Characterization of MLS(b) resistance among Staphylococcus aureus and Staphylococcus epidermidis isolates carrying different SCCmec types

This work characterizes MLS(b) resistance in 39 methicillin-resistant Staphylococcus aureus (MRSA) and 32 Staphylococcus epidermidis (MRSE) isolates. Of 21 erm(A) gene encoding MRSA isolates, 71.4% carried SCCmecIII, whereas of 12 isolates carrying the erm(C) gene, 83.3% carried SCCmecIV. Among the 25 MRSE isolates positive for the erm(C) gene, 80% had SCCmecIV or nontypeable cassettes. Isolates carrying these genes had MIC(90) ≥ 256 μg/mL to erythromycin and clindamycin. The msr(A) gene was associated with a low MIC(90) to these drugs. The erm(A) gene was associated with SCCmecIII in MRSA isolates, whereas the erm(C) gene was associated with SCCmecIV in both MRSA and MRSE isolates.     

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.     

Detection of methicillin resistance in Staphylococcus aureus isolates using two-step triplex PCR and conventional methods

A two-step triplex PCR assay targeting the mecA, femA, and nuc genes was developed for the detection of methicillin resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. The triplex PCR revealed the presence of the femA and nuc genes in all the S. aureus isolates examined (n=105). Forty-four clinical isolates were mecA positive and no foodborne isolates were mecA positive. The PCR results had a 98 or 99% correlation with the results of PBP2a latex agglutination tests or oxacillin susceptibility tests, respectively.     

Impedimetric detection of single-stranded PCR products derived from methicillin resistant Staphylococcus aureus (MRSA) isolates

Using electrochemical impedance spectroscopy (EIS) the sensitive and specific detection of the antibiotic resistance gene mecA has been demonstrated. The gene sequence was obtained from clinical Staphylococcus aureus isolates. Initially a mecA specific probe was selected from hybridisation tests with a 3' and 5' version of a previously published probe sequence. When immobilised on a gold electrode in PNA form it was possible to detect hybridisation of mecA PCR product electrochemically at concentrations as low as 10nM. By incorporating an undecane-thiol and 1.8 nm glycol spacer into the PNA probe it was possible to extend the limit of detection for mecA to 10 pM. Most published studies on EIS and nucleic acid detection report the use of short artificial DNA sequences or novel signal amplification schemes which improve sensitivity whereas this study reports the successful detection of long DNA fragments produced by PCR following extraction from clinical isolates. Finally, using screen printed electrodes the paper demonstrates hybridisation monitoring of mecA in an "on-line" assay format under ambient conditions which paves the way for rapid mecA detection in point of care scenarios.     

Gene acquisition at the insertion site for SCCmec, the genomic island conferring methicillin resistance in Staphylococcus aureus

Staphylococcus aureus becomes resistant to methicillin by acquiring a genomic island, known as staphylococcal chromosome cassette mec (SCCmec), which contains the methicillin resistance determinant, mecA. SCCmec is site-specifically integrated into the staphylococcal chromosome at a locus known as the SCCmec attachment site (attB). In an effort to gain a better understanding of the potential that methicillin-sensitive S. aureus (MSSA) isolates have for acquiring SCCmec, the nucleotide sequences of attB and surrounding DNA regions were examined in a diverse collection of 42 MSSA isolates. The chromosomal region surrounding attB varied among the isolates studied and appears to be a common insertion point for acquired foreign DNA. Insertions of up to 15.1 kb were found containing open reading frames with homology to enterotoxin genes, restriction-modification systems, transposases, and several sequences that have not been previously described in staphylococci. Two groups, containing eight and four isolates, had sequences found in known SCCmec elements, suggesting SCCmec elements may have evolved through repeated DNA insertions at this locus. In addition, the attB sequences of the majority of MSSA isolates in this collection differ from the attB sequences of strains for which integrase-mediated SCCmec insertion or excision has been demonstrated, suggesting that some S. aureus isolates may lack the ability to site-specifically integrate SCCmec into their chromosomes.     

耐甲氧西林金黄色葡萄球菌的两种新SCCmec型别及其抗药性研究

为探明本地区耐甲氧西林金黄色葡萄球菌(Staphylococcusaureus)的耐药性、流行病学分布状况及携带的葡萄球菌染色体mec盒(SCCmec)型别,用K-B琼脂扩散法、E-test和多位点PCR,对临床分离的金黄色葡萄球菌菌株进行了SCCmec分型及耐药性测定。结果发现了两种新的SCCmec型别,新1型含Ⅱ型的mecA上游特异性位点B和位于mecA内的M位点以及Ⅲ型的下游位点F,缺乏Ⅱ型上游位点C和下游位点D、G;新2型含Ⅰ、Ⅱ型的上游特异性位点A、B和两个Ⅲ型的下游位点F、H,同样缺乏位点C、D、G,可能分别为原有Ⅱ型和Ⅰ、Ⅱ型与Ⅲ型的基因重组株;且携带有新SCCmec型别的MRSA菌株,其流行病学分布特点及抗药性也与国外已报导的菌株不同,多分自门诊病人,且耐药性高,抗药谱广,值得引起高度重视和关注。     

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。     

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.     

Staphylococcus aureus isolates belonging to different multilocus sequence types present specific virulence gene profiles

To characterize 73 Staphylococcus aureus isolates from infections in an orthopedic hospital in Rio de Janeiro, we investigated the SCCmec types, the clonality by pulsed field gel electrophoresis and multilocus sequence typing, and the presence of virulence genes. Twenty-eight (38.3%) methicillin-resistant (16 SCCmec type IV and 12 type III) isolates were detected. Most (83.5%) of the isolates were included in five lineages: sequence type (ST) 239 (SCCmecIII), 1, 5, 30, and 1462 (SCCmecIV and/or methicillin-susceptible isolates). Virulence genes fnbB, bbp, and pvl were related to STs 239, 30, and 30/1462, respectively. Isolates from STs 1, 5, and 30 presented specific virulence profiles, irrespective of methicillin resistance.     

Detection of the genes of pyrogenic toxins of superantigens in clinical isolates of methicillin resistant Staphylococcus aureus

The content of methicillin resistant S. aureus (MRSA) genes, coding the synthesis of staphylococcal enterotoxins A, B, C (sea, seb, sec) and the toxin of the toxic shock syndrome (tst-H) which was classified with pyrogenic toxins of superantigens (PTSAgs), was studied with the use of PCR amplification. The study revealed the specific features of the content of genes sea and sec, detected in epidemic strains, identified earlier and found to circulate in Russian hospitals. Among the isolates, genetically related to international epidemic strain EMRSA-1, isolates containing no gene sea were detected, while among the isolates genetically related to strain EMRSA-2, isolates containing not only gene sea, but also gene sec were detected, which was indicative of the tendence of this epidemic strain in the direction of further acquisition of pathogenicity genes. As revealed in further studies, among the cultures obtained in bacteriemia, 88% contained gene sea. Two out of three isolates obtained from patients with the symptoms of toxic shock also contained this gene. The differences in the content of genes PTSAgs (sea, seb, sec and tst-H) could serve as a genetic criterium for the differention of isolates circulating in a hospital, as well as for a more complete characterization of the epidemic strains MRSA. The determination of the given genetic markers in genetic strains in circulating strains will make it possible to prognosticate the structure, severity and outcomes of hospital infections. The conditions of PCR amplification for the determination of genes sea, seb, sec and tst-H, as well as multiplex PCR for the determination of genes sea and seb, were developed.     

Determination of susceptibility of Staphylococcus aureus to methicillin by luciferim-luciferase assay of bacterial adenosine triphosphate

M c W alter , P.W. 1984. Determination of susceptibility of Staphylococcus aureus to methicillin by luciferin-luciferase assay of bacterial adenosine triphosphate. Journal of Applied Bacteriology , 56 , 145–150.
Susceptibility of 50 strains of Staphylococcus aureus to methicillin was determined by disc diffusion, minimum inhibitory concentration (MIC) test and luciferin-luciferase assay of bacterial adenosine triphosphate (ATP). ATP concentration time curves calculated on eight strains (four sensitive, four resistant) incubated at 30C indicated 2.5 h as the optimum time for determination of methicillin susceptibility. The ATP concentration and relative light unit (RLU) value of each test broth (antibiotic-containing), expressed as a percentage of its own control broth (antibiotic-free) indicated values of < 30% for ATP and < 40% for RLU to be indicative of methicillin sensitivity. Single time point ATP assays carried out on the 50 strains after 2.5 h at 30C correlated exactly with disc diffusion and MIC. Luciferin-luciferase assay of bacterial ATP appeared to be a reliable, rapid technique for determining the susceptibility of Staph. aureus to methicillin.     

Synergistic effects of anacardic acids and methicillin against methicillin resistant Staphylococcus aureus

The synergistic effects of 6-alk(en)ylsalcylic acids, also known as anacardic acids, in combination with methicillin against Staphylococcus aureus ATCC 33591 (MRSA) was investigated. The double bond in C15-anacardic acids is not essential in eliciting the antibacterial activity but is associated with increasing the activity. The synergistic effects decreased with increasing the number of double bonds in the alkyl chain. On the other hand, the antibacterial activity of anacardic acids possessing different alkyl chain lengths against the same MRSA strain was found to be a parabolic function of their lipophilicity and maximized with the alkyl chain length of C10 and C12. Notably, the synergistic effects were noted to increase with increasing the alkyl chain length.     

SCCmec遗传元件及其在耐甲氧西林金黄色葡萄球菌分子分型中的应用

携带mec基因簇的葡萄球菌盒式染色体(Staphylococcal chromosome cassette mec, SCCmec)遗传元件的获得是耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus, MRSA)耐药的主要原因。SCCmec由一个mec基因簇、一个染色体重组酶(ccr)基因簇及3个J区组成。mec基因簇含有mecA及其调控基因,mecA基因编码的耐药决定簇使MRSA对β-内酰胺类抗生素耐药;ccr基因簇编码的重组酶负责SCCmec元件的整合与切离;J区差异大,导致不同来源MRSA菌株携带SCCmec的大小不一,在组成上也具有多样性。这些特征为利用SCCmec元件进行MRSA分型创造了条件。文章介绍了SCCmec元件的结构和功能,综述了基于SCCmec的MRSA分型研究。     

Comparison of disk-diffusion method and PCR for detection of methicillin resistance in Staphylococcus aureus strains

The aim of the study was to compare the disk-diffusion (oxacillin 1 microg, cefoxitin 30 microg) method and PCR for detection of methicillin-resistance in S. aureus. The investigation were carried out on 120 S. aureus strains isolated from clinical materials of patients hospitalized in the University Hospital at the L. Rydygier Collegium Medicum in Bydgoszcz, University of Nicolaus Copernicus in Toruń. Of the 120 S. aureus strains tested, 60 (50%) were mecA-positive by PCR. Consistency of results between oxacillin disk-difussion method and PCR amounted 92.5% and cefoxitin disk-diffusion method and PCR--98.3%. The oxacillin disk-difussion method falsely identified 3 (2.5%) strains as MSSA (sensitivity 95.0%) and 4 strains as MRSA (specificity 93.3%) in comparison with PCR. The cefoxitin disk-diffusion method falsely identified 2 (1.6%) strains as MSSA (sensitivity 96.7%) and there were no false resistant results (specificity 100%). Our results showed that in disk-diffusion tests, cefoxitin is a better than oxacillin for the identification of MRSA.     

Evaluation of a rapid culture-based screening test for detection of methicillin resistant Staphylococcus aureus

The performance of a culture based assay, BacLite Rapid MRSA for the rapid detection (5 hours) of methicillin resistant Staphylococcus aureus (MRSA) from specimens (n = 377) obtained from nares, throat, wounds and perineum was investigated. Compared to culture based reference methods (chromogenic MRSA ID (bioMerieux)), selective enrichment broth, PBP2' latex agglutination (Oxoid) and VITEK 2 identification (bioMerieux), an overall sensitivity of 71% with a 82% specificity and a negative predictive value (NPV) of 95% was provided. The Baclite test is rapid and easy to use and has the advantage of a culture-based detection method for MRSA.