Staphylococcus aureus strains which are not identified by rapid agglutination methods are of capsular serotype 5.

A total of 183 recent Staphylococcus aureus clinical isolates were tested with three commercially available rapid agglutination methods. The capsular polysaccharide type and resistance to oxacillin of these isolates were also determined. Seven isolates were not identified correctly by agglutination methods. All isolates not identified by the rapid methods were of capsular serotype 5, and of these isolates, six were resistant to oxacillin. The results suggest that these agglutination kits can be improved by the use of antibodies reactive with S. aureus capsular polysaccharide.     

Identification of the capsular polysaccharides in Staphylococcus aureus clinical isolates by PCR and agglutination tests

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. The predominance of two capsular polysaccharides, types 5 and 8, on the surface of clinical isolates led to the development of a conjugate vaccine (StaphVAX) based on capsular polysaccharides types 5 and 8 conjugated to a carrier protein. We have studied the capsular phenotypes and genotypes of 195 isolates representative of all clinical syndromes that encompassed both hospital and community-acquired infections. These isolates were mainly detected in France between January 2001 and December 2004. In this population, most of clinical isolates (87%) expressed either capsular polysaccharide type 5 (42%) or 8 (45%), whereas 13% were nontypeable by the serotyping method with antibodies specific to capsular polysaccharide type 5 or 8. These 26 nontypeable strains were further serotyped and were demonstrated to express the cell wall surface antigen 336, a polyribitol phosphate N-acetylglucosamine, which resembles cell wall teichoic acid. Among methicillin-resistant Staphylococcus aureus (MRSA) strains, we found a predominance of serotype 5 for 64% of strains, whereas MSSA isolates were predominantly capsular serotype 8 (60%). All S. aureus clinical isolates included in the present study have been investigated by PCR method, demonstrating that all isolates carried either the cap5 or the cap8 locus.     

Disulfide-bonded outer membrane proteins in the genus Legionella.

Staphylococcus aureus has been classified into at least eight different capsular types by using polyclonal rabbit antisera specific for their associated capsular polysaccharides. We produced and characterized monoclonal antibodies reactive with two serologically distinct capsular types, types 5 and 8, which account for more than 70% of all S. aureus bacteremias. These type-specific, monoclonal antibodies reacted with S. aureus clinical isolates possessing the homologous capsular type and exhibited no cross-reactivity against S. aureus clinical isolates possessing the heterologous capsular type, nontypeable S. aureus clinical isolates, Staphylococcus epidermidis clinical isolates, or a variety of gram-negative organisms. The anti-type 8 monoclonal antibodies also reacted with purified capsular polysaccharide derived from the prototype type 8 S. aureus strain.     

New latex reagent using monoclonal antibodies to capsular polysaccharide for reliable identification of both oxacillin-susceptible and oxacillin-resistant Staphylococcus aureus.

A new latex agglutination test (Pastorex Staph-Plus, Sanofi Diagnostics Pasteur), consisting of a mixture of latex particles coated with fibrinogen and immunoglobulin G for the detection of clumping factor and protein A and latex particles sensitized with monoclonal antibodies directed to Staphylococcus aureus serotype 5 and 8 capsular polysaccharides, was compared with three commercially available rapid agglutination methods for the identification of 220 isolates of S. aureus (61 oxacillin resistant) and 128 isolates of coagulase-negative staphylococci. The sensitivity for identification of S. aureus was high with the Pastorex Staph-Plus test (98.6%) compared with those of the other tests, which ranged from 91.8 to 84.5%. Test sensitivities for the identification of oxacillin-resistant S. aureus were as follows: Pastorex Staph-Plus, 95.1%; Pastorex Staph, 73.8%; Staphyslide, 72.1%; and StaphAurex, 49.2%.     

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.     

Genetic and serologic evaluation of capsule production by bovine mammary isolates of Staphylococcus aureus and other Staphylococcus spp. from Europe and the United States

Bovine mastitis caused by Staphylococcus aureus is responsible for major economic losses to the dairy industry, and more-effective therapeutic or preventive approaches are sorely needed. The predominance of staphylococcal capsular polysaccharide types 5 and 8 among human isolates from many sources is well documented, but there seems to be a greater variation in the distribution of capsular serotypes among isolates from cows. A total of 636 isolates of S. aureus from cases of bovine mastitis in Sweden, Denmark, Finland, Iceland, Ireland, and the United States were investigated for production of capsular polysaccharide types 5 and 8. Approximately half of all the European isolates tested were of serotype 8, although variation among countries and among isolates of clinical and subclinical origin was observed. Sweden had the highest frequency (87%) of serotypeable isolates, and Finland had the lowest (48%). Capsule types 5 and 8 accounted for only 42% of the U.S. isolates tested. A few isolates showed weak reactivity with CP5 antiserum in a colony blot assay, and an enzyme-linked immunosorbent assay inhibition method confirmed that the levels of capsule produced by these strains were <10% of those produced by control strains. Fifty isolates that failed to react with capsular antisera all possessed the genes for production of capsular polysaccharide type 5 or 8. These results underscore the variability in capsule production by bovine isolates of S. aureus from different geographic regions. This information is important for the rational design of a capsule-based vaccine to prevent S. aureus bovine mastitis.     

Evaluation of MicroScan MIC panels for detection of oxacillin-resistant staphylococci.

Clinical isolates of staphylococci (420 Staphylococcus aureus isolates and 248 coagulase-negative staphylococci) were tested by both MicroScan MIC panels (MicroScan, West Sacramento, Calif.) and an oxacillin agar screen (Mueller-Hinton agar [Difco Laboratories, Detroit, Mich.] containing 6 micrograms of oxacillin per ml and 4% NaCl) to evaluate the ability of MicroScan to detect oxacillin-resistant strains. MicroScan panels and oxacillin agar screen plates were incubated at 35 degrees C for 24 h and at 30 degrees C for an additional 24 h. Endpoints were recorded at 24 and 48 h. By MicroScan, 23 (5.5%) and 30 (7%) S. aureus isolates and 161 (65%) and 162 (65%) coagulase-negative staphylococci were oxacillin resistant at 24 and 48 h, respectively. At both 24 and 48 h, 23 (5.5%) S. aureus isolates and 162 (65%) coagulase-negative staphylococci were resistant by the oxacillin agar screen. Five strains for which the oxacillin MIC was 2 or 4 micrograms/ml and eight strains resistant to oxacillin only at 48 h were further evaluated by broth macrodilution testing for oxacillin with and without clavulanic acid, by oxacillin and amoxicillin-clavulanic acid disk diffusion, and by oxacillin agar screen comparing Mueller-Hinton agars purchased from Difco and BBL Microbiology Systems, Cockeysville, Md. By this additional testing, all 10 S. aureus isolates and 1 of 3 coagulase-negative staphylococci examined produced increased amounts of beta-lactamase. One coagulase-negative staphylococcus appeared to be truly intermediately oxacillin susceptible. There was no significant difference in the rate of detection of oxacillin resistance between MicroScan and the agar screen. MicroScan panels should be incubated for 24 h only, because prolonged incubation caused strains producing excessive amounts of beta-lactamase to appear to be falsely oxacillin resistant.     

Evaluation of MicroScan rapid gram-positive panels for detection of oxacillin-resistant staphylococci.

The ability of MicroScan rapid panels (Baxter MicroScan, West Sacramento, Calif.) to detect oxacillin resistance in 92 clinical isolates of Staphylococcus aureus and 103 coagulase-negative staphylococci was evaluated by comparing results with those of MicroScan 24-h MIC panels and, to resolve discrepancies, oxacillin agar screening. Both panels were interpreted by the MicroScan WalkAway-96 system. Rapid panels detected 96.7% of resistant S. aureus isolates and 72% of resistant coagulase-negative staphylococci, 22 of which did not grow in the panels.     

Choice of the NaCl concentration for optimizing the detection of methicillin resistance in Staphylococcus using the gel diffusion method

The detection of oxacillin resistance is evaluated by the disk diffusion method in a collection of 374 Staphylococcus sp strains. The disk diffusion assay is performed with 5-microgram oxacillin disk and a 10(8) CFU/mL inoculum on Mueller-Hinton agar plates supplemented with 2 or 5% NaCl and incubated at 37 degrees C for 24 h. Strains are considered resistant in accordance to the French recommendations (any growth around the disk is observed). The detection of mecA gene is performed by PCR almost for resistant and discordant strains. Results are concordant for 246 of 256 Staphylococcus aureus strains (182 susceptible and 64 resistant strains) and for 105 of 118 S. epidermidis isolates tested (37 susceptible and 68 resistant strains). Six mecA-negative strains (3 S. aureus and 3 S. epidermidis) give false resistant results on agar with 2 and 5% NaCl. Seventeen isolates are discordant on 5% NaCl: 7 mecA-negative S. aureus strains are susceptible on 2% NaCl agar but resistant at 5% (3% false-positive results), 10 mecA-positive S. epidermidis strains are resistant on 2% NaCl agar but susceptible at 5% NaCl (5% false-negative results). The detection of meticillin resistance is improved on agar supplemented with 2% NaCl.     

Correlation between esterase electrophoretic types and capsular polysaccharide types 5 and 8 among methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus.

The relationship between capsular polysaccharide types 5 and 8 and esterase electrophoretic types (zymotypes) in 160 French clinical isolates of Staphylococcus aureus was studied. Methicillin-susceptible strains of capsular types 5 and 8 were represented by 11 zymotypes, indicating a high polymorphism. Methicillin-resistant strains were mainly distributed in only two distinct populations. The predominant population was represented by strains of zymotype 6 and capsular type 5, and the second population was represented by strains of zymotype 14 and capsular type 8.     

In vitro activity of antimicrobial agents against oxacillin resistant staphylococci with special reference to Staphylococcus haemolyticus

One hundred and sixty seven isolates of staphylococci isolated from the inpatients of a tertiary care referral hospital in South India were speciated and activity of oxacillin, glycopeptides, linezolid and quinupristin/dalfopristin against these isolates was tested by broth microdilution method. Of the 114 coagulase negative staphylococci (CoNS), 49.1 % were S. haemolyticus, isolated predominantly from urine (64.6%), while the rest belonged to 11 other species. More than half the isolates of S. aureus (52.8%) and 68.4% of the CoNS were oxacillin resistant. All the strains were uniformly susceptible to vancomycin, linezolid and quinupristin/dalfopristin; but 25.6% isolates of S. haemolyticus showed reduced susceptibility to teicoplanin (MIC: 8-16 mg/L). Our study demonstrates the high prevalence of oxacillin resistance among hospital isolates of S. aureus and CoNS in India. Vancomycin, along with the newer agents like linezolid and quinupristin/dalfopristin remains the drug of choice for treating multi drug resistant staphylococcal infections.     

Validation of multiplex PCR strategy for simultaneous detection and identification of methicillin resistant Staphylococcus aureus

Multiplex polymerase chain reaction (PCR) strategy is described for rapid identification of clinically relevant methicillin resistant Staphylococcus aureus (MRSA) that targets mecA and coagulase genes. In this study, 150 staphylococcal clinical isolates were used that included 40 isolates of MRSA, 55 isolates of methicillin susceptible S. aureus (MSSA), 44 isolates of methicillin susceptible coagulase negative Staphylococcus spp. (MS-CoNS) and 11 isolates of methicillin resistant coagulase negative Staphylococcus spp. (MR-CoNS). Out of 55 S. aureus strains, three strains demonstrated mecA gene, which appeared to be oxacillin sensitive by disc diffusion. When (MS-CoNS) were evaluated, 10 isolates classified as oxacillin sensitive phenotypically, yielded positive results in PCR method. The results for mecA detection by PCR were more consistent with disk susceptibility tests in case of MRSA (100%) and MSSA (95%) isolates. In contrast to above results with MRSA and MSSA, mecA detection by PCR in MS-CoNS showed less correlation with disk susceptibility tests (77%). The results for coag detection by PCR were consistent with phenotypic tests in all isolates.     

Evaluation of Rapid ATB Staph for 5-hour antimicrobial susceptibility testing of Staphylococcus aureus. Groupement pour le Dépistage, L'Etude et la Prévention des Infections Hospitalières-Groep ter Opsporing, Studie en Preventie van Infecties in de Ziekenhuizen.

The accuracy of Rapid ATB Staph (bioMérieux, La Balme-Les Grottes, France) for detection of oxacillin resistance and for detection susceptibility to 11 other antimicrobial agents in 553 and 519 Staphylococcus aureus isolates, respectively, was evaluated by comparing results with those produced by oxacillin agar screen and agar dilution methods, respectively. Further characterization of isolates with discrepant results for oxacillin testing was done by PCR detection of the nuc and mecA genes. By oxacillin agar screening, there were 307 oxacillin-resistant and 246 oxacillin-susceptible isolates. Rapid ATB results were obtained in 5 h for 515 (93.2%) of the isolates tested. Rapid ATB showed 97.0% sensitivity for detection of oxacillin resistance, confirmed by the presence of the mecA gene. After repeat testing of isolates flagged by the ATB software as possible errors, sensitivity increased to 99% for oxacillin-resistant isolates. Essential agreement with agar dilution testing for susceptibility to amoxicillin-clavulanic acid, gentamicin, erythromycin, clindamycin, and ciprofloxacin, as estimated by Youden's J statistic, was > 0.90. Subpopulations of isolates with significantly increased MICs of amikacin, rifampin, and minocycline, indicating borderline susceptibility, were detected by Rapid ATB and categorized as resistant. Rapid ATB Staph showed adequate accuracy for detection within 5 h of the oxacillin- and multiple-drug-resistant S. aureus isolates currently prevalent in Belgium.     

Evaluation of the BBL Crystal MRSA ID System for detection of oxacillin resistance in Staphylococcus aureus.

AIMS: To evaluate the BBL Crystal MRSA ID System for detection of oxacillin resistance in Staphylococcus aureus. METHODS: 52 methicillin resistant S aureus (MRSA) from five different countries and 85 methicillin susceptible S aureus (MSSA) were included. The species was confirmed by tube coagulation and detection of the clumping factor using the Staphaurex Plus. Clonal non-identity of the MRSA isolates was shown by pulsed field gel electrophoresis. MIC values (oxacillin) were determined using the Etest. Polymerase chain reaction was carried out to detect the mecA gene. The BBL Crystal MRSA ID System was carried out according to the manufacturer's instructions. RESULTS: The BBL Crystal MRSA ID System showed fluorescence in 45 of 52 MRSA (sensitivity 86.5%; negative predicitive value 92.2%), and the specificity was 97.6% (positive predicitive value 95.7%). Two of seven MRSA that failed to show fluorescence had MIC values (oxacillin) of 4 mg/litre. CONCLUSIONS: The BBL Crystal MRSA ID System is a valuable test for detecting oxacillin resistance in S aureus. Its major advantage is the short time (4-5 hours) required to perform the test when organisms are grown on tryptic soy agar or sheep blood agar. Difficulties may arise in borderline resistant isolates.     

Comparison of phenotypic methods and DNA hybridization for detection of methicillin-resistant Staphylococcus aureus.

One hundred thirty-eight Staphylococcus aureus isolates from patients with severe staphylococcal infections were collected in 15 French hospitals. Detection of the mec gene was performed by dot blot hybridization with a specific DNA probe. Dot blot results were used to characterize the isolates as methicillin susceptible (77 isolates) or resistant (61 isolates). The isolates were screened for methicillin resistance by an agar spread method on Mueller-Hinton plates containing oxacillin (2 and 10 micrograms/ml) and were incubated at 37 degrees C, with 10(8) CFU as the inoculum. MICs of oxacillin and methicillin were determined by the agar dilution method on Mueller-Hinton plates without NaCl, by using 10(5) CFU per spot, after 24 and 48h of incubation at 30 or 37 degrees C. Moderately elevated MICs were found for 20 isolates (14.5%). The mec gene was detected in six (30%) of the isolates expressing a low level of resistance to methicillin and/or oxacillin. As determined by comparison with probe hybridization results, the spread plate method with oxacillin at 2 micrograms/ml was more sensitive (sensitivity, 100%) and specific (specificity, 100%) than agar dilution with either methicillin or oxacillin in identifying methicillin resistance or susceptibility. Determinations of methicillin and oxacillin MICs by the agar dilution method had a specificity of 99 to 100% depending on the conditions of incubation, but the sensitivity was below 85% whatever the duration or temperature of incubation.     

Type 5 and 8 capsular polysaccharides are expressed by Staphylococcus aureus isolates from rabbits, poultry, pigs, and horses.

A total of 103 Staphylococcus aureus isolates from rabbits (n = 37), poultry (n = 33), pigs (n = 27), and horses (n = 6) and 14 Staphylococcus intermedius isolates from wild animals were serotyped for capsular polysaccharide types 5 and 8 by an enzyme-linked immunosorbent assay using polyclonal rabbit antibodies. About 98% of the S. aureus isolates were typeable. Type 5 was predominant in the poultry (75.8%) and pig (66.7%) isolates, whereas type 8 was more frequent among the isolates from rabbits (59.5%) and horses (83.3%). By contrast, none of the 14 S. intermedius isolates was typeable.     

Prevalence of capsular polysaccharide types 5 and 8 among Staphylococcus aureus isolates from cow, goat, and ewe milk.

Monoclonal antibodies to Staphylococcus aureus capsular polysaccharide types 5 and 8 were used to serotype by enzyme-linked immunosorbent assay 212, 54, and 33 isolates from cow, goat, and ewe milk, respectively. Capsular types 5 and 8 accounted for 69.4% of bovine isolates and 71.5 and 78.8% of goat and ewe isolates, respectively. Type 5 was predominant in strains from bovine sources (51.4%), whereas type 8 was prevalent in strains from caprine (68.5%) and ovine (75.8%) sources.     

Impact of prolonged incubation on disk diffusion susceptibility test results for Staphylococcus aureus

Because strains of Staphylococcus aureus that are resistant to penicillinase-resistant penicillins may be difficult to detect in the clinical laboratory, a variety of changes in methodology have been suggested to increase their detection. In 1984, the West Los Angeles Veterans Administration Medical Center experienced an increase in clinically significant strains of oxacillin-resistant S. aureus. To insure that such strains would not be missed by the disk diffusion test methods employed for routine testing, changes in methodology were insituted. These included interpreting zone diameters around oxacillin disks at 48 h of incubation. We collected 139 isolates from patients thought to have oxacillin-resistant S. aureus based on these test results and later retested the isolates using microdilution MIC testing. Only 85 isolates (61%) had microdilution oxacillin MICs of greater than or equal to 8.0 micrograms/ml, whereas 54 (39%) had oxacillin MICs of less than or equal to 2.0 micrograms/ml. A review of medical records revealed that in 1 year there were 98 patients with isolates appearing resistant by disk diffusion but not confirmed by microdilution MICs; many patients were placed in isolation and treated with specific antimicrobial agents. We conclude that incubation of oxacillin disk diffusion tests for longer than 24 h in conjunction with disregard for resistance to other classes of antimicrobial agents may result in an unacceptably high degree of false resistance results. Because the resistance of S. aureus has important therapeutic and infection control implications, it is necessary to recognize problems that may result in ambiguous or inaccurate susceptibility results.     

Detection of new methicillin-resistant Staphylococcus aureus clones containing the toxic shock syndrome toxin 1 gene responsible for hospital- and community-acquired infections in France

Methicillin-resistant Staphylococcus aureus (MRSA) clones harboring the toxic shock syndrome toxin 1 (tst) gene have been detected in France and in Switzerland since 2002. During a passive survey conducted between 2002 and 2003, we collected 103 tst-positive S. aureus isolates from 42 towns in France, of which 27 were resistant to methicillin. The tst-positive MRSA belonged to two clones: a major clone comprising 25 isolates of sequence type (ST) 5 and agr group 2 and a minor clone comprising two isolates of ST30 and agr3. The tst-positive MRSA clones were associated with both hospital-acquired (12 cases) and community-acquired (8 cases) infections. The MRSA clones were mainly isolated from children (overall median age, 3 years). They caused a variety of clinical syndromes, including toxic shock syndrome and suppurative infections. Both clones were found to harbor a type IV staphylococcal chromosomal cassette mec (SCCmec) and to have similar antibiotic resistance profiles (usually resistant to oxacillin, kanamycin, and tobramycin and with intermediate resistance to fusidic acid). The origin of these clones is unclear. The tst-positive agr2 MRSA clone has the same sequence type (ST5) of two pandemic nosocomial MRSA clones, namely, the Pediatric clone and the New York/Japan clone. These findings suggest that all these clones are phylogenetically related. The pulsotype of the tst-positive MRSA clones differed from that of methicillin-sensitive S. aureus (MSSA) clones by a single band involving the SCCmec element. These findings suggest that the tst-positive MRSA clones may have emerged from their respective MSSA counterparts.     

Correlation of penicillin Binding protein 2a detection with oxacillin resistance in Staphylococcus aureus and discovery of a novel penicillin binding protein 2a mutation

We compared a rapid slide latex agglutination test (LAT; Oxoid, Basingstoke, United Kingdom) that detects penicillin binding protein 2a (PBP2a) with MicroScan conventional panels (Dade Behring, West Sacramento, CA) for detection of oxacillin resistance in Staphylococcus aureus. The PBP2a LAT demonstrated 99% agreement with MicroScan oxacillin MIC results for 388 isolates of S. aureus. All 249 oxacillin-resistant isolates gave strong positive reactions in the LAT (100% sensitivity). Three of the 139 oxacillin-susceptible isolates were also strongly positive and one was weakly positive in the LAT (97.1% specificity). The three oxacillin-susceptible isolates with strongly positive reactions were further characterized. The mecA gene was detected in all three by PCR; one isolate was determined to be resistant to oxacillin by reference broth microdilution testing (MIC, 8 microg/ml), one isolate was inducibly resistant to oxacillin (MIC of 16 microg/ml after overnight induction), and one isolate remained susceptible regardless of the method used for testing. Sequence analysis of a 2.1-kb gene fragment of the mecA gene from the susceptible isolate revealed a one-base substitution at nucleotide position 1449 which results in a Met-to-Ile change for amino acid residue 483. This amino acid substitution has not been previously reported and may be associated with a change in the function of PBP2a resulting in oxacillin susceptibility. An additional 487 isolates were tested in parallel with the both the LAT and MicroScan panels using criteria in which only strong (3 to 4+) or repeatedly weak (1 to 2+) LAT reactions were considered positive, and the results showed 99.4% agreement. The PBP2a LAT provided rapid and reliable detection of oxacillin resistance and proved a useful adjunct to the phenotypic method. Both methods provided reliable detection of oxacillin-resistant S. aureus and facilitated the discovery of a novel, functionally impaired form of PBP2a.