Survey of the methicillin resistance-associated genes mecA, mecR1-mecI, and femA-femB in clinical isolates of methicillin-resistant Staphylococcus aureus.

The restriction site polymorphism of the chromosomal femAB region and the first appearance of the regulatory element mecR1-mecI associated with the methicillin resistance determinant (mec) were analyzed in 192 initially methicillin resistant (Mcr) Staphylococcus aureus clinical isolates collected between 1965 and 1990 in the Zurich area. Forty-three of the strains lost the resistance spontaneously. All isolates that were still Mcr hybridized with mecA, the gene for the low-affinity penicillin-binding protein PBP 2'. Mcr strains isolated before 1977 lacked sequences that hybridized with mecR1-mecI, a regulatory element controlling the expression of mecA; exceptions to this were one strain isolated in 1966 and one strain isolated in 1972. The size of the EcoRV fragment carrying femA, a chromosomally encoded factor involved in pentaglycine side chain formation of the peptidoglycan and essential for the expression of methicillin resistance, was conserved in all strains but one, which was susceptible to methicillin even though it carried a functional mecA gene. The methicillin susceptibility of this particular strain was presumably due to a spontaneous femA-like mutation. The 192 strains belonged to seven different EcoRV restriction fragment patterns recognizable with a 10.5-kb probe covering the femAB region. Some 93% of the 149 Mcr strains belonged to pattern A, and the remaining Mcr strains shared patterns A' and B. The 42 isolates which spontaneously lost their resistance upon storage and revival represented all seven different patterns. This strong conservation of femA suggests an important role for femA in cell wall metabolism and methicillin resistance.     

Novel polymorphisms in mec genes and a new mec complex type in methicillin-resistant Staphylococcus aureus isolates obtained in rural Wisconsin

We determined allelic polymorphisms in the mec complexes of 524 methicillin-resistant Staphylococcus aureus isolates by partial or complete sequencing of three mec genes, mecA, mecI, and mecR1. The isolates had been collected over a 10-year period from patients living in rural Wisconsin, where the use of antibiotics was expected to be lower than in the bigger cities. Of the 18 mutation types identified, 16 had not been described previously. The five most common mutations were a mutation 7 nucleotides (nt) upstream from the start site (G-->T) in the mecA promoter (34.7%), an E246G change encoded by mecA (2.2%), a cytosine insertion at codon 257 in mecA (13.2%), an N121K change encoded by mecI (7.8%), and a major mecI-mecR1 deletion designated as a class B1 mec complex deletion type (25.4%). There was a high degree of conservation of the amino acid sequence of MecR1. Strains with the same mutations had variable resistance to oxacillin, and the median MIC for isolates that harbored the 7-nt-upstream mutation was lower than that for strains harboring other mutations. Our data suggest that the mecA promoter mutation plays a more important role in determining methicillin resistance than mutations in mecI and mecA do. Eighty-five percent of the tested isolates (n = 148) with the mecA promoter mutation and the class B1 mec complex deletion belonged to the same major clonal group, identified as MCG-2, and harbored the type IV staphylococcal cassette chromosome mec DNA. There was also a wide range of oxacillin MICs for strains with wild-type mecA, mecI, and mecR1 sequences, suggesting that the genetic backgrounds of clinical strains are significant in determining susceptibility to methicillin.     

Detection of mecA, mecR1 and mecI genes among clinical isolates of methicillin-resistant staphylococci by combined polymerase chain reactions

The distribution of the mec genes mecA, mecR1 and mecI that regulate the expression of methicillin resistance was investigated by PCR in 145 staphylococci of hospital origin. Determination of alterations and deletions in parts of the genes was achieved using 11 sets of primers in combined reactions. Methicillin-resistant Staphylococcus epidermidis strains appeared relatively stable, with 57.9% of isolates containing the whole regulatory region. Alterations within the mecA gene were detected more often in other coagulase-negative staphylococci, which also had a higher percentage with deletions of regulatory genes. Among methicillin-resistant S. aureus, a genetically heterogeneous population was identified, with several alterations and deletions of mec genes.     

The distribution of mecA, mecR1 and mecI and sequence analysis of mecI and the mec promoter region in staphylococci expressing resistance to methicillin.

The presence and sequences of genes that regulate the expression of methicillin resistance was investigated in 42 isolates of Staphylococcus aureus and 102 isolates of coagulase-negative staphylococci (CNS). PCR was used to detect mecA and the regulatory genes mecR1 and mecI. In a selected group of isolates, the sequences of mecI and the mec promoter region were also determined and compared with the sequences obtained from pre-MRSA strain N315. The genetic diversity of the collection was assessed by pulsed-field gel electrophoresis (PFGE). mecA was present in 21 S. aureus and 44 CNS. mecR1 was associated with mecA in all S. aureus and in all CNS, except two isolates of Staphylococcus haemolyticus. mecI was present in 48% of mecA-positive S. aureus and 50% of mecA-positive CNS. In six S. aureus isolates, mecI contained a termination codon at nucleotide 202 which would truncate the MecI protein. No mutation was found in the mecI gene of the four other S. aureus and 15 CNS sequenced. Seven isolates of Staphylococcus simulans had a single nucleotide substitution in the mec promoter region. Expression of methicillin resistance could be explained for all mecA-positive staphylococci with mutations within mecI or in the mec promoter region or in which mecI was deleted. However, the 'wild type' sequences observed in four S. aureus and eight CNS suggest that there is another mechanism for overcoming the repression of resistance caused by mecI.     

Presence of mec genes and overproduction of beta-lactamase in the expression of low-level methicillin resistance among staphylococci

Detection of methicillin-resistant staphylococci is critical for the management of infected patients in the hospital. A total of 55 nonreplicated clinical isolates of staphylococci (31 Staphylococcus aureus and 24 coagulase-negative staphylococci; CNS) collected during a one-year period and expressing low-level resistance to methicillin (oxacillin MIC of 2-4 mg/l for S. aureus and 0.5-4 mg/l for CNS) were studied. mec determinants and overproduction of beta-lactamase were investigated and pulsed-field gel electrophoresis (PFGE) was applied as a typing method. Twenty-four S. aureus isolates and 19 CNS carried the mecA gene. The presence of mecR1/mecI and blaR1/blaI genes correlated with the expression of low-level methicillin resistance in CNS. Four mecA-negative isolates (2 S. aureus and 2 CNS) overproduced beta-lactamase. PFGE revealed the presence of 2 major clonal types in mecA-positive S. aureus isolates, and 3 in CNS. Low-level methicillin resistance of staphylococci is correlated with the presence of the mecA gene and overproduction of beta-lactamase.     

Evaluation of a new 3-h hybridization method for detecting the mecA gene in Staphylococcus aureus and comparison with existing genotypic and phenotypic susceptibility testing methods.

A new 3-h hybridization assay for detection of the staphylococcal mecA gene and the Staphylococcus aureus nuclease gene was evaluated by comparing the assay with existing genotypic and phenotypic methods. A total of 275 S. aureus strains were tested, including 257 epidemiologically unrelated strains (135 mecA-positive and 122 mecA-negative; collection I), and 18 strains with known borderline resistance to methicillin (collection II). Complete agreement was obtained for both collections when comparing the new assay with genotypic methods. We further evaluated a range of phenotypic susceptibility methods recommended in Europe and/or USA using the presence of the mecA gene as the defining standard. For collection I a high degree of agreement was found for both Etests (256 strains) and the oxacillin screen plate test (255 strains); the degree of agreement was lower for agar dilution methicillin (250 strains) and oxacillin 1 microg discs (239 strains). For the borderline strains a high degree of agreement was only obtained by the oxacillin screen plate test (17 of 18 strains). The other tests were less accurate, in the following order: agar dilution methicillin, Etest methicillin, Etest oxacillin and oxacillin discs with disagreement for four, five, nine and 13 strains, respectively. In conclusion, the new hybridization assay is a rapid and exact method for detecting the mecA gene and the S. aureus nuclease gene. This study confirms that phenotypic tests for methicillin resistance in S. aureus strains creates both false-susceptible and false-resistant results, especially for borderline resistant strains.     

Genetic organization of mecA and mecA-regulatory genes in epidemic methicillin-resistant Staphylococcus aureus from Australia and England

The mecA gene that encodes methicillin resistance in Staphylococcus aureus may be regulated by the mecR1 and mecI genes, and this region has been referred to as the mec complex. An analysis of these regulatory genes in 35 epidemic methicillin-resistant S. aureus (MRSA) isolated in England and Australia has found that they contain three classes of mec complex. Firstly, the Class A mec complex with complete mecR1 and mecI genes. Secondly, a new variant of Class A, the Class A1 mec complex, with a 166 bp deletion in the membrane-spanning domain of mecR1 and a complete mecI gene. Thirdly, the Class B mec complex, in which the penicillin-binding domain of mecR1 and the whole mecI gene are deleted by the insertion of a partial sequence of IS1272. Seven MRSA isolated in England and Australia over different time periods had the Class A mec complex. However, the isolates did not have closely related pulsed-field gel electrophoresis (PFGE) patterns. The Class A1 mec complex was found in 12 Australian isolates and the English epidemic MRSA, EMRSA-1. All these organisms were isolated in the early 1980s and had closely related PFGE patterns. The Class B mec complex region was found in nine EMRSA and seven Australian MRSA isolated over the period from the 1970s to 2000. These isolates had related PFGE patterns. The mecA region was also compared in the isolates and all but two of the isolates had an XbaI restriction site. These results support the global spread of epidemic clones and confirm the close relationship between the Australian and English MRSA strains.     

Survey of methicillin-resistant clinical strains of coagulase-negative staphylococci for mecA gene distribution.

A total number of 125 methicillin-resistant (MIC, greater than or equal to 16) coagulase-negative Staphylococcus strains isolated in Japan were surveyed for the distribution of the mecA gene, the structural gene for penicillin-binding protein 2', which is the causative genetic element for the intrinsic resistance of methicillin-resistant Staphylococcus aureus. Screening with colony hybridization by using a cloned mecA gene probe revealed that 121 strains (96.8%) belonging to the nine coagulase-negative Staphylococcus species (S. epidermidis, S. haemolyticus, S. saprophyticus, S. sciuri, S. simulans, S. hominis, S. capitis, S. warneri, and S. caprae) carried mecA in their genome, indicating wide distribution of the gene among coagulase-negative Staphylococcus species. Most (93.4%) of the mecA-carrying strains were producers of penicillinase. Four strains, including two S. haemolyticus and two S. saprophyticus strains, did not carry mecA in spite of their resistance to methicillin. One of them was of low-level resistance (MIC, 16), but three of them had moderate- to high-level resistance to methicillin (MIC, 64). Analysis of gel electrophoretic banding patterns of penicillin-binding proteins of these strains showed absence of penicillin-binding protein 2' but some alterations in signal intensities of the other penicillin-binding proteins. The result indicated that about 3% of methicillin-resistant coagulase-negative staphylococci in these hospitals had a resistance mechanism different from that associated with the production of penicillin-binding protein 2', as has been reported in the case of a borderline methicillin-resistant strain of S. aureus.     

Genotypic versus phenotypic characterization, with respect to susceptibility and identification, of 17 clinical isolates of Staphylococcus lugdunensis

OBJECTIVES: To compare different methods for the identification and determination of susceptibility to penicillin and methicillin of Staphylococcus lugdunensis. METHODS: Seventeen clinical isolates of S. lugdunensis (identified by PCR amplification and sequencing of the rpoB gene) were studied using the ATB32-Staph, Crystal, Vitek 2 and Wider commercial systems. The clumping factor test and the tube coagulase test were also performed. Beta-lactamase production was studied by chromogenic methods. Methicillin resistance was phenotypically studied by the MRSA slide latex agglutination test, growth in MRSA agar, and the Vitek 2 and Wider systems (based on oxacillin MIC), and genotypically studied by detection of the mecA gene by PCR. RESULTS: The clumping factor test was negative in 35.3% of strains. All isolates were correctly identified to species level by the ATB32-Staph system. Species misidentification rates were 5.9%, 23.5% and 29.4% with the Crystal, the Vitek 2 and the Wider systems, respectively, mostly as Staphylococcus haemolyticus. Beta-lactamase was present in 11.8% of strains. Whereas 76.5% and 47.1% of strains exhibited oxacillin resistance (MIC range 0.5-2 mg/L) by the Vitek 2 system and the Wider system, respectively, none of the strains was positive in the MRSA slide latex agglutination test or grew in MRSA agar. All strains lacked the mecA gene. CONCLUSIONS: The clumping factor test and some commercial systems may misidentify S. lugdunensis. Oxacillin resistance detected by commercial systems is not indicative of the presence of the mecA gene. These facts, together with beta-lactamase production, may preclude adequate treatment of infections by this virulent coagulase-negative Staphylococcus.     

Evidence of a novel staphylococcal mec-encoded element (mecR) controlling expression of penicillin-binding protein 2''.

A region was identified on the methicillin resistance determinant (mec) isolated from Staphylococcus epidermidis and cloned into Staphylococcus carnosus which was responsible for a novel downregulation of the expression of methicillin resistance. The presence of this region reduced the overall expression of methicillin resistance and the synthesis of the mec-encoded penicillin-binding protein 2' (PBP 2') in S. carnosus. This region was located by Bal31 deletion mutagenesis upstream of the structural gene for PBP 2'. Deletions within this region resulted in higher levels of expression of methicillin resistance and increased levels of PBP 2' synthesis. We tentatively called this region mecR. Analysis of selected Mcr strains of Staphylococcus aureus and S. epidermidis by Southern hybridization suggested that the natural occurrence of two types of mec resistance determinants differ by the presence or absence of mecR-specific sequences.     

Detection and expression of methicillin/oxacillin resistance in multidrug-resistant and non-multidrug-resistant Staphylococcus aureus in Central Sydney,Australia

Ninety clinical Staphylococcus aureus isolates from separate patients were examined phenotypically and genotypically for susceptibility to methicillin/oxacillin. Thirty were methicillin/oxacillin susceptible and 60 were methicillin and oxacillin resistant (MRSA). The 60 MRSA isolates examined were subdivided into two groups according to their antibiotic profiles and comprised 30 non-multidrug-resistant (NMDR) isolates, resistant to less than two non-beta-lactam antibiotics, and 30 multidrug-resistant (MDR) isolates, resistant to three or more non-beta-lactam antibiotics. Phenotypic and genotypic analysis of methicillin/oxacillin showed that despite use of the guidelines published by the NCCLS for the testing of S. aureus susceptibility to methicillin/oxacillin, MIC values of some NMDR MRSA isolates fell below the NCCLS-recommended breakpoints. Etest strips failed to detect two NMDR MRSA isolates tested with oxacillin and four tested with methicillin. Lowering the NCCLS-recommended oxacillin screen agar concentration from 6 to 2 mg/L and temperature of incubation to 30 degrees C, improved the specificity and sensitivity of NMDR MRSA detection from 87% to 100%. On PFGE analysis these NMDR MRSA strains were genotypically different. Genotypic tests, such as multiplex PCR for the mecA/nuc genes and DNA hybridization for the mecA gene, or phenotypic monoclonal antibody-based tests to detect penicillin-binding protein 2a (PBP2a) offer advantages for problematic isolates in detecting or confirming low-level phenotypic heterogeneous mecA expression of oxacillin and methicillin resistance in NMDR MRSA.     

多重聚合酶链反应检测耐甲氧西林葡萄球菌

目的应用一种敏感快速的多重聚合酶链反应（ＰＣＲ）,检测耐甲氧西林的金黄色葡萄球菌（ＭＲＳＡ）和凝固酶阴性葡萄球菌（ＭＲＣＮＳ）。方法临床分离的北京地区金黄色葡萄球菌１２３株,ＭＲＣＮＳ１２２株,用溶壁素和蛋白酶Ｋ制备模板ＤＮＡ,设计葡萄球菌甲氧西林耐药的决定基因,金黄色葡萄球菌独有的一个辅助基因和细菌中均有的１６ＳｒＲＮＡ基因引物,通过多重ＰＣＲ技术对标本进行扩增。结果１２３株金黄色葡萄球菌的ｆｅｍＡ基因１００％（１２３／１２３）阳性,ｍｅｃＡ基因阳性的占１８．７％（２３／１２３）,１２２株ＭＲＣＮＳ的ｆｅｍＡ１００％（１２２／１２２）阴性,ｍｅｃＡ阳性的占２４．６％（３０／１２２）。１６ＳｒＲＮＡ基因片断在多重ＰＣＲ中作为内部参照避免了假阴性结果的出现。结论建立的多重ＰＣＲ技术检测ＭＲＳＡ和ＭＲＣＮＳ具有敏感、快速、特异的特点,是一种可靠的实验诊断手段。     

Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit

The EVIGENE MRSA Detection Kit was evaluated on coagulase-negative staphylococci (CoNS) from agar plates and on staphylococci directly from positive spiked blood cultures. For the CoNS study, a total of 242 isolates were tested, and of these 237 gave valid test results. For the 237 valid tests, all gave correct mecA classification. For the blood culture procedure, a collection of 51 mecA-positive Staphylococcus aureus, 21 mecA-negative S. aureus, 31 mecA-positive CoNS and 28 mecA-negative CoNS were used for the simulated blood cultures. For the S. aureus strains, all gave valid test results and correct mecA classification. One of the MRSA isolates gave a very faint nuc signal, and another four isolates gave results close to the cut-off of the kit; however, these were still clearly positive when read by the naked eye. For the CoNS isolates, 51 of the 59 strains gave valid results. All of these 51 strains gave correct mecA status. Thus the EVIGENE MRSA Detection Kit can provide fast and accurate determination of methicillin resistance in CoNS. This preliminary study of the blood culture procedure indicates that it is possible to achieve determination of methicillin resistance in staphylococci 8 h after positivity of the blood culture, making same-day detection of methicillin resistance possible.     

Rapid detection of mecA in methicillin resistant Staphylococcus aureus using cycling probe technology.

A novel method has been developed for the detection of the mecA gene, that confers the principle mechanism of methicillin resistance in staphylococci. Cycling Probe Technology (CPT) is a rapid, simple, isothermal method for the detection of specific target sequences. CPT utilizes a unique chimeric DNA-RNA-DNA probe sequence that provides an RNase H sensitive scissile link when hybridized to a complementary target DNA sequence. In the presence of target DNA, the cycling reaction converts full-length chimeric probe into cleaved probe fragments, which accumulate and are quantified. A cycling probe designed for detection of a specific sequence within the mecA gene was used to develop a culture confirmation assay for methicillin resistant Staphylococcus aureus. The CPT assay was used to screen 238 S. aureus isolates and the results were in complete agreement with detection of the mecA gene by polymerase chain reaction (PCR). Detection of mecA should be considered the gold standard for determining methicillin resistance in S. aureus. This study demonstrates the feasibility of using CPT to meet this need.     

Role of penicillinase plasmids in the stability of the mecA gene in methicillin-resistant Staphylococcus aureus

The stability of methicillin resistance (Mcr) in three independent clinical isolates, MR108, MR6, and MR61, of methicillin-resistant Staphylococcus aureus (MRSA) was studied. The Mcr phenotype was stably maintained in the progeny of all three MRSA clones carrying penicillinase plasmids. However, when the clones were tested after elimination of the plasmids, methicillin-susceptible (Mcs) subclones appeared at various frequencies. Seven Mcs subclones were classified into two groups based on their stabilities. Five Mcs subclones, which were derived from homogeneous strains MR108 and MR61, were stably susceptible. They lost penicillin-binding protein 2' production, and moreover, the mecA gene was deleted in four of five subclones. Two subclones were derived from heterogeneous strain MR6. They were very unstable, and more than half of their progeny were Mcr revertants. However, the remainder were stably Mcs and had lost penicillin-binding protein 2' and the mecA gene. We propose that penicillinase plasmids, which are present in most MRSA strains, play an important role in the stability and phenotypic expression of the mecA gene.     

Mechanisms of heteroresistance in methicillin-resistant Staphylococcus aureus.

Characteristic for methicillin-resistant (Mcr) staphylococci is the heterogeneous expression of the intrinsic methicillin resistance. The majority of the cells express resistance to low concentrations of methicillin, and a minority of the cells express resistance to much higher concentrations. We show here (i) that the presence of the mecA encoding region on plasmid pBBB79 was sufficient to render a methicillin-susceptible (Mcs) Staphylococcus aureus strain heteroresistant and (ii) that this Mcr strain segregated highly resistant subclones which retained the high-resistance phenotype under nonselective growth conditions. The Mcr strain with only mecA on plasmid pBBB79 thus behaved identically to a Mcr strain carrying the complete mec determinant integrated at its proper chromosomal site. (iii) Curing a such highly resistant subclone from plasmid pBBB79 yielded an Mcs strain that was as susceptible as the original Mcs parent strain. (iv) Comparisons were made between the original parent and the cured Mcs strain by backcrossing pBBB79 into them and looking at their progeny. Transductants derived from the formerly highly resistant cured strain became resistant to high concentrations of methicillin, whereas transductants derived from the original parent strain were resistant to lower concentrations of methicillin and showed the typical heterogeneous resistance. We deduced therefrom that the high-level resistance expressed by the minority of the population of Mcr S. aureus was due to a chromosomal mutation(s) (chr*) involving neither mecA nor the additional 30 kb of mec-associated DNA. Moreover, we could show that this postulated mutation chr* was not linked to the femAB operon, which is known to affect methicillin resistance levels.