Evaluation of laboratory tests for detection of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

Few studies evaluating susceptibility testing of methicillin-resistant staphylococci have included isolates of Staphylococcus epidermidis, a known pathogen in many types of serious infections. We tested 175 S. epidermidis and 95 Staphylococcus aureus isolates to determine the most sensitive procedures for detecting methicillin-resistant staphylococci. Reference procedures included agar dilution with methicillin and 4% NaCl in the agar and broth microdilution with methicillin and 2% NaCl in cation-supplemented Mueller-Hinton broth. After 24 h of incubation, the results from both methods correlated well and were within 1 log2 dilution for all isolates tested. Only one-half of all resistant isolates (92 of 183) were detected at 18 h by using the standard disk diffusion technique with 5-micrograms methicillin disks, and even fewer were detected with 10-micrograms methicillin disks and newly recommended zone-size criteria. However, the standard disk diffusion method with 4% NaCl in the agar increased the sensitivity and specificity for identification of the proper phenotype to greater than 92%. The spread plate and new spot techniques, both using agar with 4% NaCl, were also sensitive methods. Of 47 S. epidermidis isolates tested against oxacillin, 6 (13%) were oxacillin susceptible but methicillin resistant. Two automated systems, the Automicrobic system (Vitek Systems) and MicroScan (American MicroScan), as well as two broth screening systems available from Remel and Austin Biological Laboratories, failed to detect several resistant isolates, depending on the species.     

Genetic diversity among methicillin-resistant Staphylococcus epidermidis (MRSE)

We selected 106 methicillin-resistant Staphylococcus epidermidis (MRSE) and 22 methicillin-susceptible S. epidermidis (MSSE) hospital isolates--each with a different PFGE pattern--for more detailed documentation of genetic diversity. The 106 MRSE isolates showed extensive variation in the SmaI DNA fragments hybridizing with the DNA probe for mecA, the molecular size of which varied from as low as 20 kb up to over 500 kb. Parallel variation was also observed in the size of DNA fragments hybridizing with the chromosomal genes orfX and gyrA, and this was also observed in MSSE isolates. In contrast, SmaI fragments associated with the housekeeping genes murE and aroE, both located distantly from orfX, showed little size variation. Typing for the mec complex and ccr identified 10 different SCCmec structures and a large number of strains (21 isolates) that were non-typeable. The majority of strains studied (36%) carried a SCCmec type IV-like structure, including strains with non-related PFGE profiles. On the other hand, closely related strains often carried different types of SCCmec. The findings indicate that the acquisition and/or loss of mobile genetic elements, including various structural types of SCCmec, may occur frequently in the vicinity of the orfX gene on the S. epidermidis chromosome.     

Treatment of meningitis due to methicillin-resistant Staphylococcus epidermidis with linezolid

Methicillin-resistant Staphylococcus epidermidis (MRSE) can cause nosocomial meningitis in the presence of prosthetic devices. Vancomycin is the treatment of choice, but its penetration into the cerebrospinal fluid is poor, especially in cases without severe meningeal inflammation. We successfully used linezolid to treat a case of posttraumatic MRSE meningitis with a low-level inflammatory response. Therapeutic effectiveness was documented microbiologically and by the simultaneous measurement of linezolid levels in serum and cerebrospinal fluid.     

糖尿病足感染中耐甲氧西林表皮葡萄球菌SCCmec分型临床研究

目的 研究分离于糖尿病足感染(DFI)溃疡创面的耐甲氧西林表皮葡萄球菌(MRSE)的SCCmec分型特点及不同型别的耐药特征.方法 2008年1月-2010年6月从天津某医院390例DFI溃疡中分离出70株表皮葡萄球菌,提取细菌的DNA,用聚合酶链反应(PCR)方法检测mecA基因,并对MRSE进行SCCmec分型;采...     

Molecular characterization of methicillin-resistant Staphylococcus epidermidis clones: evidence of geographic dissemination

Denmark and Iceland are countries where the frequency of methicillin-resistant Staphylococcus aureus is very low due to strict infection control and restrictive antibiotic use policies. In contrast, methicillin-resistant S. epidermidis (MRSE) continues to be isolated as a nosocomial pathogen. The molecular typing by pulsed-field gel electrophoresis (PFGE) of 136 MRSE isolates from five hospitals in Denmark and 94 MRSE isolates from one hospital in Iceland collected in 1997 and 1998 defined 40 different patterns. Closely related PFGE types were found in isolates recovered in Iceland, Denmark, Mexico, Uruguay, Greece, and Cape Verde, evidencing for the first time the geographic clonal dissemination of MRSE strains. The large majority (87.4%) of the MRSE isolates studied were multiresistant.     

Infection and/or colonization by methicillin-resistant Staphylococcus epidermidis (MRSE).

We analyzed the parameters predictive of identification of methicillin-resistant Staphylococcus epidermidis (MRSE) in sample performed in hospitalized patients. One hundred six Staphylococcus epidermidis strains (60 MRSE and 46 MSSE) were collected. Three variables were independently linked to MRSE isolation in multivariate analysis: hospitalization during the month preceding the current admission; on-going antimicrobial therapy before sampling, and on-going infection at the time of sampling. MRSE isolation was associated with a poor vital prognosis. The air and surfaces sampling in the rooms of two patients with nasal MRSE carriage yielded the same strains as those carried by the patient, and could play a role in the epidemiological chain of hospital-acquired MRSE infections.     

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.     

Extreme genetic diversity of methicillin-resistant Staphylococcus epidermidis strains disseminated among healthy Japanese children

For the past few years, we have been observing the dissemination of methicillin-resistant staphylococci in the community. From 2001 to 2003, an evaluation of nasal samples from 1,285 children in five day-care centers and two kindergartens in three districts in Japan revealed that methicillin-resistant coagulase-negative staphylococci (MRC-NS) have been widely disseminated in the Japanese community. Their prevalence is much greater than community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). Forty-nine children (3.81%) were colonized with MRSA, whereas 390 children (30.35%) were colonized with MRC-NS. These MRC-NS strains predominantly harbored a pair of cassette chromosome recombinase types A2 and B2 (ccrAB2). Of these, 40.8% harbored type IVa staphylococcal cassette chromosome mec (SCCmec) elements, a distinct/characteristic type of SCCmec in pandemic clones of CA-MRSA. Interestingly, there was also a high frequency of nontypeable strains which possessed atypical structures compared to previous SCCmec types. Among the MRC-NS, the majority of strains (63.59%) were methicillin-resistant Staphylococcus epidermidis (MRSE). Their genotypes, as judged from pulsed-field gel electrophoresis (PFGE), were highly diverse. They were so diverse that there was no sign of an immediate transmission of any MRSE clone among children in the same institutions. In a previous report, we expounded that a few CA-MRSA clones with distinct SCCmec types were disseminated among children in the same institutions. Au contraire, with the case of CA-MRSE, there was no single genotype of CA-MRSE disseminated among children even in the same institution or class.     

Phage typing of Staphylococcus epidermidis.

Thirteen phages were isolated from lysogenic cultures of Staphylococcus epidermidis from a clinical laboratory and used to type 223 clinical isolates of this organism. The 18 phages isolated in The Netherlands were used to type these same cultures. No correlation was observed between phage type, biotype, or clinical source of isolation. At phage concentrations of 100 times the routine test dilution, 35.0% of the cultures were typable with out phages and 21.5% were typable with the phages from The Netherlands. When only cultures in biotype 1 were considered, 43.3 and 24.1% of 141 cultures were typable with our phages and those from The Netherlands, respectively. The lytic reactions obtained with our phages were generally stronger and easier to read and the lytic patterns were, almost invariably, shorter. The typability of untypable cultures was increased 12.0% by incubation at 45 C prior to phage typing and 20% by heat shock (55 C for 5 min) prior to typing. Phage typing 5 subcultures of 20 typable cultures on 5 successive days showed that the lytic patterns were reproducible. The present status of phage typing S. epidermidis and the work needed to obtain a set of typing phages for epidemiological studies of infections by this organism are discussed.     

Detection of anti-teichoic acid immunoglobulin G antibodies in experimental Staphylococcus epidermidis endocarditis.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of rabbit immunoglobulin G (IgG) antibodies to purified cell wall teichoic acids from the Staphylococcus aureus Lafferty strain and three strains of coagulase-negative staphylococci. Significant immunological cross-reactivity occurred only between the teichoic acid of S. aureus and one coagulase-negative preparation. The ELISA was used to determine the serum IgG response to Staphylococcus epidermidis in a rabbit model of aortic valve endocarditis. Blood samples were drawn before inoculation and then every 5 days until death or sacrifice at 32 to 35 days postinoculation. Valve vegetations were culture positive at autopsy in 16 (59%) of the 27 catheterized rabbits. Antibody titers in this culture-positive group and the culture-negative group began to rise as early as day 6. Although both groups demonstrated an antibody response, the culture-positive group attained a significantly higher titer on days 26 and 31. Antibodies also rose in a control group of rabbits without a heart catheter but which were inoculated with bacteria. Again, the antibody titer was significantly less than that for the culture-positive group. This ELISA may be useful for the diagnosis of coagulase-negative staphylococcal infections in humans.     

Serum opacity factor of Staphylococcus epidermidis.

Three Staphylococcus epidermidis strains produced a factor giving rise to opacity in different sera but not in albumin. Serum opacity factor was resistant to age and heat and active in acidic media.     

Cervical adenitis caused by Staphylococcus epidermidis.

Staphylococcus epidermidis, a human commensal, is a common cause of bacteremia in immunocompromised patients with indwelling medical devices. We report a case of isolated cervical adenitis caused by S. epidermidis in an immunocompetent patient and comment on the presumed pathogenesis.     

Detection of methicillin-resistant Staphylococcus aureus by polymerase chain reaction]

The low-affinity penicillin-binding protein (PBP 2') is associated with methicillin-resistance of Staphylococcus aureus and its structural gene (mecA) not present in methicillin-susceptible S. aureus could be detected by the polymerase chain reaction (PCR) method, in which a 533 bp region of mecA was amplified and detected by agarose gel electrophoresis. Survey for the mecA gene in 210 clinical isolates of S. aureus revealed that, while there was a gross correlation between the presence of the mecA gene and the resistance level to beta-lactams, three strains of mecA (+) tested showed beta-lactam susceptibility similar to those of mecA (-) strains. These three strains did not produce a detectable amount of PBP 2' constitutively nor inducibly, which was the cause of their high susceptibility to beta-lactams. One of them yielded a typical methicillin-resistant variant at a low frequency with a concomitant recovery of PBP 2' production when the bacterial cells of high density were spread onto an agar plate containing 10 micrograms/ml of oxacillin. These findings suggested that typical methicillin resistant S. aureus occurred during chemotherapy with beta-lactam antibiotics even when resistant strains could not be detected by the susceptibility test and thus all mecA (+) strains including those with high susceptibility should be precisely detected.     

Comparison of epidemiologic markers for Staphylococcus epidermidis.

Cultures of Staphylococcus epidermidis from the eyes or nose of the same individual were compared by their antimicrobial phenotype, Staph-Ident (Analytab Products, Inc., Plainview, N.Y.) profile number, phage type, and plasmid profile to determine which parameters provide the most compelling data for their identity. None of the parameters alone provided this type of information. The most conclusive data for the identity of strains resulted when two cultures had the same long phage type and identical or similar plasmid profiles. The presence of a large, slowly migrating plasmid band(s) in a culture that agreed with its pair in all other parameters and, in all likelihood, was the same strain casts doubt in some instances on the reliability of the plasmid profile alone for strain identification in an epidemiologic study.     

Gastrointestinal carriage of methicillin-resistant Staphylococcus aureus.

Nasal and rectal cultures were taken from all patients with methicillin-resistant Staphylococcus aureus identified on routine cultures obtained because of clinical indications. Of 117 patients studied over a 3-year period, 70 (60%) had rectal colonization and 62 (53%) had nasal colonization. Rectal colonization, probably reflecting gastrointestinal carriage, may be a source of transmission of methicillin-resistant S. aureus in hospitalized patients and may be difficult to eradicate.     

Opsonic requirements of Staphylococcus epidermidis

The opsonic requirements of 18 strains of Staphylococcus epidermidis were compared in pooled normal human serum and in peritoneal dialysate from patients undergoing continuous ambulatory peritoneal dialysis (CAPD). A serum concentration of 2.5% gave optimal opsonisation. The opsonisation of all strains was antibody- and complement-dependent, and there were no significant differences in the pattern of their opsonic requirements. Peritoneal-dialysis effluent from uninfected patients was a poor source of opsonins because of the low levels of immunoglobulin G and of the C3 component of complement it contained. Growth of S. epidermidis in peritoneal-dialysis effluent rather than in broth did not alter its opsonic requirements. Strains from patients undergoing CAPD and suffering from peritonitis were not more resistant to opsonisation and phagocytic killing than those from other sources.     

Emergence of homogeneously methicillin-resistant Staphylococcus aureus.

Forty-seven clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), collected between 1986 and 1990 from 29 institutions, were analyzed for susceptibility to various antibiotics. Twenty-six strains were homogeneously methicillin resistant (i.e., greater than or equal to 10% of the cells in these strains were able to grow on Mueller-Hinton agar containing 50 micrograms of methicillin per ml). The MICs of gentamicin, clindamycin, trimethoprimsulfamethoxazole, methicillin, and imipenem for homogeneous MRSA strains were higher than those for heterogeneously resistant strains. Both types of strains were, for the most part, susceptible to vancomycin and trimethoprim-sulfamethoxazole. Ciprofloxacin-resistant MRSA strains were not isolated prior to 1988 but made up 40% of the post-1987 strains. The level of methicillin resistance correlated well with the imipenem MIC, suggesting that susceptibility to imipenem may serve as a marker to identify and monitor the prevalence of homogeneous MRSA strains.     

Rapid identification of Staphylococcus epidermidis

A panel of Minitek sugar disks, consisting of trehalose, mannitol, xylose, and sucrose, was evaluated for its ability to identify blood culture isolates of Staphylococcus epidermidis (SE). Using a heavy suspension of organism in Mueller-Hinton broth, 50 microL was pipetted onto each disk in wells of a flat-bottomed microtiter tray. The tray was covered, incubated in a moist chamber in non-CO2 at 35 degrees C, and examined after 5 and 24 hours. A color change of yellow or orange was positive; no color change (red) was negative. Expected reactions for SE were as follows: negative trehalose, mannitol, and xylose; positive, sucrose. On evaluation of 227 coagulase-negative staphylococci (CNS) at 5 and 24 hours, the panel had a sensitivity of 94 and 96%, specificity of 92 and 89%, predictive value of positive tests of 97 and 96%, and predictive value of negative tests of 84 and 87%. This panel offered an inexpensive and convenient method for differentiating SE from the other CNS in five hours.     

Staphylococcus epidermidis biofilms: importance and implications.

The coagulase-negative staphylococci and, in particular, Staphylococcus epidermidis, have emerged as major nosocomial pathogens associated with infections of implanted medical devices. These organisms, which are among the most prevalent bacteria of the human skin and mucous membrane microflora, present unique problems in the diagnosis and treatment of infections involving biofilm formation on implanted biomaterials. Epidemiological data that address whether invasive S. epidermidis strains can be traced to commensal organisms or an endemic occurrence of distinct strains with enhanced virulence have important implications for the implementation of appropriate infection control measures. An extracellular polysaccharide adhesin represents a key virulence determinant in S. epidermidis and is required for biofilm formation. Production of this adhesin, which is encoded by the ica operon, is subject to phase variable regulation (ON <---> OFF switching). Recent advances in understanding the molecular events controlling polysaccharide adhesin synthesis and the potential clinical implications of its phase variable regulation are outlined. Further research in this area may contribute to the development of novel strategies for therapeutic intervention. Finally, in addition to antibiotic prophylaxis, preventive strategies to control S. epidermidis medical device-related infections are focusing on the development of improved biomaterials and physical electrical barriers to impede bacterial colonisation.