validly described species but improved diagnostic methods are needed to distinguish between them. In addition, evidence from 16S ribosomal RNA sequencing suggests that the genus is still heterogeneous.

Physiology and genetics of antibiotic production and resistance.

Actinomycetes have the genetic capability to synthesize many different biologically active secondary metabolites and of these compounds, antibiotics predominate in therapeutic and commercial importance. Intensive research often centres on the use of molecular techniques to investigate the physiology and genetics of antibiotic biosynthesis with a view to improving production. The isolation of clones of Streptomyces hygroscopicus, the producer of geldanamycin, which synthesizes geldanamycin in S. lividans, is reported. Molecular approaches using genes for elongation factors (tuf) were used in attempts to increase the fermentation yield of kirromycin, whilst probes for aphD and sph, genes for streptomycin phosphotransferases, were used to gather information on streptomycin genes in soil. Actinomycete populations in soil and earthworms may help in developing a strategy for discovering additional antimicrobials in soil. The relationship of proline metabolism to the secondary metabolite undecylprodigiosin and the carbon regulation of spiramycin biosynthesis in S. ambofaciens is also reported.

Apparent cytomegalovirus epithelial keratitis in a cardiac transplant recipient.

A 44-year-old immunosuppressed man developed initial symptoms of intermittent irritation of the left eye three months after cardiac transplantation. Symptoms increased, with decreased vision, photophobia, and lacrimation. Slit lamp examination showed slightly raised, swollen, grayish epithelium in a broad multibranching dendritic pattern associated with fine and medium punctate epithelial erosions that stained slightly with fluorescein. Histopathologic study of the corneal epithelial scraping demonstrated swollen epithelial cells with intranuclear and intracytoplasmic viral inclusions. Viral cultures manifested a cytopathic pattern characteristic of cytomegalovirus 14 days after inoculation on human embryonic lung cells (MRC-5). Pretransplantation cytomegalovirus IgM and IgG serologic titers were negative (less than 1:16 for IgG, no IgM noted) until the onset of symptoms. Subsequently, IgM titers rose against cytomegalovirus consistent with concurrent infection.

Gas-permeable lifecell tissue culture flasks give improved growth of Helicobacter pylori in a liquid medium.

Experiments were conducted to investigate the microaerobic culture of Helicobacter pylori in a liquid medium by using gas-permeable Lifecell tissue culture flasks. Growth in Lifecell tissue culture flasks was 1.2 to 1.6 log units greater than that in glass control bottles. These results were comparable to those reported by the use of gyrated media.

Menaquinone composition of mycolic acid-containing actinomycetes and some sporoactinomycetes.

The menaquinones of 141 actinomycetes representing the genera Caseobacter, Mycobacterium, Nocardia, Rhodococcus and some related taxa lacking mycolic acids were examined by mass spectrometry. The mycolic acid-containing strains were assigned to four groups on the basis of the predominant isoprenologue detected: Rhodococcus coprophilus, R. equi, R. erythropolis, R. globerulus, R. rhodnii, R. rhodochrous and R. ruber contained dihydrogenated menaquinones with eight isoprene units; Nocardia asteroides, N. brasiliensis, N. carnea, N. otitidis-caviarum and N. transvalensis contained tetrahydrogenated menaquinones with eight isoprene units; Caseobacter polymorphus, R. bronchialis, R. rubropertinctus and R. terrae and representatives of twenty-one approved species of Mycobacterium contained dihydrogenated menaquinones with nine isoprene units; a single strain of 'Mycobacterium album', contained unsaturated menaquinones with nine isoprene units. Actinomycetes containing meso-diaminopimelic acid, arabinose and galactose in the wall peptidoglycan but lacking mycolic acids were recovered in two groups: tetrahydrogenated menaquinones with eight isoprene units were the main components from 'Nocardia' autotrophica and Pseudonocardia thermophila whereas Saccharopolyspora hirsuta and Pseudonocardia spp. contained tetrahydrogenated menaquinones with nine isoprene units. Promicromonospora citrea and 'skin coryneforms' with LL-diaminopimelic acid and glycine in the wall peptidoglycan also contained tetrahydrogenated menaquinones with nine isoprene units as the major isoprenologue. In contrast, representatives of the genera Kitasatoa, Microellobosporia, Streptomyces and Streptoverticillium were characterized by the presence of complex mixtures of tetra-, hexa- and octa-hydrogenated menaquinones with nine isoprene units. The menaquinone data correlate well with other developments in actinomycete systematics and confirm earlier suggestions that menaquinone analyses are of value in both the classification and identification of actinomycetes. Indeed, the data suggest that minimal descriptions of wall chemotype IV taxa should ideally include information on menaquinone composition.

Numerical taxonomy of staphylococci.

Over two hundred staphylococci from human and animal sources and representatives of established species of Staphylococcus, Micrococcus and Planococcus were compared in a numerical phenetic survey using 115 unit characters. Data were analyzed using the Jaccard coefficient and the unweighted pair group method with averages algorithm. Cluster composition was not markedly affected by test error, estimated as 3.49%. The staphylococci were assigned to eighteen clusters containing four or more strains and to three single member clusters. Most of the clusters were distinct and homogeneous though two were divided into subclusters. Some of the clusters and subclusters were equated with the established taxa S. aureus, S. capitis, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. hyicus, S. saprophyticus, S. sciuri subspecies lentus, S. sciuri subspecies sciuri, S. simulans, S. warneri and S. xylosus, the remaining ones may represent the nuclei of additional centres of variation. The numerical data also cast doubts upon the reliability of some of the tests recommended for the identification of coagulase-negative staphylococci.

A probability matrix for identification of some Streptomycetes.

The character state data obtained for clusters defined at the 77.5% SSM similarity level in the phenetic numerical classification described by Williams et al. (1983) were used to construct a probabilistic identification matrix. The 23 phena included were the major clusters (19 Streptomyces, 2 Streptoverticillium and 'Nocardia' mediterranea) and one minor cluster (Streptomyces fradiae). The characters most diagnostic for these clusters were selected using Sneath's CHARSEP and DIACHAR programs. The resulting matrix consisted of 41 characters x 23 phena. Identification scores, determined by Sneath's MATIDEN program were used to evaluate the matrix. Theoretical assessment was achieved by determination of the cluster overlap (OVERMAT), the identification scores for the Hypothetical Medium Organism of each cluster (MOSTTYP), and the scores for randomly selected cluster representatives using the classification data of Williams et al. (1983). The matrix was evaluated practically by the independent re-determination of the characters for the same cluster representatives, which also provided a measure of test error. Finally it was used to identify unknown isolates from a range of habitats. The results showed that the matrix was theoretically sound. Test error was within acceptable limits and did not distort identifications. Of the unknown isolates, 80% were clearly identified with a cluster. It is suggested that the matrix could form the basis for a more objective identification and grouping of the large number of Streptomyces species which have been described.

Comparison of automated and rapid manual methods for the same-day identification of Enterobacteriaceae.

The Vitek AMS automated instrument method for identification of Enterobacteriaceae was compared with two rapid manual methods intended for the same purpose, the Micro ID System and the API 20E Same-Day procedure, on a series of 400 consecutive fresh clinical isolates. Results were compared with identifications obtained using the API 20E System with overnight incubation and supplemental tube biochemicals (when needed). Both the final (8-hour) and a manually requested, presumptive 5-hour result from the AMS were compared with the 4-hour results provided by the Micro ID and the 5-hour results provided by the API. The Micro ID system proved to be the most rapid and accurate of the three test systems by correctly identifying 96.8% (387/400) of isolates. The API 20E using 5-hour readings identified 90.7% (363/400) of isolates, although 96.8% (387/400) could be identified if supplemental overnight tests were employed to separate profile codes with "good likelihood, but low selectivity." The AMS correctly identified 88.8% (355/400) isolates after 5 hours, and 95.0% (380/400) following 8 hours incubation.