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Nucleotide sequence and genetic characterization of staphylococcal bacteriophage L54a int and xis genes.

The nucleotide sequence of a staphylococcal bacteriophage L54a DNA fragment containing genes involved in site-specific recombination was determined. Mutations generated by in vitro mutagenesis were used to map and characterize the int and xis genes. The site-specific recombination functions are tightly clustered within a 1.75-kilobase stretch of DNA fragment with the gene order of attP-int-xis. The int and xis genes are transcribed divergently. The Int protein deduced from the nucleotide sequence has a molecular weight of 41,000. Int is a basic protein with 354 amino acids of which 72 are basic and 38 are acidic. The Xis protein consists of only 59 amino acids with a molecular weight of 7,180. Unlike the Xis proteins of the lambdoid bacteriophages which are all basic proteins, L54a Xis is an acidic protein containing 13 acidic and 8 basic amino acids. The Int protein is required in both integrative and excisive reactions, whereas Xis is only required in excisive reaction. A well-conserved 40-residue region, including three perfectly conserved residues found in 15 site-specific recombinases of the integrase family that have been characterized, was also found in the L54a Int protein.

Amino Acid Sequence↗

Transformation of a conditionally peptidoglycan-deficient mutant of Staphylococcus aureus with plasmid DNA.

A simple and reliable method for polyethylene glycol-induced plasmid transformation of a temperature-sensitive peptidoglycan-deficient mutant of Staphylococcus aureus is described. The procedure uses strains carrying the tofA372 mutation grown under conditions that yield osmotically fragile cells capable of efficient wall regeneration. The peptidoglycan-deficient cells were transformed with plasmids pE194 and pI258 at frequencies comparable with those obtained with protoplasts prepared with lysostaphin treatment. A readily portable tofA372 mutation was constructed by isolating an insertion of the erythromycin resistance transposon Tn551 adjacent to tofA372. tofA372 was shown by protoplast fusion and transformation analyses to be in the gene order hly-421-omega [Chr::Tn551]1059-tofA372-uraB232-omega [Chr::Tn916]1101-thrB106 on the chromosome of S. aureus NCTC 8325.

Bacteriophages↗

Cloning and expression of cadD, a new cadmium resistance gene of Staphylococcus aureus.

A cadmium resistance gene, designated cadD, has been identified in and cloned from the Staphylococcus aureus plasmid pRW001. The gene is part of a two-component operon which contains the resistance gene cadD and an inactive regulatory gene, cadX*. A high degree of sequence similarity was observed between cadD and the cadB-like gene from S. lugdunensis, but no significant similarity was found with either cadA or cadB from the S. aureus plasmids pI258 and pII147. The positive regulatory gene cadX* is identical to cadX from pLUG10 over a stretch of 78 codons beginning at the N terminus, but it is truncated at this point and inactive. Sequence analysis showed that the cadmium resistance operon resides on a 3,972-bp element that is flanked by direct repeats of IS257. The expression of cadD in S. aureus and Bacillus subtilis resulted in low-level resistance to cadmium; in contrast, cadA and cadB from S. aureus induced higher level resistance. However, when the truncated version of cadX contained in pRW001 is complemented in trans with cadX from plasmid pLUG10, resistance increased approximately 10-fold suggesting that the cadmium resistance operons from pRW001 and pLUG10 are evolutionarily related. Moreover, the truncated version of cadX contained in pRW001 is nonfunctional and may have been generated by deletion during recombination to acquire the cadmium resistance element.

Amino Acid Sequence↗

Staphylococcus aureus isolated in cases of impetigo produces both epidermolysin A or B and LukE-LukD in 78% of 131 retrospective and prospective cases.

Clinical symptoms of impetigo and staphylococcal scalded skin syndrome may not only be expressed as the splitting of cell layers within the epidermis but are often accompanied by some localized inflammation. Toxin patterns of Staphylococcus aureus isolates originating from patients with impetigo and also from those with other primary and secondary skin infections in a retrospective isolate collection in France and a prospective isolate collection in French Guiana revealed a significant association (75% of the cases studied) of impetigo with production of at least one of the epidermolysins A and B and the bicomponent leucotoxin LukE-LukD (P < 0.001). However, most of the isolates were able to produce one of the nonubiquitous enterotoxins. Pulsed-field gel electrophoresis (PFGE) of genomic DNA hydrolyzed with SmaI showed a polymorphism of the two groups of isolates despite the fact that endemic clones were suspected in French Guiana and France. The combination of toxin patterns with PFGE fingerprinting may provide further discrimination among isolates defined in a given cluster or a given pulsotype and account for a specific virulence. The new association of toxins with a clinical syndrome may reveal principles of the pathological process.

Adolescent↗

Thymineless bacteriophage induction in Staphylococcus aureus. II. Specific transduction of constitutive and inducible erythromycin resistance.

Specific transduction of inducible (eroA) and constitutive (eroB) erythromycin resistance is mediated by thymineless induced lysates from derivatives of Staphylococcus aureus strain 8325(N)thy. Both loci can coexist in the same cell but segregate by transduction or transformation. The gene(s) is probably integrated in the recipient chromosome and excised at thymine starvation.

Chromosomes, Bacterial↗

Ciprofloxacin-resistant methicillin-resistant Staphylococcus aureus in New York health care facilities, 1988. The New York MRSA Study Group.

The emergence in 1988 of ciprofloxacin-resistant methicillin-resistant Staphylococcus aureus (MRSA) in New York City was studied in nine hospitals and eight nursing homes. Of the 43 hospitalized patients studied, 21 were admitted from home, while nine of the 12 nursing home patients were transferred from a hospital. Twenty-four of the 55 patients had been treated previously with ciprofloxacin, and 26 had an identifiable risk factor for a nosocomial infection. MRSA was a contributing factor in at least five of the 21 deaths. MRSA resistance to ciprofloxacin was detected within three months of the drug's commercial availability, apparently emerged independently at a number of the health care facilities, and has become widespread. If such resistance is found in a health care facility, ciprofloxacin may not be useful as a first line antibiotic.

Adult↗

[Genetic transfer of methycilline resistance in Staphylococus epidermidis and Staphylococcus aureus strains in mixed cultures].

In mixed cultures of staphylococci a transfer of the resistance to methicillin and penicillinase plasmids as well as tetracycline and chloramphenicol plasmids was investigated. It was shown that the resistance to methicillin was transferred in mixed cultures from one strain of S. aureus to another and from S. epidermidis to S. aureus. In both cases transfer of methicillin resistance required, the presence of penicillinase plasmid in recipient or donor strain. In the case of other markers transmission was independent. Moreover it was shown that the transfer of resistance genes in mixed cultures was mediated by bacteriophage of the serologic group A.

Chloramphenicol↗

[Interspecies lysogenization in staphylococci: transfer of bacteriophage converting enterotoxin A from a clinical strain of Staphylococcus aureus to Staphylococcus intermedius].

The ability of lysogenization was examined of 50 S. intermedius strains and of 77 strains belonging to 14 different species of coagulase-negative staphylococci using 8 enterotoxin A converting bacteriophages isolated from S. aureus. All the examined bacteriophages showed lytic activity against at least 1 of 11 susceptible strains of S. intermedius to them. Lytic activity towards coagulase-negative staphylococci was observed for 6 of 8 examined bacteriophages. Two bacteriophages were active against 1 of 9 examined S. capitis strains, one against 1 of 11 examined S. haemolyticus strains, four against 1 of 6 examined S. lugdunensis strains, three against 1 of 6 examined S. warneri strains and one against 1 of 5 examined S. xylosus strains. Lysogenization with bacteriophage f421-1 able to convert positively enterotoxin A and staphylokinase and negatively beta-haemolysin of one S. intermedius strain was successful. S. intermedius lysogenized with phi 421-1 was able to produce both enterotoxin A and staphylokinase and lost ability to produce beta-haemolysin. Our results showed a broad lytic spectrum and interspecies host range of some S. aureus bacteriophages and the ability of interspecies transfer of bacteriophages between S. aureus and S. intermedius.

Enterotoxins↗

A gentamycin resistance plasmid in Staphylococcus aureus.

Drug resistance in Staphylococcus aureus is frequently mediated by non-chromosomal DNA molecules referred to as R plasmids. So far, resistance to aminosidic antibiotics was observed with streptomycin [8] and kanamycin-neomycin [3]. Resistance to gentamycin has not been reported in a recent survey by Lacey [7]. In this paper, we describe a strain of S. aureus resistant to gentamycin-kanamycin-tobramycin-sisomycin, and experiments which suggest that the genetic determinant of these resistance characters is plasmid borne.

Anti-Bacterial Agents↗