IS257 and small plasmid insertions in the mec region of the chromosome of Staphylococcus aureus.

Four copies of the insertion sequence IS257 are found in the mec region of the chromosome of the Australian methicillin resistant Staphylococcus aureus (MRSA) strain ANS46, two flanking a merAmerB sequence (encoding resistance to mercurial compounds), the other two flanking an integrated copy of the plasmid pT181 (tetracycline resistance). The termini of the integrated copy of the plasmid pT181 carry a direct repeat of 8 bp of plasmid sequence, but otherwise there are no similarities in the 8 bp sequences flanking the four copies of IS257 in this strain. Integrated copies of pT181 in strains R35 (a New Jersey MRSA) and GH32 (MRSA of Greek origin) have the same terminal repeat as in ANS46, suggesting either a specific site of insertion of IS257 into the free plasmid before integration into the chromosome, or a common evolutionary lineage for these geographically diverse isolates. A different 8 bp terminal repeat of plasmid sequence is found in the chromosomally integrated copy of pUB110 (flanked by a pair of IS257s) in R155, another New Jersey MRSA. This 8 bp repeat differs from that reported previously for pUB110/IS257 inserted into the plasmid pSK41, indicating insertion of IS257 into different sites of pUB110 before integration into the chromosome or into pSK41. In the plasmid pSK1, the two outer copies of IS257 of the three associated with Tn4003 (trimethoprim resistance) are also flanked by 8 bp repeats.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular aspects of methicillin resistance in Staphylococcus aureus.

All clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates examined so far contain the mecA gene, a 2130bp stretch of DNA of non-staphylococcal origin which, together with a larger block (up to 40-60 Kb) of 'foreign' DNA, is incorporated into the staphylococcal chromosome. mecA encodes for the 78 Kd penicillin-binding protein (PBP) 2A, which has very low affinity for beta-lactam antibiotics. The sequence of the mecA gene contains structural motifs characteristic of cell wall synthetic transpeptidases. It is generally assumed that the mecA gene product (PBP 2A) acts as a surrogate enzyme which takes over the task of cell wall synthesis from the normal complement of staphylococcal PBPs, since the latter are inhibited by relatively low (e.g. methicillin) concentrations of beta-lactam antibiotics. While direct biochemical evidence for a transpeptidase activity in PBP 2A is still missing, the essentiality of an intact mecA gene for the expression of high-level methicillin resistance has been clearly established by transposon inactivation experiments. On the other hand, it was already noted some time ago that an intact mecA and its gene product PBP 2A alone cannot be fully in control of the resistant phenotype, since all MRSA isolates, irrespective of their MIC values (from as low as 3 mg/L or as high as 1600 mg/L), were found to contain comparable amounts of PBP 2A. Such major disparities between cellular amounts of PBP 2A and the antibiotic MIC values suggested that a factor or factors of unknown nature ('factor X') other than the mecA gene product also played an essential role in the phenotypic expression of resistance. The same conclusion was reached in early genetic studies in which methicillin resistance could be reduced by insertional inactivation of a chromosomal site (omega 2003) within the so-called femA gene--(factor essential for the expression of methicillin resistance) outside the mecA determinant. More recently, several additional chromosomal sites were identified outside the mecA gene in which transposon inactivation reduced the level of beta-lactam resistance. The importance of these genes becomes clear if one realizes that it is the appropriate functioning of these determinants (in the genetic background of MRSA) rather than the quantity of PBP 2A in the cells that seems to determine the MIC value of an MRSA isolate. It is not clear at the present time how many such 'auxiliary genes' exist and exactly how these gene co-operate with the mecA gene in bringing about high-level beta-lactam resistance.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Physical mapping of the mec region of an American methicillin-resistant Staphylococcus aureus strain.

We mapped part of the mec region of a locally prevalent strain of Staphylococcus aureus. The mec region was found to harbor an insert of the transposon Tn554, which encodes spectinomycin and macrolide-lincosamide-streptogramin B resistance, and a 4.6-kb segment of DNA that contains the kanamycin resistance gene aadD. This 4.6-kb segment appears to be an integrated form of a previously described plasmid, pUB110, and is flanked by copies of the insertion sequence IS257. The integration event may be an example of processes that have led to accretion of resistance determinants in the mec region of S. aureus.

Tn554 inserts in methicillin-resistant Staphylococcus aureus from Australia and England: comparison with an American methicillin-resistant group.

We have compared methicillin-resistant (Mcr) Staphylococcus aureus isolates from Australia, the UK and the USA with regard to chromosomal inserts of the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon Tn554. The American isolates were known to have a distinctive Tn554 insert, designated insert 6, which was closely associated epidemiologically with the methicillin-resistance phenotype. Southern blots of DNA from Australian and London, UK Mcr isolates were hybridized with a range of probes related to Tn554. The isolates had similar or identical Tn554 inserts, and we consider them to be a single group, designated 'Australondon'. Australondon isolates were compared in detail with a deletion mutant, ANS62, that had lost the methicillin-resistance determinant mec, plus other resistance determinants resident in the mec region of the chromosome, and with an American Mcr isolate containing Tn554 insert 6. The Australondon isolates had three Tn554 inserts. Sequence analysis with the polymerase chain reaction showed that all of these inserts differed from classical Tn554 in that the 3'-terminal residues of the transposons were reverse complements of the usual GATGTA. One of the Australondon inserts, designated 6B, closely resembled Tn554 insert 6 in the sequence of its left flanking chromosomal DNA. This insert was found to abut the deletion from the mec region which results in strain ANS62. We infer that Tn554 insert 6B is part of the mec region of the chromosome in Australondon isolates, supporting the idea that insert 6 of the American isolates is also part of this chromosomal region.

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.

Amplification of a section of chromosomal DNA in methicillin-resistant Staphylococcus aureus following growth in high concentrations of methicillin.

Growth of two independently isolated strains of methicillin-resistant Staphylococcus aureus (MRSA) in increasing concentrations of methicillin (step-selection) resulted in increased resistance in these strains. When chromosomal DNA from the step-selected variants was probed using DNA sequences previously demonstrated to be associated with methicillin resistance in MRSA strains, amplification of the homologous chromosomal sequence was identified. Growth of these step-selected strains in the absence of methicillin resulted in loss of the amplified sequence, while the original sequence remained. There are differences between the two strains in the stability of maintenance of amplified sections. Prolonged storage of the variants on a high concentration of methicillin resulted in loss of amplified sections without concomitant loss of methicillin resistance. Thus amplification may be only one of at least two molecular mechanisms available to S. aureus to increase methicillin resistance in response to step-selection. Probing of cells of the highly resistant sub-population of a heterogeneously resistant MRSA strain showed that duplication of this mec-associated DNA is not involved in the mechanism of heteroresistance.

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.

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.

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.

Comparison of the pathogenicity of Mycobacterium paratuberculosis and Mycobacterium spp isolated from the wood pigeon (Columba palumbus-L).

The pathogenicity of 4 strains of mycobacteria isolated from wood pigeons and 2 strains of Mycobacterium paratuberculosis was compared in calves, mice and chickens. Three of the 4 wood pigeon strains and the 2 M. paratuberculosis strains produced clinical Johne's disease in calves. All 6 strains were pathogenic for mice and the 4 wood pigeon strains were pathogenic for chickens. The strains were not agglutinated by antisera to strains of the M. avium complex and all were mycobactin-dependent. It was concluded that the wood pigeon isolates may constitute a distinct group with the pathogenic capability of M. avium in chickens and M. paratuberculosis in calves.

Molecular relationships among serogroup B bacteriophages of Staphylococcus aureus.

The typing bacteriophages 55, 80, 83A, and 85 of Staphylococcus aureus, representative of the three major lytic groups of serological group B aureophages, have been examined for relatedness of their genomes and virion proteins. Phages 11 and 80 alpha were also examined to determine the relationship of phage 80 alpha to phages 11 and 80. Total genome hybridization measurements divided the phages into two groups. Phages 55 and 80, in the first group, had DNA homology of 50%. Phages 11, 80 alpha, 83A, and 85 formed a second group with 27 to 65% homology. Homology between the two groups was in the range of 14 to 22%. Phage 80 alpha is more closely related to phage 11 than to phage 80, though it is probably not a simple recombinant of phages 11 and 80. Restriction enzyme digestion and phage [32P]DNA hybridization analysis of the endonuclease-generated fragments from each phage DNA confirmed the findings of the DNA homology measurements. The endonuclease fragment patterns generated by EcoRI and HindIII were distinctive for each phage, confirming that none of the phages are closely related. Common sequences were present in most fragments from the phage DNAs when the labeled probe DNA was from a different phage in the same group. Cross-group probing of endonuclease fragments revealed both a diminished level of homology when similar sequences were present and the probable absence of some sequences. Virion proteins, examined by polyacrylamide gel electrophoresis, were similar in number and molecular weight for phages 11, 80 alpha, 83A, and 85, reflecting the DNA homology analyses. The virion proteins from phages 55 and 80, however, were more distinctive, and both differed from the phages in the other group.

Occurrence of chloramphenicol acetyltransferase and Tn9 among chloramphenicol-resistant enteric bacteria from humans and animals.

The occurrence of chloramphenicol acetyltransferase (CAT) among chloramphenicol resistant enteric bacteria from humans, animals (cats, dogs) and sewage was examined. The enzyme appears to be the basis of resistance in 83 and 84 per cent of bacteria of humans and sewage origin, and in 50 per cent of bacteria from animals. In order to identify type I CAT among chloramphenicol resistant isolates, total cellular DNA was probed with a 32P-labelled fragment of the CAT structural gene from the transposable element Tn9. Nineteen per cent of chloramphenicol resistant enteric bacteria of clinical origin, 11% of sewage isolates, and 11% of veterinary isolates gave positive hybridization results. The difference between bacteria of clinical and veterinary origin in respect of both parameters tested is significant and is interpreted as indicating genetic dis similarity between the two pools in regard to chloramphenicol resistance. This may reflect a lack of contact between the two pools, or host bacterial factors with select against CAT-mediated (and type I CAT more specifically) resistance to chloramphenicol.

The pathogenicity of Mycobacterium avium and related mycobacteria for experimental animals.

The pathogenicity of 40 strains of Mycobacterium avium, M. paratuberculosis, M. intracellulare and M. lepraemurium was investigated in chickens, rabbits, guinea-pigs, mice and calves. Mycobactin dependence and serological type were also determined. There was no evidence that mycobactin dependence was related to pathogenicity. Antigenic similarities were demonstrated between M. avium and M. paratuberculosis, and one isolate had the pathogenic characteristics of both species.

The occurrence and significance to animal health of Leptospira, Mycobacterium, Escherichia coli, Brucella abortus and Bacillus anthracis in sewage and sewage sludges.

Samples of sewage, sewage sludge and sewage effluent from one or more of four sewage treatment plants were examined for the presence of Leptospira, Mycobacterium, Escherichia coli, Brucella abortus and Bacillus anthracis. Brucella abortus and Bacillus anthracis were not isolated. Eleven strains of E. coli potentially enteropathogenic for calves or piglets, eight pathogenic strains of Mycobacterium and one patho;genic Leptospira strain were isolated from 101, 189 and 189 samples respectively. Sewage sludge is no;t considered to play a major part in the epidemiology of disease caused by these organisms.