Staphylococcus aureus phage typing, antimicrobial susceptibility patterns and patient data correlated using a personal computer: advantages for monitoring the epidemiology of MRSA.

Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA) is an important nosocomial pathogen. A problem encountered in the control of staphylococcal nosocomial infection is the difficulty correlating data available from antimicrobial susceptibility patterns (usually available locally) with phage typing patterns which are generally determined in a reference laboratory. Data problems are exaggerated because many patients have numerous samples taken over long periods of time. This paper describes the use of a database, 'DataEase', on a personal computer to correlate available data with patient demographics and to present the resulting information in whatever format is required. The information might be presented as the phage pattern of each isolate from patients in a particular unit per month or as a combination of the phage types and antimicrobial susceptibility patterns of isolates from a particular unit. The system can be used to generate a list of all isolates for phage typing in a format that allows the phage typing pattern to be recorded directly onto the list worksheet. In addition to providing clinically relevant reports, the system simplifies the collation of epidemiological information, data from which showed that since 1987 the incidence of MRSA has been rising in the group of hospitals served by this laboratory; MRSA of phage type 83A became a problem during 1990 and 1991; the incidence of gentamicin-sensitive phage type non-typable MRSA increased fourfold between 1988 and 1991; and the incidence of gentamicin-resistant MRSA rose sharply in 1992.

Importation of methicillin-resistant Staphylococcus aureus from Baghdad to Dublin and subsequent nosocomial spread.

We report the spread of a methicillin- and gentamicin-resistant Staphylococcus aureus strain (MGRSA) from the Middle East and its subsequent dissemination within two hospitals in Dublin. The index case, a 30-year-old male with serious blast injuries was transferred from a Baghdad hospital to a Dublin hospital in May 1985. He was heavily infected with two MGRSA strains, one of which spread and was responsible for numerous episodes of nosocomial infection. This strain was very similar to MGRSA isolates recovered in a Baghdad hospital during 1984. This imported strain has now spread to two hospitals in our group causing sepsis. This report emphasizes the difficulty of detecting an imported strain in an endemic area, but above all points to the potential for spread when there is considerable movement of patients and personnel.

Staphylococcus aureus bacteriophages mediating the simultaneous lysogenic conversion of beta-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion.

A new group of serotype F bacteriophages of Staphylococcus aureus has been found which mediates the simultaneous triple-lysogenic conversion of enterotoxin A, staphylokinase and beta-lysin. The phages were recovered fro methicillin-resistant strains of S. aureus isolated in Irish hospitals between 1971 and 1988 and from strain PS42-D, which has been used as the propagating strain for the S. aureus typing phage 42D since before 1965. The molecular mechanism of triple conversion mediated by three of these phages was determined by molecular cloning, restriction endonuclease site mapping and hybridization analysis, and compared with the mechanism of beta-lysin and staphylokinase conversion mediated by the serotype F, double-converting phase phi 13. THe genetic determinants mediating expression of enterotoxin A (entA) and staphylokinase (sak) were cloned from the DNA of the triple-converting phage and expression of the cloned determinants detected in Escherichia coli and S. aureus. The entA and sak determinants were closely linked in the phage DNA adjacent to the phage attachment site (attP) in each case and furthermore, the sak determinant of phage phi 13 was also located near its attP. The restriction maps of the entA-, sak- and attP-containing DNA regions of the three triple-converting phages were very similar to each other and to the corresponding sak- and attP- containing DNA region of phage phi 13. Hybridization analysis using a cloned beta-lysin determinant (hlb) and cloned attP-containing DNA fragments as probes demonstrated that beta-lysin conversion mediated by the triple-converting phages and phage phi 13 was caused by insertional inactivation of the chromosomally encoded hlb determinant by orientation-specific integration of phage DNA following lysogenization.

A new methicillin- and gentamicin-resistant Staphylococcus aureus in Dublin: molecular genetic analysis.

In June 1985 two new strains of methicillin- and gentamicin-resistant Staphylococcus aureus (MGRSA) were isolated in a Dublin hospital. Of these, one strain spread rapidly, affecting a total of 65 patients during the following 18 months, and subsequently spread to a second Dublin hospital. Detailed laboratory studies, including plasmid screening, plasmid restriction enzyme digest pattern analysis, hybridisation analysis, location of resistance determinants, and bacteriophage typing with a set of experimental S. aureus typing phages, demonstrated that the "new" MGRSA organisms, termed Phenotype III Dublin isolates, were completely distinct from, and unrelated to, the MGRSA strains responsible for serious nosocomial infections in Dublin hospitals during the decade before June 1985. These Phenotype III isolates were very similar to MGRSA organisms isolated in a Baghdad hospital during 1984. Data from plasmid curing, plasmid transfer and hybridisation experiments indicated that 20% of the Phenotype-III isolates expressed chromosomally encoded, high level resistance to ethidium bromide (MIC 120 micrograms/ml), and that this was possibly due to chromosomal integration of a penicillinase-like plasmid.

Cloning and expression in Escherichia coli and Staphylococcus aureus of the beta-lysin determinant from Staphylococcus aureus: evidence that bacteriophage conversion of beta-lysin activity is caused by insertional inactivation of the beta-lysin determinant.

The beta-lysin determinant (Hlb) from Staphylococcus aureus CN6708 was cloned in Escherichia coli K-12 using the bacteriophage replacement vector lambda L47.1. The Hlb determinant was localised to a 1250 base pair DNA sequence by cloning fragments from a Hlb+ recombinant phage into the plasmid vectors pACYC184 and pBR322 in E. coli K-12, and by the subsequent construction and analysis of several sub-clones, in vitro deletion and Tn5 insertion mutations. E. coli cells harbouring Hlb+ plasmids expressed readily detectable levels of beta-lysin and sphingomyelinase activity, which were located in the cytoplasm. Two polypeptides of molecular weight 38,000 and 33,000 which were encoded by the Hlb determinant were detected in E. coli minicells, but only the 33,000 dalton protein was detected in immunoblotting experiments with specific anti-beta-lysin serum. Hybridisation analysis with probes made from the cloned Hlb determinant and from DNA of the staphylokinase-converting phage phi 13, indicated that bacteriophage conversion of S. aureus to loss of beta-lysin activity is due to insertion of phi 13 DNA into or adjacent to the beta-lysin determinant. A shuttle plasmid was used to transfer the cloned Hlb determinant into a beta-lysin negative strain of S. aureus where the wild-type chromosomal determinant was inactivated by lysogenic conversion. Beta-lysin activity was readily detected in supernatants of S. aureus harbouring the cloned determinant.