Epidemiologic genotyping of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis at a university hospital and comparison with antibiotyping and protein A and coagulase gene polymorphisms

A total of 124 methicillin-resistant Staphylococcus aureus (MRSA) isolates were ascertained at the University Hospital of the Canary Islands between January 1997 and April 2000. Genotyping included pulsed-field gel electrophoresis (PFGE) (SmaI digestion) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis for the coagulase (coa) and protein A (spa) genes. Antibiotic resistance was the main phenotypic marker correlated with genotyping results. Three main PFGE types were detected: A (with 12 subtypes), B (with 2 subtypes), and C. PFGE type A1 was the most commonly found (61% of isolates) and the one responsible for all the epidemic outbreaks. Other genetics markers used (coa and spa RFLPs) were significantly correlated with the PFGE types detected (P < 0.001). These PCR-RFLP assays were useful as molecular markers for a quick, preliminary study of MRSA outbreaks.     

Discrimination of epidemic and nonepidemic methicillin-resistant Staphylococcus aureus strains on the basis of protein A gene polymorphism.

The X region of the protein A gene of Staphylococcus aureus contains a highly polymorphic sequence which is composed of repeats of 24 bp. We used amplification by PCR to investigate whether this region could be used to discriminate between epidemic and nonepidemic methicillin-resistant S. aureus (MRSA) strains. Most epidemic MRSA strains (24 of 33) harbored more than seven repeats, while most nonepidemic MRSA strains (10 of 14) contained seven or fewer repeats. It is conceivable that a longer X region results in a better exposition of the Fc-binding region of protein A, thereby facilitating colonization of host surfaces and contributing to the epidemic phenotype.     

DNA polymorphisms in methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus.

Restriction fragment length polymorphisms in methicillin-susceptible and methicillin-resistant (MRSA) strains of Staphylococcus aureus isolated in the same hospital over a 4-month period were studied by using SmaI and ApaI digestion of genomic DNA and pulsed-field gel electrophoresis. Each of the 20 methicillin-susceptible strains had a unique SmaI pattern, but the 27 MRSA strains showed only seven SmaI patterns. More than half of the SmaI fragments in all of these seven patterns were identical, as were those in the patterns from two unrelated MRSA strains. Digestion with ApaI, which cuts staphylococcus DNA into at least twice as many fragments, confirmed the results obtained with SmaI. Lastly, the plasmid contents of MRSA strains showing identical SmaI and ApaI electrophoretic patterns were not identical. These results are interpreted as supporting the hypothesis that all MRSA strains arose from a single clone and emphasize the need to use several methods in epidemiological investigations of MRSA outbreaks.     

Clonal dissemination of epidemic methicillin-resistant Staphylococcus aureus in Belgium and neighboring countries

Objectives   To determine the diversity of pulsed-field gel electrophoresis (PFGE) types among epidemic strains of methicillin-resistant Staphylococcus aureus (MRSA) recovered in Belgium, France, Germany and The Netherlands over the period 1981–94.
Methods   MRSA strains collected in a multicenter survey in Belgium ( n = 171) and from reference laboratories in neighboring countries ( n = 102) were characterized by PFGE analysis using the Sma I enzyme.
Results   In total, 32 PFGE types were found. Epidemic PFGE type 1, first recognized in 1984, accounted for 82% of Belgian strains (87% of hospitals) and 51% of European MRSA strains. Four other internationally epidemic PFGE types (types 8, 10, 11 and 12) were less widely disseminated and more recently detected (1991–94), each recovered from two or three countries. International spread of two PFGE types was linked to transfer of colonized patients to Dutch hospitals from another country where this type was frequently recovered.
Conclusions   Genotypic analysis indicated widespread distribution of several outbreak-associated MRSA strains over large European regions, which was in some instances related to interhospital patient transfer. These findings underscore the need for standardized international surveillance and control of MRSA transmission between healthcare institutions across Europe.     

Phenotypic characterization of epidemic versus sporadic strains of methicillin-resistant Staphylococcus aureus.

Forty strains of methicillin-resistant Staphylococcus aureus (MRSA) were divided on the basis of their epidemiologic behavior into two subgroups, sporadic MRSA (SMRSA) and epidemic MRSA (EMRSA) strains. The strains were examined for binding of 125I-labelled fibronectin, vitronectin, collagen, Fc fragments of immunoglobulin G, and fibrinogen. A significant difference between EMRSA and SMRSA strains was found for binding of 125I-labelled fibrinogen and for Fc fragments of immunoglobulin G, (P < 0.05). No significant difference in the binding of 125I-labelled fibronectin and collagen was found between EMRSA and SMRSA strains. The binding of 125I-labelled vitronectin to MRSA strains was found to be aspecific. Capsular serotypes of the strains were determined with monoclonal antibodies against capsular types 5 and 8. Strains could be divided into the following four groups: types 5, 8, and 5/8 and nontypeable. More nontypeable strains were found in the EMRSA group (66.6%). Significantly more EMRSA strains (79%) than SMRSA strains (44%) produced alpha-toxin (P < 0.025). Logistic regression analysis using a combination of the parameters 125I-labelled immunoglobulin G binding, capsular type, and alpha-toxin production predicted the epidemic character with a sensitivity of 83% and a specificity of 75%.     

Numerical analysis of electrophoretic protein patterns of methicillin-resistant strains of Staphylococcus aureus.

A total of 50 strains of Staphylococcus aureus, including 41 methicillin-resistant S. aureus (MRSA) strains, were characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins. The protein patterns contained 40-50 discrete bands and were highly reproducible. Partial patterns were used as the basis of a computer-assisted numerical analysis. The MRSA strains clustered into four phenons at the 83% similarity level; and further division of phenon 1, at the 86% similarity level, resulted in a total of six clusters. All of the MRSA isolates from an MRSA epidemic in the United Kingdom were found to cluster in phenon 1 together with 9 of the 12 MRSA isolates from eastern Australia and 3 other MRSA isolates from the United Kingdom. The remaining three eastern Australian isolates clustered separately in phenon 2. Phenon 3 appeared to be exclusive to strains that were both susceptible and resistant to methicillin and that reacted with group V phages, and phenon 4 comprised 11 isolates, all of which were other MRSA isolates from the United Kingdom. We conclude that computer-assisted numerical analysis by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins provides additional criteria for the study of the epidemiology and the evolution of MRSA.     

Protein A and coagulase expression in epidemic and non-epidemic Staphylococcus aureus.

Strains of Staphylococcus aureus were divided into groups on the basis of antimicrobial sensitivity and epidemiology and tested for protein A expression in a simple microtitre test, which detected the non-immunological binding of immunoglobulin to protein A on whole cells of S aureus. Isolates of the methicillin resistant strain prevalent in south east England (EMRSA) showed a low expression of protein A compared with the other strains of methicillin resistant S aureus (MRSA), other multiple resistant strains, and sensitive strains. Protein A and coagulase expression in 27 strains of MRSA from 15 countries associated with hospital outbreaks were compared with 27 strains of MRSA from 11 countries reported to be sporadic isolates. Twenty four of the 27 outbreak associated MRSA showed low expression of protein A and high expression of coagulase. Conversely, sporadic strains generally gave higher levels of protein A and a wide variety of coagulase reactions. The results suggest that many epidemic strains of MRSA may have phenotypic characteristics that distinguish them from sporadic strains.     

Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms.

Staphylocoagulase, a major phenotypic determinant of Staphylococcus aureus, exists in multiple allelic forms, in part because of the existence of gene variants within the 3'-end coding region. This region contains a series of repeating 81-bp DNA sequences which differ both in the number of tandem repeats and the location of AluI restriction sites among different isolates. Utilizing this finding, we developed a novel typing method for S. aureus based on polymerase chain reaction amplification of the variable region of the coagulase gene followed by AluI restriction enzyme digestion and analysis of restriction fragment length polymorphism (RFLP). Among 30 S. aureus isolates studied initially, a total of 10 distinct RFLP patterns were observed. There was excellent correlation of the RFLP patterns with typing of these isolates by multilocus enzyme electrophoresis at 20 chromosomal loci. This coagulase RFLP method was used to analyze an additional 39 S. aureus isolates and successfully traced the source of an outbreak of methicillin-resistant S. aureus infections at a local hospital.     

Staff carriage of epidemic methicillin-resistant Staphylococcus aureus.

Twenty-six nurses were repeatedly screened for carriage of epidemic methicillin-resistant Staphylococcus aureus (EMRSA) immediately before and after duty periods in which they solely attended six patients widely colonized with two EMRSA strains distinguishable by plasmid analysis. EMRSA carriage was detected in 13 nurses. Three EMRSA carriage patterns emerged: transient carriage in 12 nurses, when the EMRSA was isolated from noses or fingers of nurses after duty but was gone before their next day's duty; short-term nasal carriage, seen on occasion in 4 of these 12 nurses, when EMRSA carriage was detected on two consecutive screens; and persistent nasal carriage, seen in 1 nurse only, when the EMRSA was seen on more than two consecutive occasions. All but one of these incidents of carriage could be explained by close patient, rather than environmental, exposure and occurred despite an intensive control programme. Transient or short-term carriage in nurses probably resulted in transfer of the EMRSA between patients. Staff decontamination should be considered following a period of cohort nursing of EMRSA patients, especially if staff members are shortly to nurse unaffected patients. Our findings may explain some of the difficulties in controlling EMRSA.     

Evolutionary relationships between sporadic and epidemic strains of healthcare-associated methicillin-resistant Staphylococcus aureus

National surveillance of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates by pulsed-field gel electrophoresis (PFGE) typing allowed identification of rarely occurring 'sporadic' isolates with patterns significantly distinct from those of major epidemic clones of methicillin-resistant S. aureus (MRSA) circulating in Belgian hospitals. The aim of the present study was to compare the genetic background, antibiotic susceptibility profile and in vitro growth rates of 36 MRSA isolates with either 'epidemic' or 'sporadic' PFGE profiles to identify factors that could be involved in the epidemic behaviour of S. aureus . Sequence analysis of seven housekeeping genes (multilocus sequence typing) and seven surface-associated genes, combined with staphylococcal cassette chromosome mec (SCC mec ) typing and spa typing results, segregated sporadic isolates into four groups: (1) isolates phylogenetically distant from epidemic HA-MRSA clones that possessed several properties of community-acquired MRSA strains; (2) isolates derived from the same methicillin-susceptible S. aureus ancestor as epidemic isolates but possessing a distinct type of SCC mec ; and (3) and (4) isolates that were closely related to epidemic strains, either as recent descendants of these or as intermediate evolutionary steps between epidemic HA-MRSA strains and their putative ancestors. Sporadic isolates did not show slower growth in vitro than epidemic isolates. These findings suggest that the SCC mec type and insertion/deletion of other mobile genetic elements may be involved in modulating the epidemic behaviour of MRSA strains of similar genetic background, independently of fitness cost.     

Usefulness of PCR-restriction fragment length polymorphism typing of the coagulase gene to discriminate arbekacin-resistant methicillin-resistant Staphylococcus aureus strains

We compared the results of two typing methods for 678 strains of methicillin-resistant Staphylococcus aureus and methicillin-susceptible S. aureus. PCR-restriction fragment length polymorphism typing of the coagulase gene was a more reliable method than coagulase serotyping from the viewpoint of arbekacin resistance.     

Use of coagulase gene (coa) repeat region nucleotide sequences for typing of methicillin-resistant Staphylococcus aureus strains

Coagulase gene (coa) short sequence repeat region sequencing was used to measure relatedness among a collection of temporally and geographically diverse methicillin-resistant Staphylococcus aureus isolates. The results show that coa polymorphism is free of strong selective pressure and has a low index of variation that may be useful for long-term epidemiological investigations. coa typing is a useful addition to spa typing for analysis of S. aureus, including methicillin-resistant strains.     

Comparative evaluation of identification systems for testing methicillin-resistant strains of Staphylococcus aureus.

Several commercial systems are available to distinguish between Staphylococcus aureus and the coagulase-negative species of the Micrococcaceae family. Four latex agglutination systems (Accu-Staph, SeroSTAT, Staphaurex, and Staphylatex) and two hemagglutination systems (Hemastaph and Staphyloslide) were compared for their performance in the rapid identification of 232 isolates of staphylococci, including 114 of methicillin-resistant S. aureus. Accu-Staph, Staphaurex, and Staphyloslide correctly identified 100% of the methicillin-resistant S. aureus isolates; Hemastaph and Staphylatex, 99.1%; and SeroSTAT, 94.7%. Most reactions were easy to interpret, although 15% of the SeroSTAT reactions were weak. Autoagglutination occurred only with isolates of coagulase-negative staphylococci. False-positive reactions were rare and occurred only with systems which did not detect autoagglutination. Five of these six systems appear to be adequate for the rapid identification of S. aureus, including methicillin-resistant isolates.     

Location of the coagulase gene in Staphylococcus aureus.

Evidence is presented for the chromosomal location of the coagulase determinant in most strains of Staphylococcus aureus. By the use of a pour-plate technique, transduction of the capacity to produce coagulase to a coagulase-negative mutant of S. aureus was studied. The frequencies of transduction were low unless the transducing phage was exposed to ultraviolet irradiation and the recipient was lysogenised with the transducing phage. Attempts to transfer the coagulase gene from S. aureus to S. epidermidis were not successful.     

Clonal diffusion and evolution of mecA and Tn554 polymorphisms in methicillin-resistant Staphylococcus aureus in Italy

The purposes of the present study were to track the geographic spread of 69 MRSA strains in Italy recovered from 7 hospitals in four towns; to detect the clonal identities among the isolates by a combination of multiple genomic typing methods and to measure temporal trends in clonal types between 1984 and 1998. Our results showed the spread of three major clones among the MRSA isolates of 1984-1995 period: the most represented MRSA clone carried the PFGE pattern A, the mecA polymorph II and had no homology with Tn554 (II::NH::A); most of these isolates were susceptible to the macrolides,being similar to the historically " archaic" MRSA strains; the clone typed I::E::A, carried the PFGE pattern A, the mecA polymorph I and Tn554E commonly defined as "Iberian clone"; unique clone, showing an uncommon PFGE pattern E. the mecA polymorph II and the Tn554 E (II::E::E) and were characterized by a uniform susceptibility to tetracycline and rifampin. During 1997-98 the representation of this clone increased instead of the classical "Iberian clone". A new multi-resistant MRSA strain, carrying the PFGE pattern B (or B'), the mecA polymorph XI and Tn554 polymorph B (XI::B::B), called "Brazilian clone", increased from being absent (1984-95) to 48%. Our molecular data show an Italian MRSA "scenario" far from the common European trends and clearly documented the spread of an archaic clonal type (II::NH::A) in 1984-95, the arrival and rapid increase of Brazilian done in 1997-98 and the chronological and geographical spread of a unique (H::E::E) called "Italian clone", instead of the widely spread Iberian MRSA clone.     

Lysogenicity of methicillin-resistant strains of Staphylococcus aureus

The lysogenic status of 23 strains of methicillin-resistant Staphylococcus aureus, isolated at the Royal Prince Alfred Hospital, Sydney, since 1980, was studied. Twenty strains, belonging to the four predominant phage types isolated in this hospital, carried the same lysogenic phage which we have designated C. Three other phages were isolated from five strains belonging to phage type 84/85/90. The presence of phage C had little effect on the phage-typing pattern of the strains. Similarly, lysogenization with the other three phages did not result in a significant change in phage-typing patterns. However, when strain 1489, isolated in 1969, was lysogenized with these three phages, there was a change in phage-typing pattern. Lysogenization of this strain with phage 47T resulted in a marked loss of sensitivity to both group-I and group-III phages. The lysogenic status of these methicillin-resistant strains of S. aureus was compared with that of strains isolated between 1967 and 1970. There was no evidence that the strains isolated recently were either related to, or derived from, the earlier ones.     

Multiple-locus variable-number tandem-repeat assay analysis of methicillin-resistant Staphylococcus aureus strains

Our objective was to determine if a multiple-locus variable-number tandem-repeat assay (MLVA) for Staphylococcus aureus could predict pulsed-field gel electrophoresis (PFGE) types (i.e., USA types), thus allowing us to replace PFGE with a simpler and more rapid typing method. One hundred three well-characterized isolates representing 13 major lineages of S. aureus were tested by both PFGE and MLVA. MLVA was performed using a rapid DNA extraction technique and PCR primers for sdrCDE, clfA, clfB, sspA, and spa. PFGE was performed with genomic DNA fragments generated using SmaI, as per CDC protocols. Banding patterns were analyzed both visually and with BioNumerics software. All isolates were typeable with MLVA and PFGE. MLVA patterns were highly reproducible. PFGE separated the isolates into 13 types with 42 subtypes. Using any band difference to designate a novel MLVA type, MLVA produced 45 types, including 9 clusters containing multiple isolates. Using BioNumerics and a cutoff of >75% relatedness, MLVA produced 28 types, 11 of which contained >1 isolate. Epidemiologically related outbreak isolates of USA300-0114 from five states clustered in one MLVA pattern. USA100 isolates were present in several unrelated (<40%) MLVA types. A cutoff of >80% separated outbreak strains of USA300-0114 into three distinct MLVA types. MLVA did not differentiate community methicillin-resistant S. aureus (MRSA) lineages (USA300, USA400, USA1000, and USA1100) from health care MRSA lineages (USA100, USA200, or USA500). The ability of MLVA to differentiate among strains that are indistinguishable by PFGE may be of epidemiologic value and warrants further study.     

Efficacies of rapid agglutination tests for identification of methicillin-resistant staphylococcal strains as Staphylococcus aureus.

Four commercially available rapid agglutination tests for the identification of Staphylococcus aureus were compared with the tube coagulase test for the identification of 300 methicillin-resistant isolates of staphylococci. Isolates tested included 207 methicillin-resistant S. aureus and 93 coagulase-negative staphylococci, collected from five medical centers. Strain variability was documented by phage typing and antimicrobial susceptibility patterns. Results of rapid identification tests ranged between 82 and 86% sensitivity, significantly poorer than the 98% sensitivity which the tube coagulase test provided.     

Clonal analysis of methicillin-resistant Staphylococcus aureus strains from intercontinental sources: association of the mec gene with divergent phylogenetic lineages implies dissemination by horizontal transfer and recombination.

Genetic relationships among 254 isolates of Staphylococcus aureus resistant to methicillin recovered between 1961 and 1992 from nine countries on four continents were determined by analyzing electrophoretically demonstrable allelic variation at 15 chromosomal enzyme loci. Fifteen distinctive electrophoretic types, marking clones, were identified. The mec gene is harbored by many divergent phylogenetic lineages representing a large portion of the breadth of chromosomal diversity in the species, a result that is interpreted as evidence that multiple episodes of horizontal transfer and recombination have contributed to the spread of this resistance determinant in natural populations. Isolates recovered in the United Kingdom, Denmark, Switzerland, Egypt, and Uganda in the 1960s are of a single multilocus enzyme genotype and probably are progeny of an ancestral methicillin-resistant clone. There is geographic variation in the frequency of recovery of the common methicillin-resistant clones, an observation that may in part explain reported regional differences in natural history correlates of resistant organisms.     

Loss of the mecA gene during storage of methicillin-resistant Staphylococcus aureus strains

The mecA gene was lost in 36 (14.4%) of 250 methicillin-resistant Staphylococcus aureus isolates after 2 years of storage at -80 degrees C with the Microbank system (Pro-lab Diagnostics, Austin, Tex.). Further analysis of 35 of these isolates confirmed loss of the mecA gene in 32 isolates. This finding has important implications for the management of strain collections.