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.     

Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction.

A simple and reliable method using a polymerase chain reaction (PCR) was devised to identify methicillin-resistant staphylococci. By using lysates of the strain to be tested as templates and 22-mer oligonucleotides as primers, a 533-bp region of mecA, the structural gene of a low-affinity penicillin-binding protein (PBP 2'), was amplified by PCR and detected by agarose gel electrophoresis. Results obtained by this method were compared with those obtained by broth microdilution MIC determination for 210 and 100 clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, respectively. Of 99 mecA-negative S. aureus isolates, 100% of the strains were methicillin susceptible and 98% of the strains were oxacillin susceptible. Three strains (3%) of 111 mecA-positive S. aureus isolates exhibited almost the same susceptibility to beta-lactams as the mecA-negative ones and did not produce detectable amounts of PBP 2' despite the presence of the mecA gene. One of them yielded typically methicillin-resistant variants at a low frequency with concomitant recovery of PBP 2' production. The mecA gene was also found in coagulase-negative Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, and Staphylococcus caprae and conferred resistance on most of the bacteria.     

Isolation of methicillin-resistant coagulase-negative staphylococci from chickens.

Methicillin-resistant coagulase-negative staphylococci were isolated from the nares and skin of 1- to 8-week-old healthy chickens in three flocks from a farm. Isolation of methicillin-resistant coagulase-negative staphylococci was positive for 72 (25.7%) of the 280 chickens tested, with the frequency varying from 2.2 to 100% according to flock. A total of 45 appropriate isolates were selected and subjected to identification. Of the 45 methicillin-resistant coagulase-negative staphylococcal isolates selected, 37 were identified as Staphylococcus sciuri, 5 were identified as Staphylococcus epidermidis, and 3 were identified as Staphylococcus saprophyticus. The distribution of the species was different among the flocks. Comparative analysis of the SmaI-digested chromosomal DNA by pulsed-field gel electrophoresis revealed that the isolates could have originated from a single clone of each of S. sciuri and S. saprophyticus and three clones of S. epidermidis. By two methods based on the PCR technique, the mecA gene was detected in all five representative isolates of each methicillin-resistant coagulase-negative staphylococcal clone. The nucleotide sequence of a PCR fragment obtained from an isolate of S. sciuri was completely identical to the corresponding region of mecA genes reported in human methicillin-resistant Staphylococcus aureus isolates and Staphylococcus epidermidis isolates. The representative methicillin-resistant coagulase-negative staphylococcal isolates were resistant to many beta-lactam antibiotics, and some isolates were also resistant to macrolide and aminoglycoside antibiotics. This is the first evidence of the existence of methicillin-resistant coagulase-negative staphylococci from animals possessing the mecA gene.     

Staphylococcal resistance to aminoglycosides before and after introduction of amikacin in two teaching hospitals

A prospective study was conducted to determine the prevalence of aminoglycoside-resistant Staphylococcus aureus and coagulase-negative staphylococci before and after the introduction of amikacin as the sole aminoglycoside used in our burn unit, adult intensive care unit, and neonatal intensive care unit. Pharyngeal or endotracheal cultures, as well as superficial surveillance cultures, were collected weekly during the following four study periods: all units for 4 months before amikacin introduction, all units 4 to 8 months after, all units 12 to 13 months after, and the neonatal intensive care unit 30 months after. A total of 2,613 strains of coagulase-negative staphylococci and 316 strains of S. aureus were obtained from 916 patients. During the course of the study, amikacin-resistant coagulase-negative staphylococci increased from 0 to 22%, colonizing 43% of patients, whereas no amikacin-resistant S. aureus was detected. During the preamikacin survey, 68% of the coagulase-negative staphylococci and 12% of the S. aureus strains were resistant to tobramycin and gentamicin. This resistance did not decrease after amikacin was introduced. Initially, 83% of the aminoglycoside-resistant coagulase-negative staphylococci were resistant to both tobramycin and gentamicin. During the last surveillance this value dropped to 40%, and 48% of the strains had become resistant to all three aminoglycosides. Resistance to aminoglycosides, including amikacin, develops quickly in coagulase-negative staphylococci from clinical areas where these antimicrobial agents are widely used. However, aminoglycoside resistance in S. aureus is much less frequent.     

Methicillin-resistant Staphylococcus aureus: a three-year epidemiological and microbiological survey of high-risk patients

In order to assess the frequency, and epidemiological and microbiological features, of respiratory and blood stream infection due to methicillin-resistant Staphylococcus aureus in high-risk patients, all S. aureus strains cultured from reliable clinical specimens (respiratory secretions obtained by tracheo-bronchial aspirate or bronchoalveolar lavage, or blood cultures), were prospectively evaluated over a three-year period, in six inpatient wards selected on the ground of an elevated frequency of severe and/or nosocomially-acquired infections, because of the prevalence of immunocompromised patients, organ transplant recipients, or need of intensive care. Repeatedly positive cultures obtained from a single patient within 30 days were considered as one isolate. Of 507 S. aureus strains responsible for pneumonia or sepsis in the selected wards, 317 (62.5%) proved methicillin-resistant, in absence of significant variations throughout the study period, and according to the specimen origin. Methicillin-resistant S. aureus strains prevailed over sensitive ones in all examined wards (from a 95% rate of the respiratory intensive care unit, to 55.9% of the pneumology department), save the neonatal and pediatric intensive care unit (41.4%). Most of methicillin-resistant S. aureus isolates were recovered from lower airways, compared with blood cultures (p<.0001). The majority of the 317 methicillin-resistant strains were found in the general intensive care unit (42.6%), followed by the pneumology department (18%), and the respiratory intensive care unit (16.4%). Among methicillin-resistant S. aureus strains, a broad variation of sensitivity to other antimicrobial agents was observed: from 3.3% of erythromycin, to 76.9% of chloramphenicol, and 79.7% of cotrimoxazole; glycopeptide antibiotics remained effective against all cultured strains. In our three-year survey of more than 500 episodes of documented staphylococcal infection involving high-risk patients, methicillin resistance was a very common feature, observed at a greater frequency than that reported in literature studies focusing on surgical, pneumological, or intensive care settings. A long-term microbiological monitoring of high-risk inpatient wards may allow a continued update of local antimicrobial susceptibility maps, and significantly add to both chemoprophylaxis and empiric treatment strategies of patients which are either immunocompromised or hospitalized for a long period.     

Identification of coagulase-negative staphylococci other than Staphylococcus epidermidis by automated ribotyping

As routine identification of coagulase-negative staphylococci is problematic, the performance of automated ribotyping was evaluated for identification of coagulase-negative staphylococci other than Staphylococcus epidermidis. In total, 177 isolates were tested, comprising 149 isolates from blood samples, 15 isolates that were not identified by internal transcribed spacer (ITS)-PCR in a previous study, and 13 reference strains. The identification results were compared with those obtained by the API 20 Staph system, with standard phenotypic and molecular methods as reference. Most (n = 166; 93.8%) isolates were identified correctly by automated ribotyping. For 61 isolates, API 20 Staph and ribotyping were in agreement, but for 105 isolates, ribotyping provided correct identification and API 20 Staph did not. Four isolates not identified by automated ribotyping were recognised correctly by API 20 Staph. The remaining seven isolates could not be identified by either of the two methods. Automated ribotyping was able to distinguish Staphylococcus capitis reliably from Staphylococcus caprae. The results demonstrate the value of automated ribotyping for identification of coagulase-negative Staphylococcus (CoNS) isolates from human sources and may help to clarify the clinical relevance of CoNS species. In addition, automated ribotyping was able to detect polymorphisms that may be useful for epidemiological purposes within S. capitis, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus simulans, S. caprae, Staphylococcus warneri, Staphylococcus lugdunensis, Staphylococcus schleiferi, Staphylococcus sciuri, Staphylococcus pasteuri and Staphylococcus xylosus.     

DNA fingerprinting by pulsed-field gel electrophoresis is more effective than ribotyping in distinguishing among methicillin-resistant Staphylococcus aureus isolates.

Pulsed-field gel electrophoresis (PFGE) after SmaI restriction of DNA from 239 methicillin-resistant Staphylococcus aureus isolates (from 142 patients) produced 26 different fingerprints. The deduced chromosome sizes ranged from 2,200 to 3,100 kb (+/- 100 kb). A total of 81 isolates taken from 65 patients were then typed by PFGE and ribotyping with ClaI, EcoRI, and HindIII. Ribotypes were less discriminating than PFGE. Ribotyping did not discriminate isolates from a given PFGE fingerprint into different subsets. PFGE may be a more effective epidemiological tool than ribotyping for the typing of methicillin-resistant S. aureus strains.     

Presence of a novel DNA methylation enzyme in methicillin-resistant Staphylococcus aureus isolates associated with pig farming leads to uninterpretable results in standard pulsed-field gel electrophoresis analysis

Genomic DNA from methicillin-resistant Staphylococcus aureus isolates recovered from pigs and their caretakers proved resistant to SmaI digestion, leading to uninterpretable results in standard pulsed-field gel electrophoresis. This is the result of a yet unknown restriction/methylation system in the genus Staphylococcus with the recognition sequence CCNGG.     

Phenotypic and genotypic mupirocin resistance among Staphylococci causing prosthetic joint infection

Mupirocin MICs and mupA presence were determined in 108 staphylococci causing prosthetic joint infection. Zero of 35 isolates (0%) of methicillin-susceptible Staphylococcus aureus, 4/15 (27%) methicillin-resistant S. aureus isolates, 3/16 (19%) methicillin-susceptible coagulase-negative staphylococci, and 11/42 (26%) methicillin-resistant coagulase-negative staphylococci were mupirocin resistant. mupA was detected in all five high-level mupirocin-resistant staphylococci and one mupirocin-susceptible staphylococcus.     

Investigation of an outbreak caused by methicillin-resistant Staphylococcus aureus in a cardiovascular surgery unit by ribotyping, randomly amplifed polymorphic DNA and pulsed-field gel electrophoresis

An outbreak caused by rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) in an intensive care unit for cardiovascular surgery was investigated by phenotypic and genotypic methods. Fourteen isolates were collected during a 2-month period from clinical and environmental specimens in the unit recently re-opened after reconstruction. The isolates were tested for antibiotic susceptibility patterns and genotyped by automated ribotyping, randomly amplified polymorphic DNA-PCR (RAPD) analysis and pulsed-field gel electrophoresis (PFGE). Automated ribotyping applying EcoRI digestion proved to be of no value in separating the isolates. In contrast, PFGE grouped the isolates into four clusters different from the reference strain. These results fully correlated with the antibiograms. Twelve of the isolates were grouped into two clonally related clusters. RAPD analyses grouped the isolates into five clusters. Except for two isolates of one patient, which had different RAPD patterns, PFGE and RAPD analyses presented very similar results. The results verified the usefulness of PFGE in studies of MRSA epidemics. A combination of these two methods reduces the time to identification of an outbreak and increases the accuracy in detection of intraspecies differences.     

Clonality of clinical methicillin-resistant Staphylococcus epidermidis isolates in a neonatal intensive care unit

OBJECTIVE: Study of the clonality of methicillin-resistant Staphylococcus epidermidis responsible of epidemic infections in a neonatal intensive care unit. PATIENTS AND METHODS: All S. epidermidis isolates (mecA+) were collected during the epidemic period (December 2003-September 2004) from different pathological products of newborns. Isolates were characterized by genotyping in pulsed-field gel electrophoresis and by electrophoretic profiles obtained by PCR-based analysis of inter-IS256 spacer polymorphisms. RESULTS: Twenty methicillin-resistant S. epidermidis isolates were collected from newborns during the epidemic period and represented 41.6% of the total isolates of S. epidermidis, which is the first Staphylococcus species isolated from the unit. These isolates were collected from blood cultures (80%), vascular catheters (5%), pus (10%), and intra-tracheal tube (5%). Six genotypic profiles were individualized: type A, type B, type C, type D, type E, and type F, with clear dominance of type A. Five different PCR patterns were found with poor correlation to genotypes defined by PFGE. CONCLUSION: Neonatal nosocomial outbreak of methicillin-resistant S. epidermidis was caused by multiple clones of this species with predominance of one epidemic and multiresistant clone. This clone may be transmitted between babies and was able to persist in the unit. PCR IS 256 proved to be less discriminative than PFGE for typing MRSE.     

Delta toxin activity in coagulase-negative staphylococci from the bowels of neonates.

Coagulase-negative staphylococci are prominent in stools of neonates in some intensive care units and have been associated with necrotizing enterocolitis. A plausible mediator of bowel damage is delta-like toxin, which is produced in vitro by most coagulase-negative staphylococci, but factors influencing the expression of toxin in the bowel are unknown. We examined 105 coagulase-negative staphylococcus isolates from stools of neonates by using an enzyme-linked immunosorbent assay and detected delta toxin production by 92 isolates (88%). The amount present in 18-h broth cultures varied over 100-fold, from 933 to 125,000 ng/ml. All broths positive by enzyme-linked immunosorbent assay except one caused hemolysis of human erythrocytes. The threshold concentration for consistent cytotoxicity to fibroblasts was greater than or equal to 24,000 ng/ml. Only 56% of Staphylococcus epidermidis isolates were capable of producing this much toxin, and these were more often obtained from premature infants in intensive care than from healthy full-term infants (P = 0.003) and were more often resistant to multiple antibiotics (P less than 0.001). Cultures grown anaerobically seldom caused hemolysis (4 positive of 29 tested; P less than 0.001) because potency of the toxin was decreased (at least ninefold for S. epidermidis isolates). We conclude that only a portion of the fecal coagulase-negative staphylococci tested produced enough delta toxin in vitro to be cytotoxic, that such isolates have accumulated in our intensive care nursery, and that development of toxin-mediated bowel injury may also require a favorable redox potential within the host bowel.     

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.     

Molecular characterization of clinical methicillin-resistant Staphylococcus aureus isolates in South Africa

Eighty-two percent of 320 clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates from various infection sites collected throughout South Africa were separated into five major globally prevalent clusters by SmaI pulsed-field gel electrophoresis, spa, and SCCmec typing. Only one Panton-Valentine leukocidin-positive isolate was detected. This is the first detailed MRSA epidemiology study for the whole country.     

Latex agglutination-negative methicillin-resistant Staphylococcus aureus recovered from neonates: epidemiologic features and comparison of typing methods.

An unusual strain of methicillin-resistant Staphylococcus aureus (MRSA) was repeatedly isolated from infants in a newborn special care unit (NBSC) and a newborn intensive care unit. Between January 1989 and March 1990, approximately 100 isolates from infected or colonized infants were recovered. Surveillance cultures taken during this time revealed a 20% colonization rate, which was defined as recovery of MRSA from the nares, umbilicus, or groin. Isolates were identified as S. aureus by tube coagulase reactivity and heat-stable nuclease production but were unreactive in a latex agglutination assay. Representative isolates that were collected during the outbreak and that were found to share the latex agglutination assay-negative phenotype were compared by antibiogram (12 isolates), bacteriophage typing (20 isolates), capsular polysaccharide typing (30 isolates), and plasmid as well as chromosomal DNA analyses (20 isolates). All isolates known to be associated with the outbreak had nearly identical antibiograms and were notably susceptible to clindamycin. Staphylococcal bacteriophage typing was not useful in determining the relatedness of the isolates, since the majority were nontypeable. Plasmid pattern analysis revealed one large plasmid (approximately 100 kb) of equivalent size among the isolates. Capsular polysaccharide typing revealed that 14 of 30 isolates tested were type 5. Isolates identified in children at two other hospitals in the city which were also unreactive by the latex agglutination assay and clindamycin susceptible had plasmid and antibiogram patterns identical to those of isolates from the NBSC. Pulsed-field gel electrophoresis of restriction enzyme-digested genomic DNAs from the outbreak isolates demonstrated identical patterns which could be clearly differentiated from those of other unrelated MRSA. The strain from the NBSC is, therefore, unique and underscores the need for caution in interpreting the latex agglutination reactivities of MRSA isolates.     

Staphylococcus aureus reservoirs and transmission routes in a Portuguese Neonatal Intensive Care Unit: a 30-month surveillance study

Although Staphylococcus aureus is a major cause of outbreaks in neonatal intensive care units (NICUs), there are no studies on the epidemiology of S. aureus isolates responsible for infection in Portuguese NICUs. Between July 2005 and December 2007, a total of 54 methicillin susceptible S. aureus (MSSA) isolates were recovered from 16 infected infants, parents, health care workers (HCWs), and the environment in a level III NICU. Isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing. Virulence determinants were detected by multiplex polymerase chain reaction. Three major MSSA clones were endemic in the NICU, representing 70% (n=38) of the isolates: PFGE type A-ST5 (n=17); type B-ST30 (n=12); and type C-ST1 (n=9). Leukotoxins and hemolysins were present in all isolates, although none of them carried PVL. HCWs, plastic folders protecting clinical files, and mothers' nipples were identified as potential reservoirs and/or vehicles of dissemination of S. aureus. Consequently, additional infection control measures were implemented in this NICU.     

Methicillin-resistant Staphylococcus aureus producing Panton–Valentine leukocidin in a retrospective case series from 12 French hospital laboratories, 2000–2003

A retrospective analysis of hospital laboratory databases for 2000-2003 found that 0.4-1.0% of methicillin-resistant Staphylococcus aureus isolates had an antibiotic susceptibility pattern associated previously with the production of Panton-Valentine leukocidin (PVL). Of 81 isolates of this type, 35 were available for molecular testing. Each of the 35 available isolates carried the PVL genes, and 33 of these 35 isolates had an identical SmaI pulsed-field gel electrophoresis pattern.     

Comparison of MIDI Sherlock system and pulsed-field gel electrophoresis in characterizing strains of methicillin-resistant Staphylococcus aureus from a recent hospital outbreak.

An outbreak of methicillin-resistant Staphylococcus aureus infections at the University of Utah Health Sciences Center occurred over a 7-month period. While the isolates phenotypically appeared to be similar in gross morphology and have similar Vitek antibiotic susceptibility patterns, two additional methods of strain characterization were evaluated to enhance the epidemiological investigation: pulsed-field gel electrophoresis and gas chromatography with the MIDI Sherlock system. Sherlock uses gas chromatography to qualitatively and quantitatively analyze the cellular fatty acid composition of organisms and creates two-dimensional plots based on principal-component analysis to define groups of closely related organisms. All isolates were also evaluated by digesting their chromosomal DNAs with the low-frequency-cutting enzyme SmaI and separating the restriction fragments by contour-clamped homogeneous electric field gel electrophoresis. Sample preparation for this pulsed-field gel electrophoresis included a novel cell lysis procedure involving achromopeptidase, greatly reducing the turnaround time. Isolates tested were recovered from the following: 45 suspected outbreak patients, 6 hospitalized patients believed to be unrelated to the outbreak, 6 patients from outside the hospital, and one health care practitioner implicated in the outbreak. Of 45 phenotypically similar suspect strains, 43 clustered tightly on the Sherlock two-dimensional plot. All outbreak patient isolates were also identical by pulsed-field gel electrophoresis with the exception of the same two outliers identified by Sherlock. In this epidemiologic investigation, we found an excellent correlation between the Sherlock and pulsed-field gel electrophoresis results for strain characterization of methicillin-resistant S. aureus.     

Application of molecular typing methods to characterize nosocomial coagulase-negative staphylococci collected in a Greek hospital during a three-year period (1998-2000)

A total of 143 methicillin-resistant coagulase-negative staphylococci (MR-CNS) collected between 1998 and 2000 at the University Hospital of Patras, Greece, were characterized by antibiogram and genomic typing to define the clonal types endemic in this hospital and their evolution during the 3-year period. These isolates corresponded to 93 methicillin-resistant Staphylococcus epidermidis (MRSE) and 50 other MR-CNS, which were isolated from patients in different wards, exclusively from blood and catheter tips cultures. Pulsed-field gel electrophoresis (PFGE) of SmaI macrofragments and hybridization of ClaI digests with mecA and murE DNA probes were performed. The application of these methodologies demonstrated the existence, persistence and spread of MRSE, MR-Staphylococcus haemolyticus, and MR-Staphylococcus hominis clones in this hospital, whereas the SmaI/murE hybridization pattern was shown to be a valuable tool for the MRSE identification.     

Epidemiology and susceptibility of 3,051 Staphylococcus aureus isolates from 25 university hospitals participating in the European SENTRY study

A total of 3,051 methicillin-susceptible Staphylococcus aureus (MSSA) isolates and methicillin-resistant S. aureus (MRSA) isolates in Europe were compared. MRSA isolates constituted 25% of all isolates and were more prevalent in southern Europe. MRSA isolates appeared to be more prevalent in intensive care units than in outpatient departments. Only a small minority of MSSA isolates were multidrug resistant, whereas the majority of MRSA isolates were multidrug resistant.