Relationship between cefamandole and cefuroxime activity against oxacillin-resistant Staphylococcus epidermidis and oxacillin resistance phenotype.

The activity of cefamandole and cefuroxime against oxacillin-resistant staphylococcus epidermidis was studied in vitro to determine whether there was any relationship between oxacillin resistance phenotypes and cephalosporin activity. Oxacillin resistance phenotypes were determined by efficiency-of-plating studies on Mueller-Hinton agar containing oxacillin, with and without NaCl, and incubated at 30 and 35 degrees C. On the basis of MIC and MBC determinations, cefamandole was more active than cefuroxime against oxacillin-resistant S. epidermidis. Although temperature had minimal effect on the activity of either cefamandole or cefuroxime, NaCl significantly decreased the activity of cefuroxime but not of cefamandole. Neither cephalosporin consistently produced greater than or equal to 99.9% bactericidal activity within 24 h in timed killing-curve studies. No consistent relationship was observed between cefamandole or cefuroxime activity and oxacillin resistance phenotype.     

Screening tests for the detection of methicillin resistance in Staphylococcus epidermidis.

Methods to detect resistance to methicillin in Staphylococcus epidermidis were studied in order to find a rapid screening test suitable for routine use. One hundred and forty-nine clinical isolates, 16 isolates from skin of healthy people and two reference strains were studied. Hypersecretion of beta-lactamase as a cause of methicillin resistance was eliminated in the strains studied. Tube and microtitre breakpoint, agar breakpoint and disc diffusion methods were compared. The breakpoint for methicillin resistance used was 16 mg/L in broth and 10 mg/L in agar. The discs used contained 1 and 5 micrograms oxacillin and 5 and 10 micrograms methicillin. Turbidimetric measurements in broth during incubation were carried out using the Bioscreen analysing system. The skin strains were founf to be susceptible in all tests. Using an inoculum of 10(7) cfu/mL 111/149 clinical isolates were classified as resistant after incubation for 24 h at 35 degrees C using the tube and microtitre breakpoint tests, incubation for 72 h did not increase this rate. When an inoculum of 10(5) cfu/mL was used 73% of these strains were identified within 24 h and all within 72 h with the tube breakpoint test. Using the microtitre breakpoint test, with an inoculum of 10(5) cfu/mL or lower, all resistant strains were not detected within 48 h. All agar breakpoint tests required 48 h incubation for reliable results. Only the 1 microgram oxacillin disc always separated strains found to be resistant or susceptible in the tube breakpoint test. The zone of inhibition was clearly readable after 16 h of incubation at 35 degrees C.     

Methicillin-resistant Staphylococcus epidermidis.

Staphylococcus epidermidis is frequently associated with infection of prosthetic heart valves, prosthetic orthopedic devices, and neurosurgical shunts. Penicillinase-resistant semisynthetic penicillins, such as methicillin, have been the therapeutic and prophylactic agents of choice for S epidermidis infection. However, more S epidermidis isolates are now resistant to methicillin and other penicillins. In our laboratory 41% of S epidermidis isolates were resistant to methici-lin. All of the methicillin-susceptible isolates and 82% of the methicillin-resistant isoates were susceptible to cephalothin. Cephalothin should replace methicillin as the prophylactic and therapeutic agent of choice in institutions with a high percentage of methicillin-resistant S epidermidis.     

溶血葡萄球菌对抗菌药物的敏感性和mecA基因的检测

目的　了解溶血葡萄球菌的耐药情况 ,以指导临床合理用药。方法　对 133株临床分离的溶血葡萄球菌采用微量肉汤稀释法测定 16种抗菌药物的最低抑菌浓度 (MIC) ,多聚酶链反应 (PCR)法检测mecA基因 ,并与普通药敏试验比较 ,对不相符的菌株进行抑制、诱导试验。结果　 133株溶血葡萄球菌对利奈唑胺、万古霉素、去甲万古霉素无耐药 ,对替考拉宁的耐药率为 6 .0 % ;对阿米卡星、异帕米星、米诺环素、利福平、氟氧头孢的耐药性较低 ,耐药率 <2 0 % ;对青霉素、苯唑西林的耐药率 >90 %。除米诺环素、青霉素外 ,住院患者分离菌株的耐药率高于门诊患者分离的菌株。mecA基因阳性率为 90 .2 3% ,且PCR产物经克隆测序证实为mecA特异性产物。对 2株mecA基因阳性而苯唑西林MIC≤ 0 .2 5 μg/ml的菌株进行诱导试验后其MIC≥ 0 .5 μg/ml;对 3株mecA基因阴性而MIC≥ 0 .5 μg/ml的菌株进行抑制试验后其MIC值下降 8倍以上。 结论　溶血葡萄球菌对大多数抗菌药物均有较高的耐药性 ,对糖肽类抗生素的敏感性也在下降 ,需引起临床重视。     

溶血葡萄球菌对抗菌药物的敏感性和mecA基因的检测

目的了解溶血葡萄球菌的耐药情况,以指导临床合理用药.方法对133株临床分离的溶血葡萄球菌采用微量肉汤稀释法测定16种抗菌药物的最低抑菌浓度(MIC),多聚酶链反应(PCR)法检测mecA基因,并与普通药敏试验比较,对不相符的菌株进行抑制、诱导试验.结果 133株溶血葡萄球菌对利奈唑胺、万古霉素、去甲万古霉素无耐药,对替考拉宁的耐药率为6.0%;对阿米卡星、异帕米星、米诺环素、利福平、氟氧头孢的耐药性较低,耐药率<20%;对青霉素、苯唑西林的耐药率>90%.除米诺环素、青霉素外,住院患者分离菌株的耐药率高于门诊患者分离的菌株.mecA基因阳性率为90.23%,且PCR产物经克隆测序证实为mecA特异性产物.对2株mecA基因阳性而苯唑西林MIC≤0.25 μg/ml的菌株进行诱导试验后其MIC≥0.5 μg/ml;对3株mecA基因阴性而MIC≥0.5 μg/ml的菌株进行抑制试验后其MIC值下降8倍以上.结论溶血葡萄球菌对大多数抗菌药物均有较高的耐药性,对糖肽类抗生素的敏感性也在下降,需引起临床重视.     

Comparison of PCR detection of mecA with methicillin and oxacillin disc susceptibility testing in coagulase-negative staphylococci

The provisional BSAC method for the detection of methicillin sensitivity in coagulase-negative staphylococci (CNS) requires incubation of isolates for 48 h and raises the problem of timely reporting of susceptibility data. The forthcoming withdrawal of methicillin raises another difficulty. We evaluated 42 clinically significant CNS blood culture isolates by PCR, methicillin and oxacillin disc testing and by using methicillin Etests. Our results suggest that, although oxacillin disc susceptibility testing is a reasonable first line step, optimal and timely detection of resistance or susceptibility may require a combination of phenotypic and genotypic methods.     

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.     

Evaluation of a latex agglutination assay for rapid detection of oxacillin resistant Staphylococcus aureus

Oxacillin resistance in Staphylococcus aureus is mediated by the mecA gene, resulting in production of penicillin-binding protein 2a (PBP2a), which is not present in the oxacillin susceptible strains. We evaluated the ability of a 30-min latex agglutination (LA) test (Seiken, Tokyo, Japan) to detect production of PBP2a in 315 clinical isolates of S. aureus. The LA results were compared with results of susceptibility testing using the Vitek GPS-SV test card. The latex test was positive for all 206 isolates determined to be methicillin resistant by Vitek (sensitivity 100%), the latex test was negative for 108 of 109 isolates determined to be oxacillin susceptible by Vitek, and the latex test was positive for 1 isolate determined to be susceptible by Vitek (specificity 99.1%). The discrepant isolate was negative for the mecA gene by polymerase chain reaction (PCR). The LA test is a rapid and reliable method for detecting oxacillin resistant S. aureus.     

Analysis of Diversity of Mutations in the mecI Gene and mecA Promoter/Operator Region of Methicillin-Resistant Staphylococcus aureus and Staphylococcus epidermidis

Genomic diversity of mutation in the mecI gene or mecA promoter/operator region was analyzed for clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE). In most MRSA strains, a single base substitution was detected in either the mecI (three different positions) or the mecA promoter (two different positions), while a 28-base deletion in mecI was found in one strain. In contrast, no mutation was detected in these gene sequences of MRSE strains.     

Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecA(LGA251)

The recent finding of a new mecA homologue, mecA(LGA251) , with only 70% nucleotide homology to the conventional mecA gene has brought the routine testing for mecA as a confirmatory test for methicillin-resistant Staphylococcus aureus (MRSA) into question. A multiplex PCR was designed to differentiate mecA(LGA251) from the known mecA together with detection of lukF-PV and the spa gene fragments, enabling direct spa typing by sequencing of the PCR amplicons. The PCR analysis and subsequent spa typing were validated on a large collection (n=185) of contemporary MRSA and methicillin-sensitive S. aureus isolates, including 127 isolates carrying mecA(LGA251) . The mecA(LGA251) gene was situated in staphylococcal cassette chromosome mec type XI elements, and sequence variation within a 631-bp fragment of mecA(LGA251) in 79 isolates indicated a very conserved gene sequence. Following a successful validation, the multiplex PCR strategy was implemented in the routine testing of MRSA for national surveillance. Over a 2-month period, among 203 samples tested, 12 new MRSA cases caused by isolates carrying mecA(LGA251) were identified, emphasizing the clinical importance of testing for these new MRSA isolates.     

Detection of mecA-mediated resistance using reference and commercial testing methods in a collection of Staphylococcus aureus expressing borderline oxacillin MICs

Phenotypic methods for detecting mecA-mediated resistance in Staphylococcus aureus include both oxacillin and cefoxitin susceptibility tests; many laboratories perform multiple tests. Conflicting oxacillin and cefoxitin susceptibility results are most likely to occur for isolates that either have reduced susceptibility to oxacillin by a non-mecA-mediated mechanism or are mecA positive but are very heteroresistant. To understand the performance of oxacillin and cefoxitin tests for such isolates, we tested 135 S. aureus isolates using either cefoxitin or oxacillin and compared the results with mecA polymerase chain reaction. These strains either expressed borderline oxacillin MICs (1-4 microg/mL) and had undetermined mecA status or were mecA positive but were not detected by oxacillin broth microdilution (BMD) or disk diffusion (DD) in original testing. For 24-h readings, performance of cefoxitin tests (sensitivity/specificity) were DD (99/100), Etest using < or =6 microg/mL as susceptible (99/98), and Phoenix MIC using < or =4 microg/mL as susceptible (98/100). Using 6 microg/mL of cefoxitin as a screen test in both BMD and agar dilution also worked well (98/98-100). Sensitivity/specificity of oxacillin methods were oxacillin agar screen (BBL: 80/86; Remel, Lenexa, KS: 85/50), DD (91/59), BMD (85/88), MicroScan (89/96), VITEK Legacy (82/93), VITEK 2 (91/73), and Phoenix, (67/96). These results suggest that a cefoxitin test can be used alone to predict mecA-mediated resistance in S. aureus.     

多重PCR检测临床分离金黄色葡萄球菌lukS/F-PV和mecA基因

目的建立一种新的多重PCR方法,检测临床分离金葡菌的杀白细胞素毒力基因(lukS/F-PV)和甲氧西林耐药基因(mecA)。方法收集我院2006年1—12月从临床多种标本中分离鉴定的不重复金葡菌,应用多重PCR技术,优化反应条件,同时扩增葡萄球菌属特异性基因16S rRNA、金葡菌种特异性基因nuc、lukS/F-PV基因和mecA基因,扩增产物经凝胶成像系统分析。结果217株金葡菌经多重PCR检测,31株是16S rRNA+nuc+lukS/F-PV+mecA基因型,3株是16SrRNA+nuc+lukS/F-PV基因型,135株是16S rRNA+nuc+mecA基因型,40株是16S rRNA+nuc基因型,其中5株仅扩增出16S rRNA基因,3株仅扩增出nuc基因。lukS/F-PV基因和mecA基因测序显示与GenBank中的已有序列具有高度同源性,其阳性率分别为15.7%(34/217)和76.5%(166/217);34株lukS/F-PV基因阳性的分离株中,31株为MRSA,占91.2%(31/34)。结论本方法适用于快速检测和鉴定产杀白细胞素MRSA,lukS/F-PV基因阳性的菌株以MR...     

pH-dependent oxacillin tolerance of Staphylococcus aureus

A proportion of clinical isolates of Staphylococcus aureus exhibit resistance to bactericidal activity of certain antibiotics, despite normal susceptibility to inhibition. This phenomenon is termed "tolerance." The methodology used to determine tolerance varies greatly. To clarify the relationship between laboratory methodology and tolerance, we determined the minimal bactericidal concentrations and minimal inhibitory concentrations for 20 clinical isolates by two methods. Inocula were prepared by either 3-h growth of the organism in Mueller-Hinton broth or overnight (22 to 24 h) cultures in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.). All inocula were plated for colony counts and tested for pH. An American Type Culture Collection (Rockville, Md.) reference strain was included in all tests for standardization. Tolerance was defined by the strictest criterion, i.e., a minimal bactericidal concentration/minimal inhibitory concentration ratio of greater than or equal to 100. With the first method, none of the 20 isolates displayed tolerance (mean inoculum pH, 7.15). When inocula were grown in Trypticase soy broth with overnight incubation, 35% (7 of 20) showed tolerance (mean inoculum pH, 6.22). There was a significant association between the decreased bactericidal capacity at high oxacillin concentrations and overnight incubation in Trypticase soy broth (P less than 0.01). We suggest that tolerance in staphylococci is in some way related to the pH value of the inoculating culture. Such pH-induced tolerance may have a clinical corollary in sequestered infections where the pH is acidic.     

Bactericidal activity of oxacillin against beta-lactamase-hyperproducing Staphylococcus aureus.

The bactericidal activity of oxacillin against beta-lactamase-hyperproducing strains of Staphylococcus aureus for which the MIC by MicroScan was 1 or 2 micrograms/ml after incubation for 24 h was evaluated by MBC studies and kill kinetics methods. MBC and kill kinetics tests were both performed using Mueller-Hinton broth (MHB), with and without 2% NaCl supplementation, and incubation at 30 and 35 degrees C. When MBC testing was performed with salt-supplemented MHB, the oxacillin MBC/MIC ratio was greater than 8 for 17 and 16 of 17 S. aureus isolates at 30 and 35 degrees C, respectively. With unsupplemented MHB, the MBC/MIC ratio was greater than 8 for nine and six strains at 30 and 35 degrees C, respectively. Five representative strains were selected for kill kinetics studies under the four different test conditions. Oxacillin appeared more bactericidal by the kill kinetics method than by MBC testing. Moreover, salt supplementation did not affect the results of kill kinetics studies as dramatically as it did the MBC results. Thus, bactericidal testing results are markedly influenced by the technique employed, and further in vivo studies are necessary to fully evaluate the efficiency of oxacillin against beta-lactamase-hyperproducing strains of S. aureus.     

Synergistic effect of quinolones and oxacillin on methicillin-resistant Staphylococcus species.

Various combinations of antistaphylococcal antimicrobial agents have been tested against 17 selected Staphylococcus isolates, including methicillin-susceptible and methicillin-resistant strains of S. aureus and coagulase-negative Staphylococcus species. With the checkerboard technique the following combinations were tested: oxacillin-ofloxacin, oxacillin-temafloxacin, oxacillin-fleroxacin, vancomycin-fleroxacin, gentamicin-fleroxacin, and rifampin-fleroxacin. Against methicillin-resistant staphylococci the combination oxacillin-quinolone tested at 35 degrees C always showed a fractional inhibitory concentration (FIC) index of less than 0.75, which is interpreted as synergistic or additive. Equal or more synergistic effects were observed at 30 degrees C. In contrast, when methicillin-susceptible Staphylococcus species were tested, the FIC for the combination oxacillin-quinolone was always 1 or 2, which is considered to be indifferent. For the other mentioned combinations the FICs were also 1 or 2. Killing kinetics showed synergistic or additive bactericidal activity for the combination oxacillin-ofloxacin against methicillin-resistant Staphylococcus species, killing 1.5 to 2.8 log10 CFU more of these per ml than did the most active drug after 24 h of incubation. This difference was not observed for methicillin-susceptible strains. In vitro evidence for the potential clinical use of quinolones in treating infections due to methicillin-resistant staphylococci in combination with a beta-lactamase-resistant penicillin is provided.     

靶向mecA基因纳米胶体金探针快速检测耐甲氧西林金黄色葡萄球菌方法的建立

目的 用以纳米胶体金颗粒为载体的DNA探针杂交技术,建立快速、简便和特异的检测耐甲氧西林金黄色葡萄球菌(MRSA)的方法.方法 以直径60 nm的胶体金纳米颗粒与巯基修饰的2条mecA基因特异性寡核苷酸探针共价结合,制备成纳米胶体金探针.直接提取金黄色葡萄球菌的基因组DNA,经超声破碎后与纳米胶体金探针进行液相杂交.杂交产物经高速离心后再用反向色谱反应盘检测有无胶体金颗粒沉淀产生.用该法和PCR扩增试验同时检测临床分离株的mecA基因,以PCR法为金标准,评价纳米胶体金探针液相杂交法的准确性.结果 在95株临床试验菌株中PCR法共检出71株MRSA,24株MSSA.在71株MRSA中,纳米胶体金探针杂交试验有69株为阳性,敏感度为97.2%,最低检测限为4.72 fmol/L.而在24株甲氧西林敏感的金黄色葡萄球菌(MSSA)中,纳米胶体金探针杂交试验无一株阳性,特异度为100%.95株临床试验菌株中纳米胶体金探针杂交试验正确检出93株,准确度97.9%.结论 纳米胶体金探针杂交试验与PCR检测MRSA具有很好的一致性.应用胶体金探针杂交技术检测MRSA具有快速、简便、准确的特点,有望成为MRSA的快速诊断的新方法.     

Homology of mecA gene in methicillin-resistant Staphylococcus haemolyticus and Staphylococcus simulans to that of Staphylococcus aureus.

A penicillin-binding protein of molecular weight 76,000 inducible by beta-lactams was detected in methicillin-resistant Staphylococcus haemolyticus and Staphylococcus simulans. DNA from these strains hybridized to the mecA gene from Staphylococcus aureus; however, the chromosomal HindIII fragments containing the mecA genes were 3.4 kilobases in S. haemolyticus and 4.3 kilobases in S. simulans.