Identification of a functional capsule locus in Streptococcus mitis.

The polysaccharide capsule of Streptococcus pneumoniae is a hallmark for virulence in humans. In its close relative Streptococcus mitis, a common human commensal, analysis of the sequenced genomes of six strains revealed the presence of a putative capsule locus in four of them. We constructed an isogenic S. mitis mutant from the type strain that lacked the 19 open reading frames in the capsule locus (Δcps mutant), using a deletion strategy similar to previous capsule functional studies in S. pneumoniae. Transmission electron microscopy and atomic force microscopy revealed a capsule-like structure in the S. mitis type strain that was absent or reduced in the Δcps mutant. Since S. mitis are predominant oral colonizers of tooth surfaces, we addressed the relevance of the capsule locus for the S. mitis overall surface properties, autoaggregation and biofilm formation. The capsule deletion resulted in a mutant with approximately two-fold increase in hydrophobicity. Binding to the Stains-all cationic dye was reduced by 40%, suggesting a reduction in the overall negative surface charge of the mutant. The mutant exhibited also increased autoaggregation in coaggregation buffer, and up to six-fold increase in biofilm levels. The results suggested that the capsule locus is associated with production of a capsule-like structure in S. mitis and indicated that the S. mitis capsule-like structure may confer surface attributes similar to those associated with the capsule in S. pneumoniae.

The Bacillus cereus group: novel aspects of population structure and genome dynamics.

AIMS: To provide new insights into the population and genomic structure of the Bacillus cereus group of bacteria. METHODS AND RESULTS: The genetic relatedness among B. cereus group strains was assessed by multilocus sequence typing (MLST) using an optimized scheme based on seven chromosomal housekeeping genes. A set of 48 strains from different clinical sources was included, and six clonal complexes containing several genetically similar isolates from unrelated patients were identified. Interestingly, several clonal groups contained strains that were isolated from similar human sources. Furthermore, comparative whole genome sequence analysis of 16 strains led to the discovery of novel ubiquitous genome features of the B. cereus group, such as atypical group II introns, IStrons, and hitherto uncharacterized repeated elements. CONCLUSIONS: The B. cereus group constitutes a coherent population unified by the presence of ubiquitous and specific genetic elements which do not show any pattern, either in their sequences or genomic locations, which allows to differentiate between the member species of the group. Nevertheless, the population is very dynamic, as particular lineages of clinical origin can evolve to form clonal complexes. At the genome level, the dynamic behaviour is indicated by the presence of numerous mobile and repeated elements. SIGNIFICANCE AND IMPACT OF THE STUDY: The B. cereus group of bacteria comprises species that are of medical and economic importance. The MLST data, along with the primers and protocols used, will be available in a public, web-accessible database (http://mlstoslo.uio.no).

Characterization of a broad range antimicrobial substance from Bacillus cereus.

AIMS: The aim of this research was to isolate and characterize an antimicrobial substance from the Bacillus cereus type strain ATCC 14579. METHODS AND RESULTS: A substance with antimicrobial activity was isolated from B. cereus ATCC 14579. The substance was produced during late exponential growth and well into the stationary phase with a maximum 9 h after inoculation. The inhibitory substance was purified by reverse-phase HPLC and shown to be highly active against closely related Bacillus spp. Clinically relevant species such as Staphylococcus aureus and Micrococcus luteus were also inhibited. The substance was characterized as a bacteriocin-like inhibitory substance (BLIS) with a molecular mass of ca 3.4 kDa. The BLIS was very heat stable, and sensitive only to pronase E and proteinase K. Antimicrobial activity was stable and high in the pH range of 2.0-9.0, and relatively unaffected by organic chemicals. CONCLUSIONS: An antimicrobial substance produced by the B. cereus type strain ATCC 14579 was characterized, with a wide spectrum of activity and the potential to be applied as a control agent against pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first report of a substance with antimicrobial activity from the B. cereus type strain.

BceS1, a new addition to the type III restriction and modification family.

The nucleotide sequence of an 11-kb chromosomal BglII fragment from Bacillus cereus American Type Culture Collection (ATCC) 10987 strain revealed two closely adjacent open reading frames organized in an operon, of which the deduced amino acids showed identity to the type III restriction and modification (R/M) subunits described in Gram-negative bacteria. An enhanced transcription level was revealed when the culture was grown in the presence of foreign DNA. A cell-free extract from this culture restricted pUC19, whereas from a plain medium the restriction was very weak. The in vitro methylation protected pUC 19 from restriction. The R/M system was designated BceS1 as this endonuclease required ATP and Mg2+ as cofactors like other type III endonucleases. BceS1 is the first chromosomal type III R/M system characterized in a Gram-positive bacterium.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.

Genetic diversity of Bacillus cereus/B. thuringiensis isolates from natural sources.

The genetic diversity and relationships among 154 Bacillus cereus/B. thuringiensis isolates recovered from soil samples from five geographic areas in Norway were investigated with multilocus enzyme electrophoresis (MEE). Cluster analysis revealed two major groups (designated cluster I and cluster II) separated at genetic distance greater than 0.55. Cluster I included 62 electrophoretic types (ETs) originating from all five locations, whereas, in cluster II, all but one isolate were from the same location. The isolates were also serotyped with B. thuringiensis flagellar antisera, and 28 distinct serotypes were identified. In general, serotyping did not show correlation to the genetic diversity of the isolates. The presence of IS231- and IS240-like transposable elements was detected in 14% of the strains of cluster II only. Parasporal crystals were observed in three strains; ten other strains were toxic to Trichoplusia ni. We conclude that B. cereus/B. thuringiensis from soil exhibit a high degree of recombination.

Yeast-suspension as soiling matter in disinfectant testing.

Using the Kelsey-Sykes capacity-test, it was found that a sterile yeast suspension used to simulate 'dirty' conditions, gave an increased effect of Chloramine T against the fungi Candida albicans, Aspergillus fumigatus, Geotrichum candidum and Penicillium sp. compared with the effect under 'clean' conditions. This effect was not found with the fungus Rhodotorula rubra nor on the various bacteria tested. The enhanced effect was found with respect to both Chloramine T and Chloramine B, but not with the sodium hypochlorite solution when tested on C. albicans. This effect was due to a diffusible factor from the yeast cells. The factor was evident in the solution after heating of the yeast-cell suspension and in unsterilized yeast-cell suspension left at room temperature for 2 h or more. The effect of Chloramine T on the fungi C. albicans and A. fumigatus was reduced as expected when the yeast suspension was replaced by 20% normal horse serum. The results indicate that using sterile yeast suspensions in this type of test, may erroneously give high fungicidal effects of Chloramine, and thus lead to an incorrect use-dilution concentration, especially if the determination is made on the basis of the effect observed only under dirty conditions.

An investigation of the bactericidal and fungicidal effects of certain disinfectants by use of a capacity test.

The bactericidal and fungicidal effects of five disinfectants and one combination of two disinfectants were tested using a modified Kelsey-Sykes method in which living microorganisms suspended in a sterilized yeast suspension ("dirty" conditions) and in sterile distilled water ("clean" conditions) were added to the disinfectants in three stages. Six bacteria and two fungal organisms were employed as test microbes. Results showed that formaldehyde was virtually inactive at the dilution tested (1/50), whereas phenol and the combination propylene-phenoxetol + benzalkonium chloride were moderately effective, the latter compound being better. Glutaraldehyde was manifestly the most effective of the disinfectants tested, followed by tricresol. At a 1/50 dilution, chloramine proved to have a surprisingly strong fungicidal effect on Candida albicans and Aspergillus fumigatus under "dirty" conditions, whereas the same fungal organisms proved rather resistant to chloramine under "clean" conditions. The same was demonstrated--though less markedly--when higher dilutions of chloramine were used. The search for an explanation is still in progress. At relatively high dilutions, chloramine also proved quite effective against most of the test bacteria, especially under "clean" conditions. It is recommended that the yeast suspension be replaced by normal horse serum 20% v/v when testing the efficacy of chloramine against fungal strains under "dirty" conditions.

Autoclaving of lubricated dental instruments.

Test organisms forced mechanically into lubricated, rotating dental instruments (handpieces) were all killed during autoclaving at 134 degrees C for 8 min, even when protected by serum and oil. The test organisms were: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, and spores of Bacillus stearothermophilus. Also when testing the sterility of autoclaved simulated instrument surfaces (brass cylinders and pieces of a cotton fabric) which had been inoculated with bacteria and dried before they were sprayed with oil, there was no growth of the test organisms. In addition to the other test organisms, spores of Bacillus subtilis and Gram-positive, anaerobic bacteria isolated from used handpieces that had been exposed to several autoclavings were used. Some of the handpieces that had been left to dry after use in the dentist's office before they were autoclaved, were shown not to be sterile. Therefore, the authors suggest that autoclaving of the instruments should take place shortly after use and prescribed cleaning.

[A microbiological investigation of the effectiveness of Micro Megas E-spray]. / En mikrobiologisk undersøkelse av effektiviteten av Micro-Megas E-spray

The disinfecting effect of Micro Megas E-spray was tested using a microbiological technique which also included a practical test. Contra-angels and straight handpieces which were sprayed after being used for treatment on patients, and then dried and incubated in a liquid medium, showed a marked growth of microorganisms. The spray had a weak and barely significant growth inhibiting effect on contaminated, simulated instrument surfaces. using Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus as test bacteria. It is concluded that the spray is not suitable for distinfection of contra-angels and straight handpieces.