Peter R. Stewart

The Cloning of Chromosomal DNA Associated with Methicillin and Other Resistances in Staphylococcus aureus

Competitive hybridization was used to detect the deletion of chromosomal DNA accompanying the loss of resistance to methicillin (and concomitantly, to cadmium, mercury and tetracycline) from a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA). The method was also used to screen a partial plasmid library of chromosomal HindIII fragments from the MRSA strain. Eight recombinant plasmid clones were identified as containing DNA included in the deletion. These clones were used as probes to screen a phage library of the total DNA of the same MRSA strain, resulting in the isolation of overlapping recombinant phage clones carrying 24 kb of the deleted DNA. Two of the cloned HindIII fragments were associated closely with methicillin resistance, as shown by probing DNA from an independent methicillin-sensitive/resistant transduced strain pair and from two MRSA strains following growth in the presence of high concentrations of methicillin. The endonuclease map of the cloned DNA indicates the presence of four copies of a direct repeat less than 1 kb in size. The map is also consistent with the presence in the chromosome of sequences for mercury resistance (mer A mer B) and for tetracycline-resistance plasmid pT181.

Energetic efficiency and maintenance energy characteristics ofSaccharomyces cerevisiae (wild type and petite) andCandida parapsilosis grown aerobically and micro-aerobically in continuous culture

Saccharomyces cerevisiae andCandida parapsilosis were grown aerobically and micro-aerobically in continuous culture and the energetic efficiency (YATP) and the maintenance energy requirements compared. The bioenergetic parameters were determined using a gas-balance technique, and from cell yield decrements at low dilution rates. The data show that for aerobic cultures, cell yield related to ATP generation (YATP) for the two yeasts is similar and close to the value reported originally by Bauchop and Elsden (1960). However, the efficiency of substrate utilization is greater in the case ofC. parapsilosis since the apparent P/O value under aerobic conditions is 1.8 as compared to about 1 forS. cerevisiae. The maintenance energy coefficients based on ATP requirement for aerobic cultures of both yeast were greater than the corresponding values for micro-aerobic cultures; similarly cultures of a petite mutant ofS. cerevisiae had a lower maintenance energy requirement than comparable aerobic wild-type cultures. It is suggested that these differences may reflect the energy requirement for the upkeep of a greater degree of functional complexity in aerobic, wild-type cells, as compared with micro-aerobic or respiratory deficient cells.

The species concept and its application to tailed phages.

SummaryA recently proposed polythetic definition of virus species appears easily applicable to bacteriophages. Criteria for classification of tailed phages are evaluated. Morphology, DNA homology, and serology are the most important criteria for delineation of species, but no single criterion is satisfactory. Dot-blot hybridization and seroneutralization may suggest false relationships by detecting common sequences in the DNA of otherwise unrelated phages. Species of tailed phages can be defined by a combination of morphology and DNA homology or serology. A procedure for identification of novel phages is outlined. Phage names should include elements of host names.

Inhibition of bacterial RNA polymerase by the cyanobacterial metabolites 12-epi-hapalindole E isonitrile and calothrixin A

The alkaloid 12-epi-hapalindole E isonitrile, from a cyanobacterial Fischerella species, and the indolophenanthridine calothrixin A, from Calothrix, inhibited Escherichia coli RNA polymerase competitively with respect to ATP, and non-competitively with respect to UTP. The inhibition was dependent on the order of addition of the inhibitors. The K(I) values, with ATP as the variable substrate, were 1.3+/-0.2 mM and 0.23+/-0.11 mM, respectively. Based on comparisons with the sensitivity of whole cells to these inhibitors, it is concluded that other targets in addition to RNA polymerase may also be implicated in their action.

Mercury and tetracycline resistance genes and flanking repeats associated with methicillin resistance on the chromosome of Staphylococcus aureus

Sections of a cloned 27 kb segment of chromosomal DNA, associated with resistance to four antimicrobial agents in a clinical isolate of methicillin‐resistant Staphylococcus aureus (MRSA), were tested for their ability to determine resistance when transformed into a sensitive laboratory strain of S. aureus. This was achieved by inserting the sections into a newly constructed shuttle vector, amplifying the recombinant DNA In E. coli, and transforming protoplasts of the sensitive S. aureus strain. Two sections of the cloned DNA were found to determine resistance separately to mercuric ion and to tetracycline, in both S. aureus and Escherichia coli.

Induced deletions within a cluster of resistance genes in the mec region of the chromosome of Staphylococcus aureus

Variants of a methicillin-resistant Staphylococcus aureus showing loss of or reduced resistance to the antibiotic were isolated at frequencies of 0.1-100% from cultures which had been starved, grown at elevated temperature, or given small doses of UV radiation. Three types of variant were identified on the basis of population distribution of resistance to the antibiotic, and field-inversion gel electrophoresis of digests of the chromosome cut with the rare-cutting restriction endonuclease SmaI. Type I variants are methicillin-sensitive and have a deletion in the mec region of the chromosome. Type II variants have reduced methicillin resistance and rearranged DNA elsewhere in the chromosome. Type II variants show reduced methicillin resistance and no detectable change in the chromosome. Type I deletions were mapped using cloned fragments from the mec region. In 13 of the 16 independently isolated deletion mutants, one of the deletion endpoints appears to correlate with the positions of insertion sequences or transposons found in this region of the staphylococcal chromosome.

The expression in Staphylococcus aureus of cloned DNA encoding methicillin resistance.

A 4 kb fragment of chromosomal DNA was cloned from a clinical strain of methicillin-resistant Staphylococcus aureus. It comprises part of a section of the chromosome that was lost when the strain was cured of resistance to methicillin and to other antimicrobial agents. The fragment mediates an increased level of methicillin resistance when inserted into a shuttle vector and transformed back into the sensitive strain generated when the original DNA was deleted.

Physical mapping of the mec region of an Australian methicillin-resistant Staphylococcus aureus lineage and a closely related American strain

Methicillin-resistant (Mcr) staphylococci contain chromosomal DNA that is absent from Mcs cells. This extra DNA harbours the methicillin resistance determinant mec and often other resistance determinants. The mec region can differ substantially in structure among different isolates. We present studies on the mec region of a group of Staphylococcus aureus isolates prevalent in Australia and London. Southern hybridization analyses of a prototype Australian isolate, ANS46, and an isogenic Mcs deletion mutant, ANS62, allowed the physical map of the region to be extended to 55 kb. The DNA corresponding to the deletion, which includes mec and resistance determinants for mercury, cadmium (Cd) and tetracycline, amounted to 41 kb. It was bounded precisely at one end by the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon, Tn554. Near the other end was an element with homology to Tn554, psi Tn554, which carried the Cdr determinant. The mec region of an American Mcr isolate, R35, was found to be virtually the same as that of ANS46, except that it lacked Tn554. Another class of American Mcr isolates, prevalent since 1987, differs markedly from ANS46 in mec region organization. However, this other American class also contains an insertion of Tn554 in the mec region, and the attachment site for this insertion was found to have significant homology to attachment sites for the Tn554 and psi Tn554 insertions in the mec region of the Australian strain. These results suggest possible roles of Tn554 and Tn554-like elements in the evolutionary variation of the mec region.

The virion proteins and ultrastructure of Staphylococcus aureus bacteriophages.

The number and size of the major virion polypeptides have been determined by SDS-PAGE for the 22 Staphylococcus aureus phages of the International Typing Set, plus phages 11 and 80 alpha. Virion ultrastructure was examined by electron microscopy after negative staining with ammonium molybdate. In addition, serogroup B phages were disrupted and fractionated into head, tail-tube and baseplate components and major polypeptides assigned to these substructures. The number and size of the polypeptides correlated closely with the division of aureophages into four serogroups (A, B, F, L), although serogroup L was represented in the set by only a single phage (187). Apart from serogroup B, however, the polypeptide patterns did not reflect differences between lytic groups. Within serogroup B, polypeptide analysis yielded characteristic patterns for lysogroups I, II and III. Ultrastructural analyses confirm the data provided by polypeptide analysis. Thus, phages from the four serogroups can be identified on the basis of tail-tube length alone, although the differences between phage 187 and members of lysogroups I and III in serogroup B were less than 20 nm, or approximately 12% of the total length. Serogroup A virions differed from those of the other serogroups in that all members of the typing set in this group had elongate, rather than isometric, heads.

Lethal and mutational effects of solar and UV radiation on Staphylococcus aureus.

Strains of Staphylococcus aureus, an opportunistic pathogen commonly found on human skin, were exposed to sunlight and UV C radiation, and the lethal and mutational effects measured. Sunlight killed cells with an inactivation constant of 3×10-5 per joule per square metre; UV C was much more lethal, giving an inactivation constant of approximately 0.1 per joule per square metre. Some strains tested showed a sensitivity to sunlight that was dependent on the growth phase of the cells, exponentially growing cells showing a greater sensitivity. Mutational effects of irradiation were measured by the appearance of mutants sensitive to methicillin following irradiation of a multiresistant strain. Mutants appeared at a frequency of 10-3; this high frequency of mutation in the region of the mec gene has also been observed when multiresistant strains are subjected to nutritional or thermal stress. Mutants showed the same chromosomal alteration (seen in pulse-field gel electrophoresis of Smal-digested DNA) whether induced by solar or UV C irradiation.