Reduced cadmium transport determined by a resistance plasmid in Staphylococcus aureus.

The presence of a plasmid harboring a gene for Cd2+ resistance led to markedly reduced Cd2+ uptake via the energy-dependent Mn2+ transport system in Staphylococcus aureus strain 17810R. Cd2+ uptake by the resistant strain via this high-affinity system was seen only at very low Cd2+ concentrations. At high concentrations, Cd2+ was taken up by the resistant strain via a different low-affinity uptake system. Cd2+ uptake via this system was energy dependent but was not blocked by Mn2+. Loss of the plasmid from the resistant strain resulted in Cd2+ sensitivity and unblocking of Cd2+ transport via the Mn2+ carrier in the plasmidless derivative strain 17810S. The energy-dependent Cd2+ uptake by the sensitive strain was inhibited by Mn2+ with kinetics indicating competitive inhibition. It is suggested that the second, low-affinity uptake system for Cd2+ in the resistant strain is the energy-dependent cadmium/proton antiporter, which at low Cd2+ concentrations functions in net Cd2+ efflux.     

Characterization of a small cryptic plasmid isolated from a methicillin-resistant strain of Staphylococcus aureus

The nucleotide sequence of a small (1613 bp) plasmid, pOX2000, isolated from a methicillin-resistant strain of Staphylococcus aureus has been determined. The sequence contains only one large ORF and the predicted amino acid sequence shows homology to the REP proteins of some other small staphylococcal plasmids. In addition there are two palindromic sequences, palA and palJ, that are similar to but not identical with the palindromes known from other staphylococcal plasmids to be involved in lagging strand initiation and possibly leading strand termination, respectively. Preliminary functional analysis of pOX2000 has been carried out by assessing the effect of interrupting the sequence at three unique restriction endonuclease sites. The plasmid pOX2000, and its relationship to other small staphylococcal plasmids, is discussed.     

A cadmium resistance plasmid, pXU5, in Staphylococcus aureus, strain ATCC25923

A 25.9-kb plasmid, pXU5, encoding high level cadmium resistance was isolated from Staphylococcus aureus strain ATCC25923. A labelled cadA probe from plasmid pI258 hybridised to a 2.3-kb EcoRI fragment of pXU5. pXU5 was incompatible with an S. aureus incompatibility group 1 plasmid.     

A second gene in the Staphylococcus aureus cadA cadmium resistance determinant of plasmid pI258.

Two open reading frames on a 3.7-kb BglII-XbaI fragment which encodes the Staphylococcus aureus cadA cadmium (and zinc) resistance determinant of plasmid pI258 were identified (G. Nucifora, L. Chu, T. K. Misra, and S. Silver, Proc. Natl. Acad. Sci. USA 86:3544-3548, 1989). The [35S]methionine-labelled protein products of the 727-amino-acid CadA ATPase and of the 122-amino-acid CadC polypeptide in Escherichia coli were identified by using the T7 RNA polymerase-promoter expression system. A truncated CadA polypeptide (402 amino acids) did not confer resistance in S. aureus but was expressed in E. coli under control of the T7 RNA polymerase-promoter. Removal of 678 nucleotides from the 5' end of the published sequence (which includes the cadA promoter) abolished resistance to cadmium, whereas a 146-nucleotide-shorter deletion was without effect. The cadC gene is needed in addition to cadA for full resistance to cadmium in S. aureus and Bacillus subtilis. cadC functions both in cis and in trans.     

Energy-dependent efflux of cadmium coded by a plasmid resistance determinant in Staphylococcus aureus.

Resistance of Staphylococcus aureus strain 17810R to Cd2+ appears to be due to a plasmid-coded Cd2+ efflux system. Complete efflux of Cd2+ after transfer of preloaded cells into Cd2+-free medium occurred in the resistant strain 17810R, but not in the plasmidless derivative strain 17810S. Net efflux was blocked by 2,4-dinitrophenol, N,N,-dicyclohexylcarbodiimide (DCCD), and incubation at 4 degrees C. The inhibition of Cd2+ efflux by DCCD paralleled a stimulation of net uptake in the resistant cells by this agent. Cd2+ efflux by the resistant strain was accompanied by a reversal of inhibition of respiration, whereas in the sensitive strain, inhibition of respiration was not reversed after transfer to Cd2+-free medium. Net Cd2+ uptake by strain 17810R was inhibited by p-chloromercuribenzoate. In Cd2+ contrast, Cd2+ uptake by the plasmidless strain 17810S was affected neither by p-chloromercuribenzoate nor by DCCD when added alone, but was blocked by a combination of these two agents. Valinomycin had no effect on the reduced Cd2+ uptake by the resistant strain, whereas nigericin stimulated uptake to values comparable to those of the untreated sensitive cells. With sensitive cells, valinomycin reduced Cd2+ uptake by about 50%, whereas nigericin was without effect. A possible mechanism of Cd2+ movements in both strains is discussed.     

A dyadic plasmid that shows MLS and PMS resistance in Staphylococcus aureus

Out of a collection of 56 Staphylococcus aureus clinical strains from 1971 to 1990 in Japan, we found one 1971 isolate, strain MS8968, harboring plasmid pMS97. A transductant strain, MS15009(pMS97), showed inducible resistance to a group of drugs, the so-called MLS antibiotics in the presence of a low concentration of erythromycin (EM). However, in the case of oleandomycin (OL), the strain showed resistance to another group of antibiotics: 14-membered macrolides (EM and OL), a 16-membered macrolide (mycinamicin I), and type B streptogramin, the so-called PMS antibiotics. Moreover, plasmid pMS97 contained an erm gene with universal primers specific for erm A, AM, B, BC, C, C′, and G and an msrA gene with primers specific for msrA. The first finding suggests that two genes encoding functionally different mechanisms for MLS and PMS resistance, erm and msrA, are present together within plasmid pMS97 originating from S. aureus.     

DNA homology between the arsenate resistance plasmid pSX267 from Staphylococcus xylosus and the penicillinase plasmid pI258 from Staphylococcus aureus

A 29.5-kb plasmid, pSX267, from Staphylococcus xylosus DSM 20267 was found to code for arsenate, arsenite, and antimony (III) resistance. The isolated plasmid was transformed into S. aureus, where the same resistances were expressed. It was of special interest to see whether pSX267 showed any DNA sequence homology with the well-studied penicillinase plasmid from S. aureus pI258, also conferring arsenate, arsenite, and antimony III resistance. By the use of the Southern blotting technique, it was found that DNA sequence homology exists in the region of arsenate, arsenite, and antimony resistance, in addition to the region where the origin of replication, the incompatibility, and the replication A function were mapped on pI258. This finding was confirmed by electron microscope heteroduplex analysis, which allowed a correlation between the genetic and physical maps of pI258 and pSX267. Duplex DNA was formed at the arsenate operon of pI258, with a length of 2.6 kb, and at the incompatibility and replication A region, comprising a length of 2.5 kb. Adjacent to this latter region, two small regions of DNA homology were present, with lengths of 0.2 and 0.27 kb. Both plasmids share approximately 20% DNA sequence homology. The DNA homology of the arsenate, arsenite, and antimony III resistance coding regions between pI258 and pSX267 indicate that these plasmid-determined resistance markers are highly conserved and distributed among different staphylococcal species.     

Antimicrobial drug resistance in Staphylococcus aureus isolated from cattle in Brazil

Isolates of Staphylococcus aureus obtained from apparently healthy cattle in the State of Paraiba, Brazil were characterized in relation to resistance to 21 antimicrobial agents. Among the 46 isolates obtained, resistance to penicillin was most frequent, followed by resistance to cadmium, streptomycin, arsenate, tetracycline, mercury, erythromycin and kanamycin/neomycin. All isolates were susceptible to fusidic acid, ethidium bromide, cetrimide, chloramphenicol, benzalkonium chloride, doxycycline, gentamicin, methicillin, mnocycline, novobiocin, rifamycin, tylosin and vancomycin. Only six isolates were susceptible to all the drugs tested. With respect to the antibiotics, multi-resistant isolates were uncommon. These results are probably a consequence of the peculiarities of local drug usage pressures. In relation to metal ions, resistance to mercury was rare while resistance to arsenate was relatively frequent, which contrasts with the situation for human Staph. aureus strains. After treatment with ethidium bromide, elimination of resistance to penicillin, tetracycline, streptomycin, erythromycin and cadmium was observed, which was consistent with the genetic determinants being plasmid-borne.     

Chlorine resistance of Staphylococcus aureus isolated from turkeys and turkey products

When tested in phosphate buffer at pH 7·0, strains of Staphylococcus aureus isolated from turkeys immediately after commercial slaughter were reduced in viability by at least 10000-fold following exposure to 1 mg/1 of free available chlorine for 2 min at 20°C. In contrast, corresponding isolates from samples taken later in the process, and apparently derived from strains capable of colonizing processing equipment ('endemic' strains), were reduced only ca 100-fold; some of these strains survived treatment with 2 mg/1 of free chlorine, when tested under the same conditions. The greater resistance of 'endemic' strains to chlorine may contribute to their persistence in the processing plant.     

Chlorine resistance of strains of Staphylococcus aureus isolated from poultry processing plants

Strains of Staphylococcus aureus which colonize defeathering machinery and become endemic within poultry processing plants appear to be resistant to the normal cleaning and disinfection processes. The resistance of endemic strains to chlorine was compared with that of isolates from the natural skin flora of poultry. Endemic strains were almost eight times more resistant and this was due primarily to their ability to grow in macroclumps but also to the production of an extracellular slime layer.     

Dynamics of antibiotic resistance of Staphylococcus aureus isolated from chronic osteomyelitis patients

The level of antibiotic resistance of 365 S.aureus strains, isolated from chronic osteomyelitis patients in 1990 and 2000, was determined. As revealed in this study, over 10 years the isolation rate of strains adapted to the antimicrobial action of all tested preparations increased in some degree. The increase of the resistance level to aminoglycosides was most significant. The proportion of polyresistant strains was 9.1% in 1990 and 30.5% in 2000. The isolation rate of methicillin-resistant S. aureus (MRSA) did not increase during these 10 years, but the range of preparations suitable for the treatment of MRSA-infected patients was shown to become essentially narrower. These data confirm the view on the possibility of further decrease in the effectiveness of the conservative treatment of chronic osteomyelitis due to the rapid growth of the antibiotic resistance of S. aureus.     

Identification and characterization of a shuttle plasmid with antibiotic resistance gene from Staphylococcus aureus

While studying antibiotic-resistant plasmids from multi-drug-resistant nosocomial Staphylococcus aureus strains, we isolated a small (2.889 kb) chloramphenicol-resistant (Cm(r)) plasmid, which was designated as pMC524/MBM. The molecular size of pMC524/MBM was close to that of pC194 (2.910 kb), a well-known Cm(r) staphylococcal plasmid. Unlike pC194, this plasmid can replicate and express itself efficiently and stably in Escherichia coli. However, Cm is needed for stable maintenance of pMC524/MBM in different hosts. In this study, the nucleotide sequences of these two plasmids were compared after sequencing of pMC524/MBM [EMBL Accession No. AJ312056 SAU312056]. Although these two plasmids have striking nucleotide sequence homology, the Plus Origin, Minus Origin, the replication protein (Rep), and the chloramphenicol acetyl transferase (Cat) have considerable variations. Possibly, these changes have modulated pMC524/MBM into an efficient shuttle-plasmid.     

Replication mutants of Staphylococcus aureus macrolide-lincosamide-streptogramin B resistance plasmid pT48

Copy-number mutants of Staphylococcus aureus macrolide-lincosamide-streptogramin B (MLS) resistance plasmid pT48 were isolated by their resistance to the non-inducing macrolide, tylosin. One mutant plasmid, pcopD3, showed a three- to five-fold cis-dominant increase in copy number, and nucleotide sequence analysis revealed that the mutant had a single base change within the replication region. All other pT48 mutants examined had the unusual phenotype of increased plasmid multimerization and elevated copy number. These mutants were effective in trans and DNA sequencing showed that plasmids with this phenotype were deleted in one of two ways. The deletions caused similar alterations to the C-terminus of the wild-type pT48 Rep protein. The two types of mutant Rep proteins terminate with the same pentapeptide sequence: Ala-Asn-Glu-Ile-Asp. The multimerization phenotype of these mutants can be explained by defective termination of rolling-circle type replication.     

Cleavage maps of a tetracycline plasmid from Staphylococcus aureus

The cleavage maps of a Staphylococcus aureus plasmid, pSN1 (2.75 megadaltons), conferring tetracycline resistance, were determined. Cleavage maps are given for HpaI and HindIII restriction endonucleases by using the single HpaII site as a reference point. Nucleases EcoRI, BamHI, SalI, and HaeIII have no sites on this plasmid.     

Drug resistance of Staphylococcus aureus strains, isolated from children with intestinal dysbacteriosis

The level of antibiotic-sensitivity of 73 S. aureus strains isolated from children with dysbacteriosis of the large intestine in an outpatient clinic was determined. The isolation rate of polyresistant strains was 44%. Methicillin-resistant S. aureus (MRSA) were isolated from 25 children (34.2%). 60% of MRSA strains could not be typed with the international set of phages. Among the strains capable of being lyzed by the phages the representatives of phage groups 3 and 4 prevailed. All MRSA strains were sensitive to vancomycin, 84-88% of the strains were sensitive to chloroamphenicol, rifampicin, spiramycin and neomycin, 80% of the strains were sensitive to fusidin and phosphomycin. The level of sensitivity of methicillin-sensitive S. aureus strains (MSSA) to different groups of antistaphylococcal antibiotics was higher. 36-64% of MRSA strains and 21-27% of MSSA strains were resistant to the action of curative bacteriophages. The suppression of obligate microflora was the risk factor in the development of staphylococcal infection of the gastrointestinal tract in children.