Development of a rapid assay methodology for antimicrobial susceptibility testing of Staphylococcus aureus

Development of a new phenotypic technique for rapid antimicrobial susceptibility testing (AST) of methicillin-resistant Staphylococcus aureus is presented. The new technique combines bacterial culturing and specific immunometric detection in a single separation-free process. The technique uses dry chemistry reagents and the recently developed two-photon excitation detection technology, which allows online detection of bacterium-specific growth. The performance of the new technique was evaluated by monitoring the growth of S. aureus reference strains and determining their susceptibility to oxacillin. In the direct analysis of clinical specimens, method specificity and tolerance to interferences caused by other bacteria present in the sample are pivotal. Other bacteria can compete with the bacteria of interest for nutrients, for example. Specificity and tolerance were studied against Staphylococcus epidermidis reference strains. The results suggest that the new technique could allow rapid AST directly from clinical samples within 6 to 8 h. Such a rapid and simple testing methodology would be a valuable tool in clinical microbiology because it would shorten the turnaround times of microbiologic analyses. Advantages of the new approach in relation to conventional methods are discussed.     

分枝杆菌药物敏感试验快速荧光检测法的研究

目的 建立一种Mycobacterium(分枝杆菌)药敏试验的快速荧光检测法。方法 用自己研制的荧光检测管检测异烟肼(INH)、利福平(RFP)、乙胺丁醇(EB)、丁胺卡那霉素(AMK)、左旋氧氟沙星(LVFX)对分枝杆菌的体外抑菌活性,并用41例临床菌株与改良罗氏法进行了配对比较,确定各药物在药敏试验中的应用浓度：结果 5种药物的应用浓度分别确定为INH：0．1μg/ml、RFP;1μg/ml、EB：2μg/ml、AMK：2μg/ml、LVFX：2μg/ml;本法进行分枝杆菌的药敏试验仅需7—10d,较改良罗氏法的28d缩短了18—21d。2种方法的药敏试验结果无显著性差异。结论 本法经济实用并缩短了分枝杆菌药敏的报告时间。     

Microbial genomics and antimicrobial susceptibility testing

Introduction: Antimicrobial susceptibility testing is key in modern clinical microbiology. With pandemic emergence of (multi-)antibiotic resistance, methods to detect and quantify resistance of clinically important bacterial species are imperative. Historically, antimicrobial susceptibility testing (AST) was mostly performed using methods relying on bacterial growth. Such methods may be time-consuming and more rapid alternatives have been actively sought for.

Areas covered: Among the new AST methods there are many that focus on detection of causal resistance genes and/or gene mutations. The approaches most used are based on nucleic acid amplification and, more recently, high-throughput (next generation) sequencing of amplified targets and complete microbial genomes. The authors provide a review of PCR-mediated and genomic AST methods used for human and veterinary pathogens and show where these approaches work well or may become difficult to interpret.

Expert commentary: Microbial genome sequencing will play an important role in the field of AST, but there remain issues to be resolved. These include the development of user friendly data analysis, reducing the duration and cost of sequencing and comprehensiveness of the databases. In addition, clinical evaluation studies need to be performed involving real-life patients.     

丝状真菌的快速鉴定及药敏试验

目的评估基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术在丝状真菌鉴定中的作用,分析常用抗菌药物对丝状真菌的药敏试验结果。方法收集100株丝状真菌,采用MALDI-TOF MS进行快速鉴定,并与显微镜检查结果进行比对;用E-test法进行丝状真菌药敏试验。结果 MALDI-TOF MS对100株丝状真菌鉴定达到种鉴定水平的有61株(得分≥2.000),达到属鉴定水平的有36株(1.700~1.999),未能鉴定的有3株(1.700);与镜检结果不一致的有1株。两性霉素B对絮状表皮癣菌的90%最低抑菌浓度(MIC_(90))为0.19μg/m L,而对黄曲霉的MIC_(90)32μg/m L。伊曲康唑对断发毛癣菌、犬小孢子菌和絮状表皮癣菌的MIC_(90)均0.38μg/m L,而对黑曲霉的MIC_(90)32μg/m L。氟康唑对大部分受试菌株的MIC_(90)均256μg/m L。伏立康唑和卡泊芬净对烟曲霉、黄曲霉、黑曲霉、红色毛癣菌、断发毛癣菌和犬小孢子菌的MIC_(90)分别≤0.38μg/m L和≤1μg/m L。结论 MALDI-TOF MS技术可快速、准确、高通量检测临床分离的丝状真菌。伏立康唑和卡泊芬净对丝状真菌具有较好的抗菌活性。     

Evaluation of direct E-test on lower respiratory tract samples: a rapid and accurate procedure for antimicrobial susceptibility testing

We compared the direct E-test susceptibility testing (DET) on respiratory samples with a standard microbroth dilution method (MBD) after quantitative cultures. A total of 152 samples from intensive care unit patients were processed by DET onto Mueller-Hinton agar. Oxacillin, piperacillin/tazobactam, cefepime, imipenem, ciprofloxacin, and amikacin were the antimicrobials evaluated. Cultures were 102 monomicrobials and 50 polymicrobials. Overall, 93.8% of the isolates were recovered by the DET. Among the 772 microorganism-antibiotic combinations evaluated, there was a total agreement with the MBD in 96.1%. There were 8 very major errors (1.03%), 15 major (1.94%), and 7 minor (0.91%). All discrepancies but one corresponded to polymicrobial cultures, and most occurred with cefepime (8 cases, 7.07%) and imipenem (7 cases, 6.18%). Readings of DET were easy to interpret and improved with transmitted light. DET on respiratory samples is a rapid technique that provides susceptibility results in 18 to 24 h comparable with these obtained by MBD.     

Staphylococcal Cassette Chromosome mec type and antibiotic susceptibility profiles of vaginal and nonvaginal MRSA clinical isolates

Methicillin-resistant Staphylococcus aureus (MRSA) causes nosocomial and community-associated infections, representing significant healthcare concerns. Limited studies have investigated cervicovaginal MRSA colonization and antibiotic susceptibility. Upon comparing clinical cervicovaginal MRSA isolates to nonvaginal isolates by Staphylococcal Cassette Chromosome mec type, presence of Panton-Valentine Leukocidin toxin, antibiotic susceptibility, and presence of associated resistance genes, no significant differences were observed between the anatomical sites, but were observed between our hospital- and community-associated MRSA isolates. There was a significant increase in erythromycin resistance in our vaginal MRSA isolates compared to previous vaginal MRSA reports and an increase in clindamycin, doxycycline, and mupirocin resistance in our nonvaginal MRSA isolates compared to previously reported community-based skin and soft tissue MRSA isolates. Additionally, this is the first report of mupirocin resistance in vaginal MRSA isolates.     

粘液型铜绿假单胞菌药敏试验不同时间段结果分析

目的探讨产粘液型铜绿假单胞菌(Pseudomonasaexuginosa,PA)药敏试验(K-B法)结果的报告时机。方法对所分离出的11株产粘液型的PA进行体外药敏试验(K-B法)和环丙沙星、舒普深的MIC测定,且记录不同时间段(24h、48h、72h)的结果。然后将此菌进行转种数代,使其粘液消失,再进行上述操作。并将上述两次结果进行对比分析。结果产粘液型PA24h抑菌环直径的毫米数与48h、72h后的结果有显著性差异(P<0.05)。48h、72h的结果与转种后、粘液消失的PA24h、48h、72h的结果无显著性差异(P>0.05)。环丙沙星和舒普深的MIC的测定结果与K-B法的结果相符。结论产粘液型的PA的体外药敏试验(K-B法)结果应该尽量在48小时后报告。     

Identification of treatment strategies for Mycoplasma genitalium-related urethritis in male patients by culturing and antimicrobial susceptibility testing

Mycoplasma genitalium was first isolated from urethral swab specimens of male patients with non-gonococcal urethritis. However, the isolation of M. genitalium strains from clinical specimens has been difficult. Co-cultivation with Vero cells is one available technique for the isolation of M. genitalium. The strains that can be used for antimicrobial susceptibility testing by broth dilution or agar dilution methods are limited. Macrolides, such as azithromycin (AZM), have the strongest activity against M. genitalium. However, AZM-resistant strains have emerged and spread. Mutations in the 23S rRNA gene contribute to the organism’s macrolide resistance, which is similar to the effects of the mutations in macrolide-resistant Mycoplasma pneumoniae. Of the fluoroquinolones, moxifloxacin (MFLX) and sitafloxacin have the strongest activities against M. genitalium, while levofloxacin and ciprofloxacin are not as effective. Some clinical trials on the treatment of M. genitalium-related urethritis are available in the literature. A doxycycline regimen was microbiologically inferior to an AZM regimen. For cases of treatment failure with AZM regimens, MFLX regimens were effective.     

毛霉目病原真菌的实验室鉴定及体外抗真菌药物敏感 性分析

摘要:目的以分子测序为参考 ,评估形态学、基质辅助激光解吸电离飞行时间质谱对毛霉目真菌的鉴定能力,分析毛霉目真菌对两性霉素B、泊沙康唑的体外药敏特点。方法收集 2018年1月-2022年2月广东省人民医院25例毛霉病住院患者标 本,进行镜检并对培养的茵落行形态学鉴定、质谱鉴定、分子测序和体外药敏试验。结果KOH 湿片法阳性率(76%)高于革兰染色法(32%),差异有统计学意义(P<0.05);形态学鉴定可将68%的菌株鉴定到属水平,3株鉴定错误,2株无法鉴定;将所 有菌株进行质谱鉴定,单用IVD和RUO数据库鉴定率分别为56%和44%,两库联合可将鉴定率提高为64%。毛霉目真菌对两性霉素B的抑制50%菌株生长的最小抑菌浓度(MIC3o)为1μg/mL,根霉属相比其他属对两性霉素B表现出较高的MIC 值,其中2株MIC>32 μg/mL,另2株MIC为8 μg/mL;根霉属对泊沙康唑的MICso为0.5 μg/mL,横梗霉属MICgo为4 μg/ mL,小克银汉霉属MICg0为2 μg/mL,有部分菌株表现出较低MIC值,同样亦有部分菌株对泊沙康唑表现出较高的MIC 值。结论 将传统镜检、培养与质谱技术和分子技术相结合,尽量将毛霉目真菌准确鉴定到种,并积极开展毛霉体外药敏试验,可为临床治疗提供参考依据。     

Antimicrobial susceptibility and molecular characteristics of 857 methicillin-resistant Staphylococcus aureus isolates from 16 medical centers in Japan (2008-2009): nationwide survey of community-acquired and nosocomial MRSA

This study is a nationwide survey of all clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates, including community-acquired MRSA (CA-MRSA), in Japan. A total of 857 MRSA clinical isolates were collected from the 16 institutions throughout Japan that participated in the survey (2008-2009). The drug susceptibility and staphylococcal cassette chromosome mec (SCCmec) typing and the presence of specific pathogenic genes were evaluated. The isolates comprised SCCmec type II (73.6%), type IV (20%), and type I (6%). The percentage of SCCmec type IV isolates was significantly higher in outpatients than in inpatients. Most of the isolated strains were sensitive to vancomycin (VCM, MIC ≤2 μg/mL), linezolid (MIC ≤4 μg/mL), and teicoplanin (MIC ≤8 μg/mL). Although most strains were sensitive to VCM, the MIC value of VCM for SCCmec type II strains was higher than that for SCCmec type IV strains. Only 4 (2.3%) of 171 SCCmec type IV strains were Panton-Valentine leukocidin (lukS/F-PV)-positive. Thus, this result indicates a unique feature of SCCmec type IV strains in Japan. The information in this study not only is important in terms of local public health but will also contribute to an understanding of epidemic clones of CA-MRSA.     

In-vitro antifungal susceptibility testing of lanoconazole and luliconazole against Aspergillus flavus as an important agent of invasive aspergillosis

Introduction

The incidence of Aspergillus infections has recently increased remarkably in certain tropical and sub-tropical countries, with Aspergillus flavus being identified as the leading cause of infections after A. fumigatus. Lanoconazole (LAN) and luliconazole (LUL) are currently approved for topical treatment of cutaneous fungal infections. We aimed the in-vitro antifungal susceptibility testing of two imidazole, LAN and LUL against A. flavus.

2017年度江苏地区碳青霉烯类耐药肠杆菌科细菌的分布特点和耐药性分析

目的　了解2017年“江苏省细菌耐药监测网”耐碳青霉烯类肠杆菌科细菌(CRE)的分布特点及其耐药性。方法　收集2016年10月1日-2017年9月30日监测网内CRE菌株,药物敏感性试验采用纸片扩散法(K-B法)和自动化仪器MIC法,结果判断参照2017年版CLSI　M100-S27标准。结果　10405株CRE菌株中,克雷伯菌属、大肠埃希菌、肠杆菌属分别为56.3%、12.8%、10.2%;标本来源和科室分布最多者为呼吸道标本(占60.1%)和重症监护室(占27.2%);全省CRE的平均检出率为8.4%,其中徐州地区高达16.8%,泰州仅为3.9%。CRE菌株对多种临床常用抗菌药物高度耐药,除阿米卡星的耐药率(40.4%)低于50%外,对其他抗菌药物的耐药率均在55.1%～96.8%;儿童分离菌株对氨曲南、阿米卡星、庆大霉素、环丙沙星、妥布霉素的耐药率均低于成人分离株,但对头孢他啶、头孢西丁、亚胺培南、美罗培南、厄他培南的耐药率却略高于成人分离株。结论　CRE菌株对多数临床常用抗菌药物呈高度耐药,严重限制临床抗感染治疗药物的选择。CRE菌株分布呈现地区和院内不同病区的差异性,如重症监护室相对集中,故应对之进行流行病学调查,并采取有效的感染防控措施以遏制其在医院中的播散。     

2012年中国CHINET碳青霉烯类耐药肠杆菌科细菌的分布特点和耐药性分析

目的了解2012年中国CHINET细菌耐药监测网耐碳青霉烯类肠杆菌科细菌（CRE）的分布特点及其耐药性。方法采用纸片扩散法（K-B法）和自动化仪器方法对上述菌株作药物敏感性试验,并按2012年版CLSI M100-S22标准判断结果。结果共收集临床分离的CRE菌1 499株,包括克雷伯菌属952株（63.5%）、肠杆菌属226株（15.1%）和埃希菌属206株（13.7%）等。标本来源和科室分布最多者分别为呼吸道标本（48.2%）和重症监护室（29.3%）。药敏试验结果显示,CRE菌株对多数临床常用抗菌药物高度耐药,除对阿米卡星和甲氧苄啶-磺胺甲口恶唑的耐药率低于50.0%外（分别为46.9%和49.8%）,对其他抗菌药物的耐药率均在70.0%~100%。儿童分离株对环丙沙星、氨基糖苷类和甲氧苄啶-磺胺甲口恶唑的耐药率低于成人分离株。结论 CRE菌株对多数临床常用抗菌药物呈高度耐药,严重限制临床抗感染治疗有效药物的选择。CRE菌株在医院中的某些科室如重症监护室和神经外科较为集中,应对之进行流行病学调查,并采取有效的感染防控措施以遏制此类耐药菌株在医院中的播散流行。     

5种抗菌药物与舒巴坦对临床分离鲍曼不动杆菌体外联合药敏试验

目的观察头孢哌酮、头孢他啶、亚胺培南、替加环素、黏菌素单药及其与舒巴坦联合用药对鲍曼不动杆菌的体外抗菌作用。方法选取2011-2012年抗菌药物耐药趋势监测研究(SMART)监测美罗培南耐药鲍曼不动杆菌23株、美罗培南敏感鲍曼不动杆菌21株,采取棋盘稀释法进行联合药敏试验,计算部分抑菌浓度指数(FIC)判定联合效应。结果所有组合体外均未出现拮抗作用,头孢哌酮、替加环素与舒巴坦联合应用对鲍曼不动杆菌的抗菌效应以协同作用为主,FIC指数≤0.5的菌株分别达56.8%及50.0%;亚胺培南、黏菌素与舒巴坦联合应用对鲍曼不动杆菌的抗菌效应主要表现为协同和部分协同作用,FIC指数1的菌株分别占61.4%和52.3%;头孢他啶和舒巴坦联合应用则主要表现为无关作用,FIC≥4的菌株占63.6%。结论 5种联合用药组合中,头孢哌酮与舒巴坦联合用药的体外协同作用效果最好,尤其对碳青霉烯敏感的鲍曼不动杆菌,可增强其体外抗菌活性;亚胺培南、替加环素与舒巴坦联合用药可增加对碳青霉烯耐药鲍曼不动杆菌的体外抗菌活性。临床对碳青霉烯耐药鲍曼不动杆菌引起的院内感染,可考虑联合应用亚胺培南/舒巴坦、替加环素/舒巴坦进行治疗。     

Direct inoculation method using BacT/ALERT 3D and BD Phoenix System allows rapid and accurate identification and susceptibility testing for both Gram-positive cocci and Gram-negative rods in aerobic blood cultures

This study describes a direct inoculation method using the automated BacT/ALERT 3D and the BD Phoenix System in combination for identification and susceptibility testing of isolates from positive blood cultures. Organism identification and susceptibility results were compared with the conventional method for 211 positive aerobic blood cultures. Of 110 Gram-positive cocci (GPCs), 98 (89.1%) isolates were correctly identified to the species level. Of 101 Gram-negative rods (GNRs), 98 (97.0%) isolates were correctly identified to the species level. The overall categorical agreement in antimicrobial susceptibility testing among the 110 GPCs was 92.7%, with 0.04% very major and 0.7% major error rates. The overall categorical agreement among 78 isolates of enterobacteria and 23 isolates of nonfermenters in GNRs was 99.5% and 91.1%, respectively, with no major errors identified. We conclude that, compared with previously reported direct inoculation methods, our method is superior in identification and susceptibility testing of GPCs.     

Antifungal susceptibility testing in Candida species: current methods and promising new tools for shortening the turnaround time

ABSTRACT

Introduction

Invasive fungal diseases (IFDs) have received attention as an emerging public health threat, are difficult to diagnose and to treat, and are associated with substantial morbidity and mortality. The standard of care in IFD management requires an early and targeted antifungal treatment, hence covers – amongst others – species identification and antifungal susceptibility testing (AFST).     

Novel method for clearing red blood cell debris from BacT/ALERT blood culture medium for improved microscopic and antimycobacterial drug susceptibility test results

Even though automation in mycobacterial culture has immensely improved the detection of organisms, identification of species and antimycobacterial susceptibility testing from blood culture bottles remain cumbersome and error-prone due to the presence of intact red blood cells (RBCs). The removal or lysis of these RBCs and excessive protein from the blood components could theoretically help improve this process. The present study reports an effective method that uses ammonium chloride (NH(4)Cl) and Triton X-100 to lyse the RBCs in blood culture medium. The method was optimized by preparing various concentrations of NH(4)Cl and Triton X-100, and incubation conditions, leading to eight protocols. The lysis protocol with a concentration of 150 mM of NH(4)Cl, 0.5% Triton X-100, and 1% potassium bicarbonate, pH 7.0, and incubation at 37 degrees C for 15 min was found to be optimal. This method not only made the culture medium clear, the protein concentration decreased from 753.5+/-39.4 to 53.2+/-4.2 mg/mL in the M. tuberculosis-spiked culture medium and in the blood culture medium inoculated with the blood from tuberculosis patients. The method had no adverse effect on mycobacteria, and no depletion of M. tuberculosis colony-forming units was found. The lysate could be used for antimycobacterial susceptibility testing with no difficulty in setting the mycobacterial concentration of inoculum to 0.5 McFarland standards. Furthermore, this method had the added advantage in the microscopy and molecular methods for the speciation of Mycobacterium sp.     

结核分枝杆菌培养和药敏试验及菌群鉴定快速荧光法的研究

目的 建立一种经济、快速的结核分枝杆菌培养、药敏和菌群鉴定的方法。方法 用我们研制的快速荧光检测法（本法）与传统的改良罗氏法（L-J法），对分枝杆菌标准菌株及临床分离菌株，同期进行培养、药敏试验和菌群鉴定的对照研究，验证本法培养结核分枝杆菌的特性，确定药敏和菌群鉴定的药物最适应用浓度。结果 本法培养比L-J法检出时间平均提前19．13d，且阳性率高。药敏试验和菌群鉴定中各药物浓度确定为INH0．1μg／ml、RFP1μg／ml、EB2μg／ml、AMK2μg／ml、LVFX2μg／ml、PNB200μg／ml和TCH2、5μg／ml；7～10d可获结果，比L-J法缩短18～21d。结论 本法明显缩短了结核分枝杆菌培养、药敏和菌群鉴定的时间，提高培养阳性率；经济、实用，适合在基层单位推广。     

Novel technologies emerging for preimplantation genetic diagnosis and preimplantation genetic testing for aneuploidy

Introduction: Preimplantation genetic diagnosis (PGD) was introduced as an alternative to prenatal diagnosis: embryos cultured in vitro were analysed for a monogenic disease and only disease-free embryos were transferred to the mother, to avoid the termination of pregnancy with an affected foetus. It soon transpired that human embryos show a great deal of acquired chromosomal abnormalities, thought to explain the low success rate of IVF – hence preimplantation genetic testing for aneuploidy (PGT-A) was developed to select euploid embryos for transfer.

Areas covered: PGD has followed the tremendous evolution in genetic analysis, with only a slight delay due to adaptations for diagnosis on small samples. Currently, next generation sequencing combining chromosome with single-base pair analysis is on the verge of becoming the golden standard in PGD and PGT-A. Papers highlighting the different steps in the evolution of PGD/PGT-A were selected.

Expert commentary: Different methodologies used in PGD/PGT-A with their pros and cons are discussed.     

A cost-saving algorithm for rapid diagnosis of Staphylococcus aureus and susceptibility to oxacillin directly from positive blood culture bottles by combined testing with BinaxNOW® S. aureus and Xpert MRSA/SA assay

We studied an algorithm combining 2 rapid methods to detect Staphylococcus aureus and its susceptibility to oxacillin directly from positive blood cultures; our goal was to reduce the cost of the procedure, while maintaining accuracy and a short turnaround time. A total of 581 blood cultures containing gram-positive cocci in clusters were tested by BinaxNOW® Staphylococcus aureus Test. Positive samples were further assessed by the Xpert MRSA/SA BC Assay. Phenotypic methods have identified coagulase-negative staphylococci in 505 samples and S. aureus in 76 samples, of which 51 were oxacillin sensitive and 25 were oxacillin resistant. Sensitivity and specificity of the BinaxNOW® Test were 92% and 99%, respectively, compared to the phenotypic method. The Xpert MRSA/SA BC Assay showed complete concordance with phenotypic identification and antimicrobial susceptibility results. The combined rapid assays produced results within 2 hours and reduced the cost by 75% compared with the Xpert MRSA/SA BC Assay if used alone on all blood bottles.