A Locked Nucleic Acid (LNA)-Based Real-Time PCR Assay for the Rapid Detection of Multiple Bacterial Antibiotic Resistance Genes Directly from Positive Blood Culture

Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1–10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.     

Adhesion of a Staphylococcus aureus strain to biomaterials does not select methicillin-resistant mutants

Bacterial adhesion to polymethylmethacrylate and to silicon elastomer, materials frequently used in clinical applications, has been investigated to assess whether adhesion selects methicillin-resistant mutants in the bacterial population in contact with the materials. The methicillin susceptibility of a susceptible Staphylococcus aureus (ATCC 25923) was measured by a modification of plate antibiogram Kirby-Bauer method, which allows optimised detection of small variations in antibiotic susceptibility. In both adherent and non-adherent bacterial subpopulations, the presence of mecA gene, which encodes for the protein PBP 2a responsible for methicillin resistance was searched for by Polymerase Chain Reaction (PCR). The contact with the two polymers did not induce in the bacteria population any phenotypic increase in methicillin resistance, or the selection of mutants carrying the mecA gene.     

Clinical applications of molecular biology for infectious diseases

Molecular biological methods for the detection and characterisation of microorganisms have revolutionised diagnostic microbiology and are now part of routine specimen processing. Polymerase chain reaction (PCR) techniques have led the way into this new era by allowing rapid detection of microorganisms that were previously difficult or impossible to detect by traditional microbiological methods. In addition to detection of fastidious microorganisms, more rapid detection by molecular methods is now possible for pathogens of public health importance. Molecular methods have now progressed beyond identification to detect antimicrobial resistance genes and provide public health information such as strain characterisation by genotyping. Treatment of certain microorganisms has been improved by viral resistance detection and viral load testing for the monitoring of responses to antiviral therapies. With the advent of multiplex PCR, real-time PCR and improvements in efficiency through automation, the costs of molecular methods are decreasing such that the role of molecular methods will further increase. This review will focus on the clinical utility of molecular methods performed in the clinical microbiology laboratory, illustrated with the many examples of how they have changed laboratory diagnosis and therefore the management of infectious diseases.     

老年患者血培养病原菌分布及耐药性调查

目的了解老年患者血液感染的病原菌种类以及对抗菌药物的耐药状况。方法对2290份老年住院患者血培养标本中186例培养阳性检出菌及药敏结果进行统计学分析。结果在分离的172株细菌中,革兰阴性菌、革兰阳性菌及真菌分别占54.7%、34.9%和10.5%;革兰阴性菌中以大肠埃希菌占绝对优势(20.9%),假单胞菌其次(10.5%),革兰阳性菌中以凝固酶阴性葡萄球菌(CNS)占绝对优势(20.9%),金黄色葡萄球菌仅为2.9%。药敏试验表明老年菌血症的病原菌对常用抗菌药物的耐药严重,尤其是CNS及假单胞菌属细菌表现为较高的耐药率及多重耐药性。结论老年菌血症病原菌以机会致病菌感染比例较高,致病菌耐药问题严重,及时监测老年致病菌的变迁和耐药发展趋势以指导临床用药至关重要。     

Detection of methicillin- and aminoglycoside-resistant genes and simultaneous identification of S. aureus using triplex real-time PCR Taqman assay

In this study we describe a triplex real-time PCR assay that enables the identification of S. aureus and detection of two important antibiotic resistant genes simultaneously using real-time PCR technology in a single assay. In this triplex real-time PCR assay, the mecA (methicillin resistant), femA (species specific S. aureus) and aacA-aphD (aminoglycoside resistant) genes were detected in a single test using dual-labeled Taqman probes. The assay gives simultaneous information for the identification of S. aureus and detection of methicillin and aminoglycoside resistance in staphylococcal isolates. 152 clinical isolates were subjected to this triplex real-time PCR assay. The results of the triplex real-time PCR assay correlated with the results of the phenotypic antibiotic susceptibility testing. The results obtained from triplex real-time PCR assay shows that the primer and probe sets were specific for the identification of S. aureus and were able to detect methicillin- and aminoglycoside-resistant genes. The entire assay can be performed within 3 h which is a very rapid method that can give simultaneous information for the identification of S. aureus and antibiotic resistance pattern of a staphylococcal isolate. The application of this rapid method in microbiology laboratories would be a valuable tool for the rapid identification of the S. aureus isolates and determination of their antibiotic resistance pattern with regards to methicillin and aminoglycosides.     

Dual-Priming Oligonucleotide-Based Multiplex PCR for the Detection of Helicobacter pylori and Determination of Clarithromycin Resistance with Gastric Biopsy Specimens

Background:  Assessment of Helicobacter pylori ( H. pylori ) clarithromycin resistance has rarely been performed routinely despite an increasing resistance rate. Our aim was to develop and evaluate the use of dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) to detect point mutations in the 23S rRNA gene responsible for clarithromycin resistance of H. pylori.
Materials and Methods:  Gastric biopsy specimens from 212 untreated patients with dyspepsia were examined by culture, histology, and DPO-based multiplex PCR. A disk diffusion test and E-test were used for performing phenotypic antibiotic susceptibility tests.
Results:  Among the biopsy specimens tested, 22.2% (47/212), 42.5% (90/212), and 41.5% (88/212) of the specimens were classified as H. pylori positive by culture, histology, and DPO-based multiplex PCR, respectively. Among 96 strains identified by either culture or DPO-based multiplex PCR, 80 strains were clarithromycin-susceptible and 16 strains (16.7%) were clarithromycin-resistant. There was 94.1% (32/34) concordance between phenotypic susceptibility tests and DPO-based multiplex PCR. In two patients with discrepant results, only DPO-based multiplex PCR detected clarithromycin-resistant strains. DPO-based multiplex PCR identified additional 49 clarithromycin-resistant or clarithromycin-susceptible H. pylori among 165 culture-negative specimens.
Conclusions:  DPO-based multiplex PCR can be used as a practical method for the detection of H. pylori infection and the determination of clarithromycin susceptibility in addition to phenotypic antimicrobial susceptibility tests.     

237例新生儿败血症的病原分布及耐药性分析

目的了解近年本地区新生儿败血症病原学及细菌耐药状况,用以指导临床治疗。方法对2006年5月至2010年4月收治的237例病例的血液进行培养,并对分离的菌株进行鉴定和药敏试验。结果革兰阳性菌为主要病原菌,排名前3位的病原菌分别是:表皮葡萄球菌(48.52%)、人葡萄球菌(16.03%)、凝固酶阴性葡萄球菌(10.13%)。对培养出的革兰阳性菌最敏感的抗生素是万古毒素、阿米卡星,对培养出的革兰阴性菌最敏感的抗生素是丁胺卡那霉素、亚胺培南。结论本地区新生儿血培养病原菌以革兰阳性菌为主,耐药菌株较多,临床医师应根据细菌鉴定及药敏试验选择敏感药物治疗。     

新生儿耐甲氧西林凝固酶阴性葡萄球菌败血症40例分析

目的：探讨耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）在新生儿败血症中的分布,临床特征及耐药性特点。方法：应用全自动血培养系统（BACTE/9050）,ATB细菌鉴定仪,从80例新生儿血培养阳性瓶中分离出40株MRCNS。应用ATB药敏系统,测定了青霉素等17种抗生素的耐药性。结果：80株细菌中,分离出40株MRCNS（占50%）,其中溶血葡萄球菌17株（42.5%)居首位,表皮葡萄球菌15株（37.5%),其他葡萄球菌8株（20%）。药敏结果显示多重耐药。结论：凝固酶阴性葡萄球菌（CNS）已成为小儿血培养的第一位检出菌,主要为溶血葡萄球菌和表皮葡萄球菌,MRCNS占主导地位（达50%左右）,成为新生儿病房院内感染的主要致病菌。药敏结果显示MRCNS呈多重耐药,万古霉素及其他糖肽类抗生素是治疗MRCNS感染的首选药物,MRCNS在新生儿病房造成院内感染的危害性以及耐药性应引起广泛关注。     

Conventional methods and future trends in antimicrobial susceptibility testing

Antimicrobial susceptibility testing is an essential task for selecting appropriate antimicrobial agents to treat infectious diseases. Constant evolution has been observed in methods used in the diagnostic microbiology laboratories. Disc diffusion or broth microdilution are classical and conventional phenotypic methods with long turnaround time and labour-intensive but still widely practiced as gold-standard. Scientists are striving to develop innovative, novel and faster methods of antimicrobial susceptibility testing to be applicable for routine microbiological laboratory practice and research. To meet the requirements, there is an increasing trend towards automation, genotypic and micro/nano technology-based innovations. Automation in detection systems and integration of computers for online data analysis and data sharing are giant leaps towards versatile nature of automated methods currently in use. Genotypic methods detect a specific genetic marker associated with resistant phenotypes using molecular amplification techniques and genome sequencing. Microfluidics and microdroplets are recent addition in the continuous advancement of methods that show great promises with regards to safety and speed and have the prospect to identify and monitor resistance mechanisms. Although genotypic and microfluidics methods have many exciting features, however, their applications into routine clinical laboratory practice warrant extensive validation. The main impetus behind the evolution of methods in antimicrobial susceptibility testing is to shorten the overall turnaround time in obtaining the results and to enhance the ease of sample processing. This comprehensive narrative review summarises major conventional phenotypic methods and automated systems currently in use, and highlights principles of some of the emerging genotypic and micro/nanotechnology-based methods in antimicrobial susceptibility testing.     

Clinical significance on MicroScan Rapid plus series using various antibiotic-resistant bacteria

MicroScan Rapid plus Neg II Series and MicroScan Rapid plus Pos Series by Siemens Healthcare Diagnostics K.K. are the panels which enable to measure identification and antimicrobial susceptibility testing quickly and we have confirmed that it is useful for detecting drug resistance bacteria. As the identification result of comparing Rapid plus series with the current panel by using 143 strains of various drug resistance bacteria, Gram positive cocci was 87. 7%, glucose fermenter was 100% and glucose non-fermenter was 77.3 in Gram negative bacilli. On the evaluation of antimicrobial susceptibility testing, Rapid plus series, in comparison with the current panel, confirmed the lower tendency of MIC value on some drugs, but it basically presented the good concordance rate. In terms of the reporting time of antimicrobial agent, non-fermenter or MRCNS reported the result as needed after 8 hours and it took a little longer time for the report of antimicrobial agent. On the other hand, 80% or higher of antimicrobial agent on panel was reported for intestinal bacteria in 4.5 hours and for MRSA in 6.5 hours. It enabled to report the testing result on the same day. Due to the results above, Rapid plus series was highly valued on the usability, such as the early detection of drug resistance bacteria and the selection of therapeutic agents.     

临床微生物检验和细菌耐药性监测探析

目的：探讨临床微生物检验和细菌耐药性监测。方法：对临床分离出的致病菌的耐药情况以及敏感性进行回顾分析,检查出各种病菌对各类抗菌药物的敏感率和耐药率,本文以葡萄球菌属、肠杆菌科、非发酵菌的代表菌种的耐药性和敏感性为例。结果：通过选取金黄色葡萄球菌、表皮葡萄球菌、溶血葡萄球菌、大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌为例进行回顾分析,它们对抗菌药物的耐药率、敏感率具体情况如文中表格所示。结论：临床病原菌的耐药性问题不容忽视,治疗时要根据药物的敏感性和耐药性选择适当的抗菌药物,合理使用抗菌药物,减轻抗菌药物的耐药寿命。     

Colorimetric microbial viability assay based on reduction of water-soluble tetrazolium salts for antimicrobial susceptibility testing and screening of antimicrobial substances

The applicability of a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt [WST-8]} via 2-methyl-1,4-naphthoquinone (2-methyl-1,4-NQ) as an electron mediator for determining the susceptibility of various bacteria to antibiotics and screening antimicrobial substances was investigated. The measurement conditions, which include the effects of the concentration of 2-methyl-1,4-NQ, were optimized for proliferation assays of gram-negative bacteria, gram-positive bacteria, and pathogenic yeast. In antimicrobial susceptibility testing, there was excellent agreement between the minimum inhibitory concentrations determined after 8 h using the WST-8 colorimetric method and those obtained after 22 h using conventional methods. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of the susceptibility of various bacteria to antibiotics. In addition, the current method was applied to the screening of bacteriocin-producing lactic acid bacteria and its efficiency was demonstrated.     

单细胞拉曼技术在病原微生物检测中的研究进展

单细胞拉曼技术是基于拉曼光谱分析原理实现非培养、无标签、快速、高效、低成本揭示物质本质的新方法。近年来,单细胞拉曼技术也开始在病原微生物检测领域崭露头角。本文结合单细胞拉曼技术基本原理这一理论基础,阐述该技术在病原微生物鉴定、药物敏感性检测中的研究进展及最新技术方向,并探讨其在临床实验室应用的可行性,为未来病原微生物检测技术提供新的方向。     

Detection of the nec1 virulence gene and its correlation with pathogenicity in Streptomyces species on potato tubers and in soil using conventional and real-time PCR

AIMS: To evaluate the virulence gene nec1 as a reliable marker for the detection of pathogenic Streptomyces species on potato tubers and in soil samples using conventional and real-time quantitative PCR assays. Methods AND RESULTS: Two pairs of conventional primers (outer and nested) and one set of primers/probe for use in real-time PCR were designed to detect the necrogenic protein encoding nec1 gene of Streptomyces scabiei strain ATCC 49173(T). The conventional PCR primers were also incorporated into a multiplex PCR assay to simultaneously detect the nec1 gene in conjunction with the potato pathogens Helminthosporium solani and Colletotrichum coccodes. The specificity of each PCR assay was confirmed by testing 32 pathogenic and nonpathogenic reference strains of Streptomyces representing 12 different species and 74 uncharacterized streptomycete strains isolated from diseased tubers. A clear correlation between pathogenicity and the detection of nec1 by PCR was demonstrated. The sensitivity and specificity of both the conventional and real-time PCR assays allowed the detection of nec1 on potato tubers in the absence of visible symptoms of common scab, and in seeded soil down to a level equivalent to three S. scabiei spores per gram soil. CONCLUSIONS: Reliable and quantitative PCR techniques were developed in this study for the specific detection of the virulence gene nec1 of pathogenic Streptomyces species on potato tubers and in soil samples, and the data demonstrated a clear correlation between pathogenicity in Streptomyces species and the presence of the nec1 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Together with the DNA extraction protocols, these diagnostic methods will allow a rapid and accurate assessment of tuber and soil contamination by pathogenic Streptomyces species.     

肠杆菌科细菌肺部感染病原菌分布与耐药性监测

目的了解浙江龙游县人民医院肠杆菌科细菌肺部感染病原菌分布及耐药性,为临床合理使用抗菌药物提供依据。方法采用纸片扩散法对病原菌进行药敏试验,并进行ESBLs检测,按CLSI 2012年标准判定药敏结果,用WHONET 5.6软件分析结果。结果痰培养共分离出371株肠杆科细菌,主要为肺炎克雷伯菌、大肠埃希菌和阴沟肠杆菌,产ESBLs菌阳性率高,对碳青霉烯类抗菌药物耐药率低,均在10.00%以下,其他药物均有不同程度耐药,测试抗菌药物的耐药率差异有统计学意义（P〈0.05）。结论临床常见肠杆菌科细菌耐药率高,开展病原菌耐药性监测,对指导临床抗感染治疗合理选择抗菌药物具有重要意义。     

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock¹. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours^{2, 4}. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes⁵. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods⁶. This assay was based on a study in which PCR was used to measure the growth of bacteria⁷. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.     

Methicillin resistance study in clinical isolates of coagulase-negative staphylococci and determination of their susceptibility to alternative antimicrobial agents

AIMS: To achieve reliable detection of methicillin resistance in clinical isolates of coagulase-negative staphylococci. METHODS AND RESULTS: Strains (105) were evaluated by normatized antimicrobial susceptibility methods, and for the presence of the methicillin resistance-determining mecA gene, using the polymerase chain reaction. Correlation between phenotypic and genotypic methods was obtained in 87.6% of the samples. Six strains, classified as methicillin-susceptible by phenotypic assays, revealed the presence of the mecA gene, indicating that methicillin resistance expression was probably repressed. Another seven isolates failed to show mecA amplification after displaying methicillin resistance in phenotypic evaluations. The susceptibility of the methicillin-resistant isolates to other antimicrobial agents was variable. CONCLUSION: Genotypic determination of the mecA gene proved to be the most reliable method for detection of methicillin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Correct assessment of methicillin resistance, such as that attained through genotyping, is essential for defining therapeutic strategies, particularly when treating severely compromised patients.     

血流感中的病原菌分布及耐药性分析

目的了解血流感染中的病原菌种类、分布及耐药性,为临床合理选用抗菌药物提供依据。方法按照无菌操作方法采集疑为血流感染患者的血液标本进行血培养,采用美国BD公司PHOENIX-100全自动细菌鉴定药敏系统进行细菌鉴定及药敏试验,并对结果进行统计分析。结果共检出1080株病原菌,其中革兰阴性杆菌630株（占58．3％）,革兰阳性球菌428株（占39．6％）,真菌22株（占2．1％）。药敏结果显示：革兰阳性球菌对万古霉素、利奈唑胺敏感率最高,对青霉素、红霉素、头孢菌素等耐药率较高。革兰阴性杆菌对碳青霉烯类、哌拉西林／他唑巴坦、头孢哌酮／舒巴坦敏感率较高,对青霉素类、头孢菌素类和喹诺酮类耐药率较高。结论引起血流感染的病原菌分布复杂,耐药率较高,临床应提高血培养送检率,根据药敏结果合理选用抗菌药物,以减少多重耐药菌的发生和传播。     

82例极低/超低出生体重儿败血症的临床分析

目的:分析极低/超低出生体重儿败血症的临床特征、病原菌及药物敏感情况,为其早期诊断及治疗提供参考。方法:对2014年1月1日至2017年12月31日阜阳市人民医院新生儿重症监护病房(NICU)确诊的82例早产极低/超低出生体重儿败血症的临床表现、实验室检查、病原菌及药敏情况进行回顾性分析。结果:极低/超低出生体重儿败血症以早发型(≤7天)为主,晚发型以院内感染为主,临床表现缺乏特异性,实验室检查中白细胞、血小板出现减低,C反应蛋白和降钙素原增高。病原菌以革兰阴性菌为主,其次为革兰阳性菌和真菌。药敏结果显示革兰阴性菌对三代头孢、氨苄西林类抗生素100%耐药,对加他唑巴坦的抗生素耐药率较低,对碳氢酶烯类抗生素敏感。革兰阳性菌对β-内酰胺类、大环内酯类、氨基糖甙类及克林霉素等耐药率较高,对万古霉素、利奈唑烷敏感。82例败血症患儿中,死亡6例,死亡率为7.3%。结论:早产极低/超低出生体重儿败血症缺乏特异性临床表现,且发病率高,应密切观察患儿临床表现及动态监测其C反应蛋白、血小板等的变化,同时及时完善细菌培养及药敏试验,有效合理使用抗生素,以减少多重耐药菌株产生,改善患儿预后。