Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌

目的 应用mecA基因PCR扩增法检测耐甲氧西林金黄色葡萄球菌（methicillin resistant staphylococcus aureus,MRSA）。方法 临床分离的70株金黄色葡萄球菌,应用mecA基因PCR扩增法鉴定MRSA,并与苯唑西林纸片扩散法进行比较。结果 70株金黄色葡萄球菌用PCR扩增法和纸片扩散法有6株鉴定有差异,4株。mecA基因阳性而纸片扩散法鉴定为敏感,1株mecA基因阳性纸片扩散法鉴定为临界耐药,1株mecA基因阴性却表现为苯唑西林耐药,2种方法符合率为91．43％。结论 mecA基因PCR扩增法可以准确、快速判定MRSA,特别是对隐匿型或低水平耐药菌株的检出有重要的价值。     

Analysis of borderline-resistant strains of methicillin-resistant Staphylococcus aureus using polymerase chain reaction.

Identification of methicillin-resistant Staphylococcus aureus by drug-susceptibility tests alone poses a serious problem, because a considerable number of clinical S. aureus isolates are borderline resistant to methicillin. To circumvent this problem, we have developed a quick and sensitive method of PCR amplification for the detection of mecA gene, which, coding for PBP2', is the specific genetic element of methicillin-resistant Staphylococcus aureus. This method made it possible to identify MRSA strains in a short time using as few as 30 cells as a starting material for template DNA. Using this method, we found that the strains of borderline methicillin-resistance could be accurately identified. We also found one S. aureus clinical strain, T3, which lacked mecA gene in spite of its resistance to methicillin.     

Staphylococci in orthopaedic surgical wounds.

From 50 infected surgical wounds of orthopaedic patients, 43 (86%) staphylococcal strains were isolated. 34 of all these staphylococci belonged to Staphylococcus aureus species (i.e. 68 % of all isolates from surgical wounds; 79% of staphylococcal isolates); 9 were coagulase-negative staphylococci (i.e. 21% of all isolates from surgical wounds; 18% of staphylococcal isolates). Among microorganisms isolated from the wounds we also found 2 (4%) of the Enterobacteriaceae family; 2 (4%) of the Pseudomonas genus; 3 (6%) of the Streptococcus genus. Thus, orthopaedic surgical wounds were infected by staphylococci (mainly S. aureus) more frequently than by other micro-organisms. All the staphylococcal strains were screened for methicillin resistance by agar disk diffusion testing and for the presence of mecA gene responsible for methicillin resistance by PCR. 32% of the S. aureus and 33% of the S. epidermidis strains resulted methicillin resistant and mecA-positive. The data confirm the diffusion of methicillin resistant S. aureus in surgical site infections and shows that the so-called "new pathogens", i.e. S. epidermidis and other coagulase-negative staphylococci, also exhibit a frequent and hazardous methicillin-resisting ability.     

Quantitative disk diffusion as a convenient method for determining minimum inhibitory concentrations of oxacillin for staphylococci strains

In this study the susceptibility of 58 coagulase-negative staphylococci (CoNS) strains and 58 Staphylococcus aureus strains to oxacillin was evaluated by a novel method called quantitative disk diffusion (DD) method. The results obtained were compared to phenotypic methods as agar dilution (AD) for oxacillin, disk diffusion (DD) for cefoxitin, and related to the presence of the mecA gene detected by PCR. Minimum inhibitory concentrations (MIC) determined by the quantitative DD method were equivalent to MICs determined in the AD method for S. aureus (Student's t test, p=0.99) and CoNS (Student's t test, p=0.97). Incongruent results between PCR mecA gene determinations and the quantitative DD method were obtained in 8 strains (5 S. aureus and 3 CoNS) where the mecA gene expression was blocked. However, oxacillin resistance was detected by the proposed method even in staphylococci strains showing low-level or heterogeneous resistance to the antibiotic while other phenotypic methods failed. The single quantitative DD method is not expensive, it can be performed in any laboratory and permits accurate identification of oxacillin resistant staphylococci.     

Comparison of Real-Time PCR with Disk Diffusion, Agar Screen and E-test Methods for Detection of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the “gold standard” comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.     

Methicillin resistance in staphylococci isolated from subclinical mastitis in sheep

One hundred ovine milk samples were subjected to bacteriological analysis to detect staphylococci. Twenty-four staphylococcal strains isolated were characterised for methicillin resistance with disk diffusion test (DDT) after incubation at 24 and 48 h, oxacillin agar screen test, Minimal Inhibitory Concentration (MIC), nitrocefin test for beta-lactamase production and PCR for the mecA gene. Nine staphylococcal strains resulted resistant in DDT; some differences in the halo diameter at double incubation period were noted; eight of these strains were resistant in MIC test; just one strain was positive to oxacillin agar screen test. All strains were mecA negative by PCR and positive by nitrocefin test. On the basis of these results methicillin-resistant strains can be classified as beta-lactamase hyperproducers.     

Multiplex PCR assay to identify methicillin-resistant Staphylococcus haemolyticus

Staphylococcus haemolyticus is the most frequently coagulase-negative Staphylococcus species associated with antimicrobial resistance isolated from nosocomial infections. We developed an accurate and simple multiplex PCR assay to identify methicillin-resistant S. haemolyticus (MRSH) isolates. We designed species-specific primers of the mvaA gene that encodes a 3-hydroxy-3-methylglutaryl coenzyme A involved in the mevalonate pathway of the microorganism. Simultaneously, mecA gene primers of methicillin resistance were also used. The PCR assay was established using 16 strains of different reference Staphylococcus species and validated with a collection of 147 clinical staphylococcal isolates that were also phenotypically characterized. Reliable results for the detection of MRSH isolates were obtained for 100% of the strains evaluated, showing that this PCR assay can be used for the routine microbiology laboratories. This is the first report using species-specific multiplex PCR to detect a single segment of S. haemolyticus associated with a segment of mecA gene.     

Additional DNA in methicillin-resistant Staphylococcus aureus and molecular cloning of mec-specific DNA.

Additional DNA was shown to be present in methicillin-resistant Staphylococcus aureus by one- and two-dimensional restriction endonuclease analyses of the chromosomal DNA. A 3.5-kilobase Bg/II fragment, which was present in methicillin-resistant strains but not in the isogenic methicillin-sensitive parental strain, was cloned into newly constructed plasmid pWDB1 in Escherichia coli. Hybridization of this 3.5-kilobase Bg/II fragment with different methicillin-sensitive and methicillin-resistant S. aureus clinical isolates indicated that the fragment represents part of the methicillin resistance determinant (mec). In addition, the fragment carries a sequence that is present in some large staphylococcal plasmids, as well as in penicillinase plasmid pI524.     

Development of a real-time Staphylococcus aureus and MRSA (SAM-) PCR for routine blood culture

The notification of "Gram-positive cocci, possibly staphylococcus" in a blood culture drawn from a seriously ill patient is responsible for a large amount of vancomycin prescribing in institutions where methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of bacteraemia. A duplex real-time TaqMan polymerase chain reaction targeting the species-specific nuc gene, and the mecA gene encoding methicillin-resistance, was developed as a tool for rapid identification and detection of S. aureus and methicillin-resistance, and optimised for immediate as-needs testing. Three different DNA extraction methods achieved varying DNA quality, with PCR inhibition the main problem. Serial blood cultures (n=120) identified as possible staphylococci on Gram stain from our clinical laboratory were examined. There was one false negative result for a methicillin-resistant Staphylococcus epidermidis, which was positive on repeat testing, and one false negative result due to DNA extraction failure for MRSA from peritoneal dialysate inoculated into blood culture medium. Sensitivity and specificity of 97% and 100%, respectively, were obtained for mecA; and sensitivity and specificity of 98% and 100%, respectively, for nuc. Detection of slow-growing coagulase-negative staphylococci as co-infecting strains may be reduced. The assay quickly and reliably identified S. aureus in mixed infection, and identified methicillin resistance in both S. epidermidis and S. aureus strains.     

Comparison of <Emphasis Type="Italic">mecA</Emphasis> Polymerase Chain Reaction With Phenotypic Methods for the Detection of Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In the present study, several conventional methods to detect methicillin-resistant Staphylococcus aureus (MRSA) were compared with polymerase chain reaction (PCR) detection of mecA gene–positive isolates. Cefoxitin E-test was also evaluated as a possible phenotypic method of MRSA detection. Oxacillin agar screen and PBP2′ latex agglutination methods were found to be more sensitive than oxacillin and cefoxitin disk-diffusion methods. Cefoxitin disk diffusion was found to be the most specific. A combination of oxacillin agar screening with cefoxitin disk diffusion, or oxacillin disk diffusion with PBP2′, improved sensitivity and specificity. Cefoxitin E-test with the current break points had low sensitivity and specificity (33.3% and 75%, respectively) for the detection of MRSA. However, changing the break points to ≤ 4 μg/ml and to ≥ 6 μg/ml for sensitive and resistant, respectively, greatly improved both. Changing the 30-μg cefoxitin disk-diffusion break points to ≤ 21 mm for resistant slightly improved sensitivity but had no effect on specificity. It was therefore concluded that the use of more than one screening method is necessary to detect all MRSA isolates in clinical settings.     

Molecular identification and genotyping of MRSA isolates

The aim of this study was to identify and characterize 97 methicillin-resistant Staphylococcus aureus (MRSA) isolates. Two conventional multiplex PCR assays, a real-time PCR assay and two PCR-based genotyping techniques including the spa - and hypervariable region (HVR)-typing methods were used to identify and characterize 97 MRSA strains isolated between April 2006 to September 2007 from the Steve Biko Academic Hospital. All MRSA isolates were positive for 16S rRNA gene, 99% were positive for the mec A gene and 4% positive for the Panton–Valentine leukocidin (PVL) gene. Staphylococcal cassette chromosome mec (SCC mec ) typing showed 67% of isolates were SCC mec II [health-care-associated MRSA (HA-MRSA)], 14% were SCC mec III (HA-MRSA) and 4% were SCC mec IVd [community-associated MRSA (CA-MRSA)]. These CA-MRSA isolates showed a prevalence of 100% for the PVL gene. Using spa typing, three distinct clusters could be identified while HVR typing revealed six different clusters. CA-MRSA isolates were clustered together using spa and HVR typing. This study showed the prevalence of the CA-MRSA strains, PVL genes, the SCC mec types and the clonality of the MRSA strains. The high prevalence of the PVL gene in CA-MRSA isolates already residing in intensive care units was alarming and indicated the emergence of new MRSA lineages with a particular fitness for community and hospital transmission.     

Rare occurrence of mupirocin resistance among clinical Staphylococcus isolates in Jordan

Staphylococcal infections have high occurrence in Jordanian patients. This study was carried out to determine the rates of high- and low-level mupirocin resistance (MupH and MupL) among staphylococci with the molecular characterization. Two hundred and thirty-two non-duplicate Staphylococcus spp. isolated from different clinical specimens were tested for mupirocin susceptibility using disk diffusion method and minimum inhibitory concentration (MIC). Resistance genes and clone relatedness was studied using polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus primers (Eric-PCR) for the latter. Plasmid curing was performed to determine the genetic location of MupA gene. Among the 232 strains, 144 (62%) were methicillin-resistant Staphylococcus aureus (MRSA), 33 (14.2%) methicillin-susceptible Staphylococcus aureus (MSSA) and 55 (23.7%) were of other coagulase-negative Staphylococcus spp. (CoNS). Of all strains tested, only 6 (2.6%) were mupirocin resistant. MecA gene was detected in both MupL and MupH strains but MupA gene was only detected in MupH. Plasmid curing improved the plasmidic location of MupA gene. Molecular typing by Eric-PCR method revealed heterogenicity of the genetic make up of our MupL and MupH strains. Staphylococci with MupA-carrying genes are present in Jordanian hospitals, but thank to the limited use of mupirocin, they remain rare.     

The combined effect of methicillin and teicoplanin against Staphylococcus aureus strains investigated in vitro

The synergy or antagonism between teicoplanin and methicillin against the methicillin-resistant strains of Staphylococcus aureus with decreased susceptibility to vancomycin was studied using agar dilution and E-tests. The investigated strains were GISA and h-GISA isolated in our laboratory as well as standard. In the used range of concentrations, a synergy was shown in the case of two strains and an antagonism was shown in the case of other two strains. For the most strains the tested combination of antibiotics showed indifference. The antagonistic effect was observed in the case of the standard strain Mu3 and the one from eight strains h-GISA isolated in our laboratory.     

Slime production and antibiotic susceptibility in staphylococci isolated from clinical samples

A total of 187 isolates from several clinical specimens were identified to species level as 129 Staphylococcus aureus strains and 58 coagulase-negative staphylococci (CNS) strains by the API Staph System (Biomerieux). Slime production was detected both by the conventional Christensen's method as well as by the Congo red agar method. Seventy-two strains of staphylococci isolates (38.5%) were found to be slime producers by Christensen's test tube method whereas 58 strains (31%) were slime positive with Congo red agar method. There was no statistically significant difference between the two methods for the detection of slime production (P > 0.05). Susceptibility of isolates against antimicrobial agents was tested by the disk diffusion method. Staphylococcal species had resistance to one or more antibiotics. Among the various antimicrobial agents, oxacillin (71.1%) and erythromycin (47.1%) showed higher resistance than most of the agents used against all isolates. Oxacillin resistant S. aureus (ORSA) and oxacillin resistant coagulase-negative staphylococci (ORCNS), 97 (75.2%) and 36 (62.1%) respectively were frequently observed in strains isolated from clinical materials. Among the ORSA strains, two strains were resistant to vancomycin. Moreover, 96 (74.4%) of 129 S. aureus strains were positive for beta-lactamase enzyme. However, 78 (81.25%) of 96 beta-lactamase positive S. aureus strains were beta-lactamase positive ORSA isolates, but none of them had vancomycin resistance.     

Accuracy of phenotypic methicillin susceptibility methods in the detection of Staphylococcus aureus isolates carrying different SCCmec types

A total of 138 isolates, 118 methicillin-resistant Staphylococcus aureus (MRSA) isolates (staphylococcal cassette chromosome type II, 20 isolates, type III, 39 isolates and type IV, 59 isolates) and 20 methicillin-sensitive S. aureus isolates were evaluated by phenotypic methods: cefoxitin and oxacillin disk diffusion (DD), agar dilution (AD), latex agglutination (LA), oxacillin agar screening (OAS) and chromogenic agar detection. All methods showed 100% specificity, but only the DD tests presented 100% sensitivity. The sensitivity of the other tests ranged from 82.2% (OAS)-98.3% (AD). The LA test showed the second lowest sensitivity (86.4%). The DD test showed high accuracy in the detection of MRSA isolates, but there was low precision in the detection of type IV isolates by the other tests, indicating that the genotypic characteristics of the isolates should be considered.     

Adhesion of a Staphylococcus aureus strain to biomaterials does not select methicillin-resistant mutants

Bacterial adhesion to polymethylmethacrylate and to silicon elastomer, materials frequently used in clinical applications, has been investigated to assess whether adhesion selects methicillin-resistant mutants in the bacterial population in contact with the materials. The methicillin susceptibility of a susceptible Staphylococcus aureus (ATCC 25923) was measured by a modification of plate antibiogram Kirby-Bauer method, which allows optimised detection of small variations in antibiotic susceptibility. In both adherent and non-adherent bacterial subpopulations, the presence of mecA gene, which encodes for the protein PBP 2a responsible for methicillin resistance was searched for by Polymerase Chain Reaction (PCR). The contact with the two polymers did not induce in the bacteria population any phenotypic increase in methicillin resistance, or the selection of mutants carrying the mecA gene.     

Evaluation of susceptibility to antibiotics of Staphylococcus aureus strains resistant to methicillin]

Methicillin-resistant strains of Staphylococcus aureus (MRSA) constitute a serious diagnostic and therapeutic problem. Over 500 strains of Staphylococcus aureus were tested for susceptibility to methicillin. By application of a screening method, 13.7% of these strains were classified as methicillin-resistant. Over 95% of these strains were isolated from hospital infections. Applying criteria of belonging of these strains to methicillin-resistance classes it was found that 49.3% belonged to class II, 31.2% to class III and 19.5% to class IV. Analysis of susceptibility to antibiotics of MRSA strains demonstrated significant differences between class II and between class III and IV in resistance to imipenem, gentamycin, erythromycin and tetracycline. All tested strains were susceptible to ciprofloxacin, ofloxacin, vancomycin and teicoplanin. The screening method (25 mg methicillin/l of TSA medium) results in obtaining of univocal results of determination of methicillin-resistance in S. aureus.     

Distribution and regulation of the mobile genetic element-encoded phenol-soluble modulin PSM-mec in methicillin-resistant Staphylococcus aureus

The phenol-soluble modulin PSM-mec is the only known staphylococcal toxin that is encoded on a mobile antibiotic resistance determinant, namely the staphylococcal cassette chromosome (SCC) element mec encoding resistance to methicillin. Here we show that the psm-mec gene is found frequently among methicillin-resistant Staphylococcus aureus (MRSA) strains of SCCmec types II, III, and VIII, and is a conserved part of the class A mec gene complex. Controlled expression of AgrA versus RNAIII in agr mutants of all 3 psm-mec-positive SCCmec types demonstrated that expression of psm-mec, which is highly variable, is controlled by AgrA in an RNAIII-independent manner. Furthermore, psm-mec isogenic deletion mutants showed only minor changes in PSMα peptide production and unchanged (or, as previously described, diminished) virulence compared to the corresponding wild-type strains in a mouse model of skin infection. This indicates that the recently reported regulatory impact of the psm-mec locus on MRSA virulence, which is opposite to that of the PSM-mec peptide and likely mediated by a regulatory RNA, is minor when analyzed in the original strain background. Our study gives new insight in the distribution, regulation, and role in virulence of the PSM-mec peptide and the psm-mec gene locus.     

MRSA中mecA及femB基因的检测与耐药相关性

研究femB、mecA基因在耐甲氧西林金黄色葡萄球菌(MRSA)中的表达与耐药的关系.运用PCR对MRSA的femB、mecA基因进行检测,MRSA耐药检测采用头孢西丁纸片法.40 株金黄色葡萄球菌(下简称金葡菌)通过头孢西丁纸片法,检出 30 株耐头孢西丁的菌株,通过PCR检测这 40 株金葡菌mecA基因,30 株MRSA全部为阳性, femB基因在 30 株MRSA中全部表达,而甲氧西林敏感的金黄色葡萄球菌(MSSA)的未表达.结果可见,PCR能快速准确地鉴定MRSA, mecA基因是MRSA的耐药基因,femB基因是MRSA的耐药相关基因.