如何准确检测耐甲氧西林的金黄色葡萄球菌和凝固酶阴性葡萄球菌?

答:检测mecA基因和其表达的青霉素结合蛋白(PBP2a)是预测葡萄球菌对苯唑西林耐药的最准确方法。罕见非mecA基因介导的苯唑西林耐药机制,纸片法测定苯唑西林为中介或耐药,应加测苯唑西林的MIC,如果MIC≥4μg/ml,即使mecA基因和PBP2a检测阴性,苯唑西林也应报告耐药。在凝固酶阴性葡萄球菌(CoNS)中头孢西丁纸片扩散法的结果与检测mecA/PBP2a的一致性好于苯唑西林纸片,可以减少在非表皮葡萄球菌的CoNS中苯唑西林纸片的假阳性问题,所以应该首选头孢西丁纸片。对金黄色葡萄球菌两种纸片的结果相近,但头孢西丁纸片更容易读取结果。如何准…     

问：如何准确检测耐甲氧西林的金黄色葡萄球菌和凝固酶阴性葡萄球菌？

答：检测mecA基因和其表达的青霉素结合蛋白（PBP2a）是预测葡萄球菌对苯唑西林耐药的最准确方法。罕见非mecA基因介导的苯唑西林耐药机制，纸片法测定苯唑西林为中介或耐药，应加测苯唑西林的MIC，如果MIC≥4ug／ml，即使mecA基因和PBP2a检测阴性，苯唑西林也应报告耐药。在凝固酶阴性葡萄球菌（CONS）中头孢西丁纸片扩散法的结果与检测mecA／PBP2a的一致性好于苯唑西林纸片，     

头孢西丁纸片法筛查耐甲氧西林金黄色葡萄球菌的评价

目的评价头孢西丁纸片法筛选耐甲氧西林金黄色葡萄球菌(MRSA)的方法.方法采用NCCLS推荐的30μg头孢西丁纸片法和苯唑西林纸片法检测MRSA,以PCR方法检测mecA基因为金标准.同时做头孢西丁对金黄色葡萄球菌的最低抑菌浓度(MIC).结果用PCR方法检测145株金黄色葡萄球菌,其中mecA基因阳性83株,阴性62株.用头孢西丁纸片法筛查MRSA阳性82株,阴性63株.敏感性为98.8%,特异性为100%.而苯唑西林纸片法检测MRSA敏感性为95.2%,特异性为96.8%.结论头孢西丁纸片法筛查MRSA与mecA基因检测具有很好的一致性,该方法可在常规工作中推广使用.     

头孢西丁纸片扩散法检测耐甲氧西林凝固酶阴性葡萄球菌的评价

目的评价头孢西丁（FOX）纸片扩散法（K-B）检测耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）的临床应用价值。方法用头孢西丁纸片扩散法检测临床分离的139株凝固酶阴性葡萄球菌（CNS）,并与苯唑西林（OXA）纸片扩散法、mecA基因检测进行比较。结果mecA基因阳性的109株CNS对FOX显示耐药,而对OXA则只有97株显示耐药。结论头孢西丁纸片扩散法与mecA基因检测高度一致,苯唑西林纸片扩散法则存在一定的差异,临床实验室应用FOX取代OXA来检测MRCNS。     

头孢西丁纸片扩散法检测耐甲氧西林凝固酶阴性葡萄球菌的评价

目的评价头孢西丁（FOX）纸片扩散法（K-B）检测耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）的临床应用价值。方法用头孢西丁纸片扩散法检测临床分离的139株凝固酶阴性葡萄球菌（CNS）,并与苯唑西林（OXA）纸片扩散法、mecA基因检测进行比较。结果mecA基因阳性的109株CNS对FOX显示耐药,而对OXA则只有97株显示耐药。结论头孢西丁纸片扩散法与mecA基因检测高度一致,苯唑西林纸片扩散法则存在一定的差异,临床实验室应用FOX取代OXA来检测MRCNS。     

头孢西丁纸片扩散法检测耐甲氧西林葡萄球菌的临床应用评价

目的用PCR法检测葡萄球菌的mecA基因为参比方法,评价头孢西丁纸片扩散法检测耐甲氧西林葡萄球菌(MRS)的临床应用价值。方法临床分离的163株葡萄球菌,分别用头孢西丁、苯唑西林纸片扩散法、PCR法检测mecA基因,以PCR扩增法检测mecA基因作为参比方法进行比较。结果163株临床分离的葡萄球菌经PCR法检测mecA基因,阳性率为81.0%,其中,耐甲氧西林金黄色葡萄球菌(MRSA)和耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)的发生率分别为74.1%和83.8%。头孢西丁、苯唑西林纸片扩散法用于检测MRSA与PCR法检测mecA基因有很好的一致性,其敏感性和特异性均为100.0%,而对于凝固酶阴性葡萄球菌,头孢西丁纸片扩散法比苯唑西林纸片扩散法检测MRS的敏感性和特异性高,敏感性分别为100.0%和94.3%,特异性分别为96.0%和88.0%。结论头孢西丁纸片扩散法检测耐甲氧西林葡萄球菌具有很高的灵敏度,但对于mecA基因阴性的凝固酶阴性葡萄球菌,用头孢西丁纸片法检测,约有5%菌株要误报为MRCNS。由于头孢西丁纸片扩散法操作简单,较苯唑西林纸片扩散法检出MRS的敏感度和特异性高,结果与PCR法检测mecA基因有很好的相关性,不需特殊仪器设备,可在临床微生物实验室常规开展。     

头孢西丁纸片扩散法筛选耐甲氧西林葡萄球菌

目的:评价头孢西丁纸片法筛选耐甲氧西林葡萄球菌(MRS)的方法.方法:采用2004年NCCLS推荐的30μg/片头孢西丁纸片法检测MRS,以PCR方法检测mecA基因为"金标准".结果:在60株临床分离的葡萄球菌中,金黄色葡萄球菌36株,凝固酶阴性葡萄球菌24株.用PCR方法检测mecA基因阳性43株(金黄色葡萄球菌25株,凝固酶阴性葡萄球菌18株),阴性17株(金黄色葡萄球菌11株,凝固酶阴性葡萄球菌6株);用头孢西丁纸片法筛查MRS耐药(+)41株(金黄色葡萄球菌23株,凝固酶阴性葡萄球菌18株),敏感(-)19株(金黄色葡萄球菌13株,凝固酶阴性葡萄球菌6株).以PCR检测mecA基因法为"金标准",头孢西丁纸片法的敏感度和特异度分别为95.3%(41/43)、100%(17/17).准确度为96.7%(58/60).结论:头孢西丁纸片法筛查MRS与mecA基因检测法具有很好的一致性,该方法适合在临床微生物实验室中推广使用.     

头孢西丁纸片扩散法检测耐甲氧西林金黄色葡萄球菌

目的　评价头孢西丁纸片扩散法检测耐甲氧西林金黄色葡萄球菌 (MRSA)在临床的应用价值。方法　用头孢西丁纸片扩散法检测临床分离的 94株金黄色葡萄球菌 ,并与苯唑西林纸片扩散法、琼脂稀释法及mecA基因检测进行比较。结果　mecA基因阳性的 77株金黄色葡萄球菌 ,头孢西丁纸片扩散法均显示耐药。结论　头孢西丁纸片扩散法与mecA基因检测高度一致 ,是筛选和确认MRSA的一种可靠的试验方法。     

头孢西丁纸片扩散法检测耐甲氧西林金黄色葡萄球菌

目的评价头孢西丁纸片扩散法检测耐甲氧西林金黄色葡萄球菌(MRsA)在临床的应用价值。方法用头孢西丁纸片扩散法检测临床分离的94株金黄色葡萄球菌，并与苯唑西林纸片扩散法、琼脂稀释法及mecA基因检测进行比较。结果mecA基因阳性的77株金黄色葡萄球菌，头孢西丁纸片扩散法均显示耐药。结论头孢西丁纸片扩散法与mecA基因检测高度一致，是筛选和确认MRSA的一种可靠的试验方法。     

苯唑西林MIC≥0.5ug/ml折点对凝固酶阴性葡萄球菌耐药性高估情况分析

目的：了解使用苯唑西林MIC（最低抑菌浓度）≥0.5ug/ml的折点判断部份凝固酶阴性葡萄球菌为耐甲氧西林葡萄球菌对此类葡萄球菌耐药性高估的情况。方法：对73株苯唑西林MIC法耐药的凝固酶阴性非表皮葡萄球菌同时用头孢西丁纸片法扩散法（K-B法）检测,分析结果。结果：9株表现为敏感（抑菌圈≥25mm）,占12%,其中6株苯唑西林MIC介于0.5-2ug/ml之间,3株苯唑西林MIC≥2ug/ml。头孢西丁敏感与耐药的两组菌对常用抗阳性球菌药物敏感性有明显差别。结论：对于苯唑西林MIC介于0.5-2ug/ml的凝固酶阴性的非表皮葡萄球菌,临床实验室应遵照美国临床和实验室标准化研究所（CLSI）指南,用头孢西丁K-B法或其它方法复核验证其耐药性,苯唑西林MIC≥2ug/ml的菌株,可直接报告为耐甲氧西林的葡萄球菌。     

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.     

Evaluation of alternative disk diffusion methods for detecting mecA-mediated oxacillin resistance in an international collection of staphylococci: validation report from the SENTRY Antimicrobial Surveillance Program

To validate the current National Committee for Clinical Laboratory Standards recommendations of the cefoxitin disk as a preferred surrogate marker to detect oxacillin resistance in staphylococcal isolates, 304 staphylococcal isolates originating from 49 sites in 16 countries in the SENTRY Antimicrobial Surveillance Program (2003) were tested. Two hundred three Staphylococcus aureus and 101 coagulase-negative staphylococci (CoNS), of which >95% were bloodstream isolates, were evaluated by comparing the results of the National Committee for Clinical Laboratory Standards broth microdilution method for oxacillin with those of the disk diffusion test using oxacillin, cefoxitin and ceftizoxime disks. Discrepancies were resolved using the PBP2a latex agglutination test. For S. aureus, the cefoxitin disk performed without interpretive error followed by the ceftizoxime disk (1% major and 0.5% minor errors; > or =20 mm = susceptible); use of the oxacillin disk test had the highest error rates with 4.4% major and 1.5% minor errors, whereas the oxacillin minimal inhibitory concentration (MIC) test was 99.0% accurate. For CoNS, the oxacillin disk test had the highest error rate with 4.0% major errors, followed by the cefoxitin (3.0% major error rate) and the ceftizoxime (1% very major and 1% minor error: > or =20 mm = susceptible) disk tests. The oxacillin MIC test was also 99.0% accurate for CoNS testing. Modification of the ceftizoxime disk diffusion breakpoints for CoNS resulted in complete intermethod categorical agreement. The overall accuracy of the four tests was as follows: modified ceftizoxime disk (99.3%) > oxacillin MIC = cefoxitin disk (99.0%) > current ceftizoxime disk (98.4%) > oxacillin disk (94.7%). In conclusion, these results confirm the superior performance characteristics of cefoxitin and ceftizoxime disk tests as surrogate markers to detect oxacillin resistance; by using an international collection of clinically significant staphylococcal isolates, we also demonstrate its wide global application.     

Phenotypic detection of mec A-positive staphylococcal blood stream isolates: High accuracy of simple disk diffusion tests

Detection of oxacillin-resistance in staphylococci by phenotypic methods remains problematic. Although standardized susceptibility test methods are adequate for Staphylococcus aureus, many are less satisfactory for the coagulase-negative staphylococci (CNS). We have studied 108 consecutive blood culture isolates of staphylococci. The mec A gene was detected by PCR in one S. aureus and 55 CNS isolates. Susceptibility testing was performed as follows: oxacillin (1-μg), ceftizoxime (30-μg), and cephalothin (30-μg) by disk diffusion; oxacillin, ceftizoxime, cephalothin, methicillin, ampicillin, ampicillin/sulbactam, penicillin, cefazolin, imipenem, and meropenem by the broth microdilution method. In addition, isolates were tested by the oxacillin agar screen plate method. The single oxacillin-resistant S. aureus strain was detected by all oxacillin susceptibility test methods and by the ceftizoxime disk and MIC methods. Two oxacillin-susceptible S. aureus were intermediate (minor error) by ceftizoxime broth microdilution (MIC, 16 μg/mL). The most sensitive, simple phenotypic methods for detection of oxacillin-resistant CNS (mec A positive) were as follows: oxacillin disk diffusion at 98%, oxacillin screen plate at 91%, oxacillin broth microdilution at 87%, ceftizoxime disk diffusion at 100%, ceftizoxime broth microdilution at 87%, and methicillin broth microdilution at 83%. These results indicate that oxacillin and ceftizoxime disk diffusion tests are the most accurate phenotypic methods in routine clinical use for detection of oxacillin-resistant CNS. Oxacillin broth microdilution MIC testing (2% NaCl supplement) would perform more satisfactorily (100% sensitivity) with an adjusted interpretive breakpoint at ⩽0.5 μg/mL, in contrast to the lower accuracy of the “so-called” reference agar screen test.     

In vitro susceptibilities of four species of coagulase-negative staphylococci.

The in vitro susceptibilities of 260 strains of coagulase-negative staphylococci to penicillin G, oxacillin, nafcillin, methicillin, cephalothin, and seven non-beta-lactam antimicrobial agents were determined and compared with the susceptibilities of 54 strains of Staphylococcus aureus with known patterns of susceptibility. Penicillin G susceptibility for S. aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis was readily determined by using beta-lactamase tests with induced cells and with a standardized microdilution test. MIC criteria for susceptibility used for S. aureus were applicable to the coagulase-negative species. Percentages of organisms susceptible were as follows: S. epidermidis, 7%; S. haemolyticus, 5%; and S. hominis, 47%. Oxacillin susceptibility for these four species was readily determined by using a modification of the microdilution test. MIC criteria for susceptibility used for S. aureus were applicable to S. haemolyticus and S. hominis, but alternate criteria were necessary for S. epidermidis. Percentages of organisms susceptible were as follows: S. epidermidis, 29%; S. haemolyticus, 36%; and S. hominis, 97%. Staphylococcus saprophyticus differed from the other staphylococcal species; all strains were beta-lactamase negative and were penicillin susceptible but had higher penicillin G MICs than did susceptible strains of the other species. There was total cross resistance among the penicillinase-resistant penicillins and cephalothin for the coagulase-negative staphylococci as well as for S. aureus; oxacillin MICs were more reliable than MICs of the other drugs or a standardized disk diffusion test for distinguishing resistant from susceptible strains. Vancomycin, rifampin, and ciprofloxacin were consistently active against all staphylococci. Erythromycin, clindamycin, gentamicin, and trimethoprim-sulfamethoxazole were more active against oxacillin-susceptible staphylococci than against oxacillin-resistant staphylococci.     

Susceptibility of Staphylococcus species and subspecies to teicoplanin.

Twenty-four Staphylococcus species and their subspecies were examined for their susceptibilities to teicoplanin by disk diffusion (30-micrograms disk) and agar dilution for the determination of MICs. Moderately susceptible and resistant clinical strains were further tested for their susceptibilities to oxacillin and vancomycin. Teicoplanin resistance was not observed in the reference strains of the various Staphylococcus species isolated from healthy volunteers or animals. However, the novobiocin-resistant species Staphylococcus saprophyticus, Staphylococcus cohnii, Staphylococcus xylosus, Staphylococcus arlettae, Staphylococcus kloosii, and Staphylococcus gallinarum were less susceptible to teicoplanin (MIC, 2 to 8 micrograms/ml) than most of the novobiocin-susceptible species were (MIC, 0.5 to 4 micrograms/ml). Clinical isolates of coagulase-negative species were generally less susceptible to teicoplanin than were reference strains. Seven percent of the Staphylococcus epidermidis clinical strains were moderately susceptible (MIC, 16 micrograms/ml) to teicoplanin. Of these strains, 70% were oxacillin resistant. For Staphylococcus haemolyticus strains, 11% were resistant (MIC, greater than 16 micrograms/ml) and 21% were moderately susceptible to teicoplanin. Of these strains, 95% were oxacillin resistant, No strains of S. epidermidis or S. haemolyticus were intermediate or resistant to vancomycin. Teicoplanin appears to be less active in vitro against oxacillin-resistant S. haemolyticus. However, teicoplanin is an effective antimicrobial agent against many Staphylococcus species.     

头孢西丁纸片评价耐甲氧西林葡萄球菌的探讨

目的 评价头孢西丁纸片法检测耐甲氧西林葡萄球菌（MRS）的特异性和灵敏度。方法 收集临床分离金黄色葡萄球菌100株、凝固酶阴性葡萄球菌（CoNS）52株，分别进行头孢西丁纸片法、添加4oANaCl的头孢西丁纸片法、添加2％NaCl的苯唑西林纸片法以及苯唑西林-盐琼脂法检测，以苯唑西林-盐琼脂法作为MRS检测标准，按CLSI2006版标准判读结果。结果 头孢西丁纸片、添加4oANaCl的头孢西丁纸片、添加2oANaCl的苯唑西林纸片检测MRS特异性均达100．0％；对于金黄色葡萄球菌3种纸片法的灵敏度分别为82．7％、79．3％、72．4％；对于凝固酶阴性葡萄球菌，3种纸片法的灵敏度分别为97．9％、95．8oA、95．8％。加盐与不加盐头孢西丁纸片法检测MRS的差异无显著性（P〉0．06），耐药株抑菌圈直径（mm）分别为10．68士6．42、13．36士7．37（P〈0．01）。结论 加盐与不加盐头孢西丁纸片法均能可靠检测MRS，前者对耐药株结果更易判读。     

Staphylococcus aureus and Coagulase-Negative Staphylococci from Blood Stream Infections: Frequency of Occurrence,Antimicrobial Susceptibility,and Molecular (mecA) Characterization of Oxacillin Resistance in the SCOPE Program

Staphylococci are major causes of nosocomial blood stream infection. The recently completed SCOPE Surveillance Program found that coagulase-negative staphylococci (CoNS) and Staphylococcus aureus were the first and second most common etiologic agents, respectively, causing nosocomial blood stream infection in the USA. The frequency of oxacillin resistance was 68% among 1553 strains of CoNS and 26% among 787 strains of S. aureus in this study. Extended susceptibility profiles were generated for a subset of 150 S. aureus and 300 CoNS against 16 antimicrobial agents. Oxacillin-susceptible strains of both CoNS and S. aureus were uniformly susceptible to β-lactam agents with the exception of ampicillin and penicillin. Oxacillin-susceptible S. aureus were also highly susceptible to the fluoroquinolones, aminoglycosides, and trimethoprim/sulfamethoxazole. The oxacillin-susceptible CoNS were less susceptible to these agents, and only glycopeptides were reliably active against oxacillin-resistant strains. PCR detection of the mecA gene was used to scrutinize current NCCLS interpretive breakpoint MICs for determining susceptibility or resistance to oxacillin. We found complete concordance between the presence or absence of mecA and the NCCLS oxacillin interpretive breakpoint categories for S. aureus. In contrast, the NCCLS breakpoints for oxacillin significantly underestimate the degree of true oxacillin resistance among CoNS. Using the presence of mecA as the reference standard, we detected 15.7% false susceptibility to oxacillin using a MIC susceptible breakpoint concentration of ≤2 μg/mL. Lowering the oxacillin MIC breakpoint to ≤0.25 μg/mL for CoNS would greatly improve the accuracy of the MIC test performance. We found that both the current oxacillin disk test and the 30-μg ceftizoxime disk test functioned quite well in predicting those strains of CoNS that contain mecA. These studies have demonstrated both a high level of antimicrobial resistance among nosocomial blood stream isolates of staphylococci as well as significant problems with the current NCCLS breakpoints for oxacillin when testing CoNS.     

一种PCR检测耐甲氧西林金黄色葡萄球菌株新方法的建立

目的建立一种PCR检测耐甲氧西林金黄色葡萄球菌株新方法。方法收集2016年1月至2018年12月入住本院ICU的重症患者的脓液、分泌物、血液等标本中分离培养病原菌株,共43个样本,且均为初次分离所得。以mecA基因检测呈阳性为"金标准",对样本分别进行实时荧光PCR检测和纸片扩散法检测。结果实时荧光PCR方法检测法准确度高于头孢西丁及苯唑西林纸片扩散法;所有耐甲氧西林葡萄球菌对青霉素和苯唑西林药耐药性最强,对红霉素和克林霉素次之,对万古霉素、利奈唑胺、替加环素、利福平均表现为敏感,对四环素、庆大霉素等具备一定的敏感性。结论新建立的荧光PCR检测法相比头孢西丁纸片扩散法和苯唑西林纸片扩散法不仅准确度高,而且特异性好,能够准确检测出耐甲氧西林金黄色葡萄球菌。