Performance of four slide agglutination methods for identification of Staphylococcus aureus when testing methicillin-resistant staphylococci.

Four commercially available slide agglutination systems for identifying Staphylococcus aureus were compared with the conventional slide (clumping factor) and tube coagulase tests. The systems evaluated included Bacto Staph Latex (Difco Laboratories, Detroit, Mich.), Staphyloslide (BBL Microbiology Systems, Cockeysville, Md.), Mini ID Accu-Staph (Carr-Scarborough Microbiologicals, Inc., Decatur, Ga.), and Staphaurex (Wellcome Diagnostics, Research Triangle Park, N.C.). A total of 338 clinical isolates, including methicillin-resistant S. aureus (n = 149), methicillin-susceptible S. aureus (n = 78), methicillin-resistant, coagulase-negative staphylococci (n = 45), and methicillin-susceptible, coagulase-negative staphylococci (n = 66), were tested by each method. The slide test for clumping factor, the 4-h tube coagulase test, Bacto Staph Latex, Staphyloslide, Mini ID Accu-Staph, and Staphaurex detected 212 (93.4%), 218 (96%), 223 (98.2%), 223 (98.2%), 221 (97.4%), and 224 (98.7%) of the S. aureus (44% methicillin-resistant) isolates, respectively. There were no false-positive results with any of the methods when the 111 strains of coagulase-negative staphylococci were tested. The results of this evaluation suggest that the four slide identification methods tested can provide rapid and accurate identification of methicillin-resistant S. aureus strains.     

Evaluation of different disk diffusion/media combinations for detection of methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci

In order to find a disk diffusion method with both high sensitivity and specificity for determination of methicillin resistance primarily for S. aureus but also for coagulase-negative staphylococci we screened several methodological variants using a material of 66 S. aureus comprising of 11 methicillin-susceptible, 18 borderline-resistant, and 37 methicillin-resistant strains. Only four of the combinations studied performed with both high sensitivity and specificity. Two of these, the Columbia agar +4.5% NaCl and Mueller Hinton agar +2% NaCl combined with a 5 microg oxacillin disk, confluent inoculum and 24 h incubation at 35 degrees C were further evaluated using 105 MRSA and 91 mecA-negative S. aureus and 193 clinical isolates of coagulase-negative staphylococci. The Columbia agar +4.5% NaCl performed excellently for both S. aureus and coagulase-negative staphylococci. For Columbia agar +4.5% NaCl using a 5 microg oxacillin disk we suggest an interpretive zone diameter of R < or =15 mm and S > or =16 mm for S. aureus and R < or =24 mm and S >or =26 mm for coagulase-negative staphylococci. The Mueller Hinton agar +2% NaCl performed well for coagulase-negative staphylococci but for S. aureus at least three (3%) very major errors were found, making this method less attractive.     

Incidence of constitutive and inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci in a community and a tertiary care hospital

The incidences of inducible clindamycin resistance at two hospitals (an inner-city hospital and a suburban community hospital) were 7 and 12% for methicillin-resistant Staphylococcus aureus, 20 and 19% for methicillin-susceptible S. aureus, and 14 and 35% for coagulase-negative staphylococci, respectively. Given the variability of inducible resistance to clindamycin found in our two hospitals, we conclude that susceptibility testing of staphylococci should include the disk diffusion induction test (D-test).     

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.     

Does nasal cocolonization by methicillin-resistant coagulase-negative staphylococci and methicillin-susceptible Staphylococcus aureus strains occur frequently enough to represent a risk of false-positive methicillin-resistant S. aureus determinations by molecular methods?

By analyzing the colonization of the anterior nares in cardiothoracic surgery patients on admission, nasal cocolonization by methicillin-susceptible Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci was detected in 8/235 (3.4%) specimens. Consequently, in a low-methicillin-resistant S. aureus (MRSA) setting, a molecular MRSA screening test targeting the mecA gene and an S. aureus-specific gene in parallel and applied directly to clinical specimens would be associated with an unacceptable positive predictive value of about 40%.     

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.     

Incidence of inducible clindamycin resistance in staphylococci: first results from Turkey

In total, 408 staphylococcal isolates were tested for inducible clindamycin resistance (ICR) by the disk-diffusion induction test (D-test). ICR was detected in 5.7% of 105 methicillin-resistant Staphylococcus aureus (MRSA) isolates, 3.6% of 111 methicillin-susceptible S. aureus isolates, 30.8% of 94 methicillin-resistant coagulase-negative staphylococcal (CoNS) isolates, and 11.2% of 98 methicillin-sensitive CoNS isolates. All MRSA isolates that were erythromycin-resistant and clindamycin-susceptible were positive by the D-test. The same results were obtained with an azithromycin instead of an erythromycin disk. All isolates were susceptible to quinupristin-dalfopristin. The cost-benefit of the d-test should be evaluated locally after determining the incidence of the different resistance phenotypes.     

Outbreak of Mupirocin-Resistant Staphylococci in a Hospital in Warsaw, Poland, Due to Plasmid Transmission and Clonal Spread of Several Strains

An outbreak of mupirocin-resistant (MuR) staphylococci was investigated in two wards of a large hospital in Warsaw, Poland. Fifty-three MuR isolates of Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. xylosus, and S. capitis were identified over a 17-month survey which was carried out after introduction of the drug for the treatment of skin infections. The isolates were collected from patients with infections, environmental samples, and carriers; they constituted 19.5% of all staphylococcal isolates identified in the two wards during that time. Almost all the MuR isolates were also resistant to methicillin (methicillin-resistant S. aureus and methicillin-resistant coagulase-negative staphylococci). Seven of the outbreak isolates expressed a low-level-resistance phenotype (MuL), whereas the remaining majority of isolates were found to be highly resistant to mupirocin (MuH). The mupA gene, responsible for the MuH phenotype, has been assigned to three different polymorphic loci among the strains in the collection analyzed. The predominant polymorph, polymorph I (characterized by a mupA-containing EcoRI DNA fragment of about 16 kb), was located on a specific plasmid which was widely distributed among the entire staphylococcal population. All MuR S. aureus isolates were found to represent a single epidemic strain, which was clonally disseminated in both wards. The S. epidermidis population was much more diverse; however, at least four clusters of closely related isolates were identified, which suggested that some strains of this species were also clonally spread in the hospital environment. Six isolates of S. epidermidis were demonstrated to express the MuL and MuH resistance mechanisms simultaneously, and this is the first identification of such dual MuR phenotype-bearing strains. The outbreak was attributed to a high level and inappropriate use of mupirocin, and as a result the dermatological formulation of the drug has been removed from the hospital formulary.     

In vitro activity of vancomycin and teicoplanin against gram-positive cocci]

The activities of vancomycin and teicoplanin against 148 strains of Gram-positive cocci were tested using agar diffusion and liquid microdilution MIC determination. Tested strains included 84 staphylococci, 32 S. aureus, 52 coagulase-negative staphylococci (CNS), 52 enterococci, and 12 streptococci. Most strains (136) were susceptible to both agents, with inhibition diameters of 17 mm or more. MRSA strains exhibited lower geometric MIC means with teicoplanin (0.90 micrograms/ml) than with vancomycin (1.79 micrograms/ml); this difference was found for methicillin-susceptible S. aureus strains (1.07 and 1.38 micrograms/ml for teicoplanin and vancomycin, respectively). In contrast, methicillin-susceptible and methicillin-resistant strains of CNS exhibited similar MICs (1.60 micrograms/ml approximately). Enterococci were more susceptible to teicoplanin (MIC 0.25 micrograms/ml) than to vancomycin (MIC 1.35 micrograms/ml). Both vancomycin and teicoplanin were thus found to be consistently effective against Gram-positive cocci; however, teicoplanin proved more effective than vancomycin against enterococci and methicillin-resistant S. aureus strains and may therefore be a valuable therapeutic alternative for these multiresistant organisms.     

Development of a real-time PCR assay for rapid identification of methicillin-resistant Staphylococcus aureus from clinical samples

A major drawback of mecA PCR to detect methicillin-resistant Staphylococcus aureus (MRSA) directly from patient materials is the high frequency of methicillin-resistant coagulase-negative staphylococci. Therefore, a reliable detection method for MRSA from clinical samples using real-time PCR was developed. The PCR assay targeting the integration site (orfX) of the staphylococcal cassette chromosome mec (SCCmec) was evaluated in MRSA SCCmec reference strains (n = 9), MRSA ST strains (n = 16) and clinical isolates of MRSA (n = 124), MSSA (n = 53), methicillin-resistant coagulase-negative staphylococci (n = 47), and methicillin-susceptible, coagulase-negative staphylococci (n = 32). The diagnostic values of the assay were 98% sensitivity and 100% specificity. Furthermore, the PCR detection method was evaluated with 60 swabs from different body sites which were incubated overnight in brain-heart infusion. The PCR gave positive results for 27 of 29 swabs which were found to contain MRSA by conventional methods. The diagnostic values of the PCR assay for these samples were 93% sensitivity and 100% specificity. To determine the in vitro sensitivity of the assay, swabs were inoculated with serially diluted bacterial suspensions. After overnight enrichment the detection limit of the PCR was less than 10 CFU/swab. This new real-time PCR assay proved to be a fast, sensitive and specific tool for MRSA detection in a routine microbiological laboratory.     

New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci

Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus-specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) and S. aureus, either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was approximately 25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.     

Detection of intrinsically resistant (heteroresistant) Staphylococcus aureus with the Sceptor and AutoMicrobic systems.

Modified procedures for the Sceptor Gram-Positive MIC Panel and the Vitek AutoMicrobic System GPS-M Card were evaluated for their ability to detect methicillin-resistant (heteroresistant) Staphylococcus aureus. A total of 398 clinical isolates (including 222 methicillin-resistant S. aureus) obtained from 10 hospitals were tested. Both systems had 2% NaCl in the oxacillin wells. Sceptor MIC panels were inoculated with an organism suspension prepared from an 18- to 24-h blood agar plate and were inoculated for a full 24 h at 35 degrees C before MICs were read. All methicillin-resistant S. aureus isolates were detected as resistant to oxacillin at greater than or equal to 8 micrograms/ml by the Sceptor method and at greater than 2 micrograms/ml by the Vitek method. All 176 oxacillin-susceptible, methicillin-susceptible S. aureus isolates were correctly distinguished from methicillin-resistant S. aureus isolates by Sceptor. However, with the Vitek system 29 methicillin-susceptible S. aureus isolates tested as falsely resistant to oxacillin and four isolates tested as falsely resistant to vancomycin. The modified testing procedure with the Sceptor system can be used reliably for accurate susceptibility testing of methicillin-resistant and methicillin-susceptible S. aureus. The Vitek GPS-M card does not accurately discriminate between methicillin-resistant and methicillin-susceptible S. aureus with an oxacillin breakpoint of greater than 2 micrograms/ml.     

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.     

Use of a primary isolation medium for recovery of methicillin-resistant Staphylococcus aureus.

Clinical specimens frequently contain methicillin-resistant Staphylococcus aureus (MRSA) isolates in low numbers or mixed with methicillin-susceptible staphylococci, which can obscure MRSA on nonselective media. By using an oxacillin-containing mannitol-salt-based selective and differential medium on 936 respiratory specimens, we recovered 45% more MRSA isolates (29 versus 20) than on nonselective media alone.     

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus Isolates from Patients Newly Identified as Nasal Carriers

We aimed to determine whether additional molecular and microbiological evaluations of methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients newly identified as nasal carriers were useful for control strategies and whether longitudinal testing during the same or repeat hospitalization changed MRSA status. Nasal swabs from patients positive by Xpert MRSA PCR and not known to be colonized in the previous year were cultured for S. aureus. Isolates were tested for resistance to a variety of antibiotics, including high-level mupirocin resistance (HLMR) and low-level mupirocin resistance (LLMR) and the presence of genes mecA and mupA and those for Panton-Valentine leukocidin (PVL), USA300, and USA400. Repeat nasal screens during the 6-month study were tested for continued presence of MRSA. Among 130 patients, cultures revealed MRSA in 85 (65.4%), methicillin-susceptible S. aureus in 19 (14.6%), and no growth in 26 (20%). MRSA isolates were USA300 positive in 13/85 (15.3%) and LLMR in 8/85 (9.4%) patients. No isolates were HLMR or mupA positive. mecA dropout was detected in 9/130 (6.9%) patients. The rate of subsequent MRSA infections in USA300-positive versus -negative patients was not different. MRSA nasal status remained concordant in 69/70 (98.6%) patients who had follow-up testing. The findings do not support expanding MRSA surveillance to include routine detection of genes for USA300, PVL, or mupA, all of which were either of low frequency or not significantly associated with MRSA infection risk in our population of newly identified nasal carriers. Repeat nasal screening for MRSA during the same or subsequent hospitalizations over 6 months could also be deferred, reducing costs associated with screening.     

Epidemiological study of staphylococcal colonization and cross-infection in two West African Hospitals

Surveillance in two medium-size (250-300 beds) hospitals located in the most populated islands of Cape Verde was undertaken in July 1997 in order to obtain data concerning nasal carriage of staphylococci. Nasal swabs (172) taken from inpatients and health care workers (HCW) from different internment services yielded 68 Staphylococcus aureus and 105 coagulase-negative staphylococcal (CNS) isolates, demonstrating extensive colonization of both inpatients and HCW by S. aureus (carriage rate 41%) and CNS (carriage rate 65%). The most frequent CNS species were S. epidermidis and S. haemolyticus. Three species--S. aureus, S. epidermidis, and S. sciuri-were recovered from wound swabs. The antibiotic susceptibility profiles of S. aureus and CNS differed sharply: all 68 S. aureus were resistant to penicillin but were fully susceptible to oxacillin as well as the other antimicrobial agents tested-gentamicin; erythromycin, except for three strains; ciprofloxacin; sulfamethoxazole-trimethoprim, except for two strains; vancomycin; and amoxicillin/clavulanate. In contrast, most (91/105) of CNS were resistant to both penicillin and oxacillin, and a variable but substantial proportion of CNS isolates also carried multiresistant traits to gentamicin, erythromycin, sulfamethoxazole-trimethoprim, and amoxicillin/clavulanate. The analysis by PFGE of the methicillin-susceptible S. aureus (MSSA) and the methicillin-resistant S. epidermidis (MRSE) strains provided evidence for extensive cross-infection and cross-colonization from HCW to patients.     

In vitro activity of evernimicin and selected antibiotics against methicillin-resistant staphylococci: a 24-country study

Objective   To collect and analyze data on susceptibility of methicillin-resistant staphylococci to evernimicin and other antimicrobial agents.
Methods   Recent clinical isolates of methicillin-resistant staphylococci from 33 laboratories in North America, Europe and South Africa were investigated.
Results   Of the antimicrobial agents tested, evernimicin had the lowest MIC₉₀s for methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci (0.75 and 1.0 mg/L, respectively). Resistance to ciprofloxacin and erythromycin was widespread, with higher levels of resistance in North America than in other regions.
Conclusions   Susceptibility surveys help to determine the antimicrobial activity of new agents. Ciprofloxacin- and erythromycin-resistant staphylococci were prevalent throughout all regions.     

Real-time PCR can rapidly detect methicillin-susceptible and methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles

We developed, validated, and implemented real-time polymerase chain reaction (PCR) detection of the femA gene for Staphylococcus aureus and the mecA gene for methicillin resistance directly from BACTEC (Becton Dickinson, Sparks, MD) blood culture bottles showing gram-positive cocci in clusters. For the 332 positive blood cultures tested, the assay had 100% sensitivity and specificity for identifying methicillin-susceptible (n=28) and methicillin-resistant (n=28) S aureus, and overall was 98% sensitive and 94% specific, with 3 uninterpretable test results when identification of coagulase-negative staphylococci was included. PCR detection yields rapid (2-3 hours) results and accurate identification of S aureus directly from signal-positive blood culture bottle samples.     

Patterns of multidrug resistance among methicillin-resistant hospital isolates of coagulase-positive and coagulase-negative staphylococci collected in the international multicenter study RESIST in 1997 and 1998

The primary purpose of the multicenter international study "RESIST" was to obtain an update on the degree of multidrug resistance among methicillin-resistant staphylococci collected from a geographically diverse sample. A total of 3,307 staphylococcal isolates were recovered from single patients and primarily from clinical specimens that were collected at 20 collaborating regional health centers located in several countries in Europe, Asia, and Latin America during a 3- to 4-month period each in 1997 and 1998. All strains were deposited at the Laboratory of Molecular Genetics at ITQB/UNL in Oeiras, Portugal, for quality control and for testing by microbiological and molecular typing techniques; the Laboratory of Microbiology at The Rockefeller University serving as organizational center. The majority of strains, 3,100, were methicillin-resistant, of which 1,749 were coagulase positive (methicillin-resistant Staphylococcus aureus, MRSA), and 1,351 were coagulase negative (methicillin-resistant coagulase negative staphylococci, MRCNS). The overall frequency of drug resistance traits among the 1,749 MRSA strains was high (over 70% and up to and over 90% of the strains) to ciprofloxacin, erythromycin, clindamycin, gentamicin, and tetracycline, and was somewhat less frequent to sulfamethoxazole-trimethoprim (45%), chloramphenicol (30%), and rifampin (38%). None of the 3,307 staphylococcal isolates showed reduced susceptibility to vancomycin except for a single methicillin-resistant coagulase-negative isolate. The great majority of staphylococci were also susceptible to the new antimicrobial Synercid. In contrast, resistance to teicoplanin was significant among methicillin-resistant strains of coagulase-negative staphylococci, particularly among Staphylococcus haemolyticus. MRSA isolates showed marked geographic variation in their patterns of multiresistance, most likely reflecting the properties of unique multiresistant MRSA clones dominant in the hospitals that provided the MRSA isolates from the various geographic areas. The multiresistance patterns of MRSA strains and strains of methicillin-resistant coagulase-negative staphylococci originating at the same country source also showed striking differences, suggesting that resistance to antimicrobial agents emerged under different antibiotic pressures in these bacterial species.     

New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci

Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin- and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA (Staphylococcus genus specific), nuc (S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin- and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin- and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.