High-resolution melting analysis for the rapid detection of fluoroquinolone and streptomycin resistance in Mycobacterium tuberculosis

Background

Molecular methods for the detection of drug-resistant tuberculosis are potentially more rapid than conventional culture-based drug susceptibility testing, facilitating the commencement of appropriate treatment for patients with drug resistant tuberculosis. We aimed to develop and evaluate high-resolution melting (HRM) assays for the detection of mutations within gyrA, rpsL, and rrs, for the determination of fluoroquinolone and streptomycin resistance in Mycobacterium tuberculosis (MTB).

Methodology/Principal Findings

A blinded series of DNA samples extracted from a total of 92 clinical isolates of MTB were analyzed by HRM analysis, and the results were verified using DNA sequencing. The sensitivity and specificity of the HRM assays in comparison with drug susceptibility testing were 74.1% and 100.0% for the detection of fluoroquinolone resistance, and 87.5% and 100.0% for streptomycin resistance. Five isolates with low level resistance to ofloxacin had no mutations detected in gyrA, possibly due to the action of efflux pumps, or false negativity due to mixed infections. One fluoroquinolone-resistant isolate had a mutation in a region of gyrA not encompassed by our assay. Six streptomycin-resistant strains had undetectable mutations by HRM and DNA sequencing, which may be explained by the fact that not all streptomycin-resistant isolates have mutations within rpsL and rrs, and suggesting that other targets may be involved.

Conclusion

The HRM assays described here are potentially useful adjunct tests for the efficient determination of fluoroquinolone and streptomycin resistance in MTB, and could facilitate the timely administration of appropriate treatment for patients infected with drug-resistant TB.     

Study on the rapid, secondary-growth mutants of Staphylococcus epidermidis (author's transl)

According to colony type, growth rate and development of secondary growth on the proteose-peptone No. 3 mannitol salt agar (PMS) and the nutrient agar (NA) media, Staphylococcus epidermidis may be classified into three groups. Group I includes strains which develop smooth colonies on both media. Group II consists of those which show rapid propagation of entire clones and develop secondary growth on the PMS medium, but grow only smooth colonies on the NA medium, and which may be called reversible mutants. Group III includes thoes which show secondary growth on both PMS and NA as well as other media, which may be called irreversible mutants. One percent proteose-peptone No. 3 and 5--7% NaCl are the essential ingredients for the induction of mutation, and mannitol can enhance it. Except the high sensitivity of the reversible mutants of human origin, the three groups of chicken origin showed similar drug susceptibility to biosynthesis inhibitors of protein and cell wall. On the HI medium, chloramphenicol inhibited secondary growth of irreversible mutants at 25.0 microgram/ml minimal antimutagenesis concentration (MAC), whereas streptomycin, penicillin, erythromycin and oxytetracycline did not at all. The irreversible mutants had higher resistance to biosynthesis inhibitors of DNA or RNA, e.g. mitomycin C (MMC), novobiocin (NOV) and rifampicin (RIF), than the other two groups. On the HI medium, MMC at the MAC of 0.16 microgram/ml, NA at 25.0 microgram/ml and NOV at 2.5 microgram/ml inhibited the secondary growth of irreversible mutants, but RIF did not. To the irreversible mutants, the MIC and MAC of NA on the PMS medium were both higher than those on the HI medium. The MACs of MMC and NOV on the PMS medium were also higher than those on the HI medium, but their geometric mean MIC remained almost unchanged on both media. Because the MACs of MMC (0.31 microgram/ml) and NA (100.0 microgram/ml) to the reversible mutants on the PMS medium were much similar to those of the irreversible mutants, it suggests that both groups had the similar mutation mechanism.     

High-resolution melt analysis for the detection of a mutation associated with permethrin resistance in a population of scabies mites

Permethrin as a topical acaricide cream is widely used to treat scabies. The neuronal voltage-sensitive sodium channel (Vssc), necessary for the generation of action potentials in excitable cells, is the target of pyrethroid acaricides such as permethrin. Pyrethroid resistance has been linked to specific mutations in the Vssc gene. Following the partial sequencing of the Vssc gene in the scabies mite Sarcoptes scabiei (L.) (Astigmata: Sarcoptidae), we compared Vssc gene sequences from permethrin-sensitive and -tolerant S. scabiei var. canis Gerlach mites, and identified a G to A single nucleotide polymorphism (SNP) in permethrin-tolerant mites resulting in an amino acid change from glycine to aspartic acid in domain III S6. The mutation is in a region of the gene where mutations have been identified in a range of pyrethroid-resistant arthropods. Results of in vitro permethrin exposure assays showed that survival rates for mites bearing the mutation were similar to those previously reported for mites from human subjects where clinical tolerance to permethrin had been observed. A real-time polymerase chain reaction-high-resolution melt (PCR-HRM) assay was developed to detect this SNP. This assay provides a useful methodology for screening for this and other mutations associated with permethrin resistance in scabies mite populations and thus facilitates surveillance for acaricide resistance.     

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.     

Rapid procedure for the detection of plasmids in Staphylococcus epidermidis.

A rapid, reproducible, mini-volume assay capable of detecting staphylococcal plasmid DNA in the range of 0.8 to 32 megadaltons has been developed. The assay employs lysostaphin-mediated lysis of cells followed by a short, low-speed centrifugation and does not require treatment with ribonuclease or protease or deproteinization with phenol. A period of only 24 h may be required to detect the presence and size of a plasmid once an organism has been isolated. This method has been used to study the plasmid ecology of Staphylococcus epidermidis and to correlate the presence or absence of plasmids with tetracycline, chloramphenicol, neomycin, penicillin, and cadmium resistances.     

High-resolution melting analysis of cDNA-derived PCR amplicons for rapid and cost-effective identification of novel alleles in barley

An original method has been established for the identification of novel alleles of eukaryotic translation initiation factor 4E (eIF4E) gene, which is required for resistance to agronomically important bymoviruses, in barley germplasm. This method involves scanning for sequence variations in cDNA-derived PCR amplicons using High-resolution melting (HRM) followed by direct Sanger sequencing of only those amplicons which were predicted to carry nucleotide changes. HRM is a simple, cost-effective, rapid and high-throughput assay, which so far has only been widely used in clinical pathology for molecular diagnostic of diseases and patient genotyping. Application of HRM allowed significant reduction in the amount of expensive Sanger sequencing required for allele mining in plants. The method described here involved an investigation of total cDNA rather than genomic DNA, thus permitting the analyses of shorter (up to 300-bp) and fewer overlapping amplicons to cover the coding sequence. This strategy further reduced the allele mining costs. The sensitivity and accuracy of HRM for predicting genotypes carrying a wide range of nucleotide polymorphisms in eIF4E approached 100%. Results of the current study are promising and suggest that this method could also potentially be applied to the discovery of superior alleles controlling other important traits in barley as well in other model and crop plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Heteroduplex analysis by capillary array electrophoresis for rapid mutation detection in large multiexon genes

Heteroduplex analysis (HA) has proven to be a robust tool for mutation detection. HA by capillary array electrophoresis (HA-CAE) was developed to increase throughput and allow the scanning of large multiexon genes in multicapillary DNA sequencers. HA-CAE is a straightforward and high-throughput technique to detect both known and novel DNA variants with a high level of sensitivity and specificity. It consists of only three steps: multiplex-PCR using fluorescently labeled primers, heteroduplex formation and electrophoresis in a multicapillary DNA sequencer. It allows, e.g., the complete coding and flanking intronic sequences of BRCA1 and BRCA2 genes from two patients (approximately 25 kb each) to be scanned in a single run of a 16-capillary sequencer, and has enabled us to detect 150 different mutations to date (both single nucleotide substitutions, or SNSs, and small insertions/deletions). Here, we describe the protocol developed in our laboratory to scan BRCA1, BRCA2, MLH1, MSH2 and MSH6 genes using an ABI3130XL sequencer. This protocol could be adapted to other instruments or to the study of other large multiexon genes and can be completed in 7-8 h.     

Genome-based analysis of virulence genes in a non-biofilm-forming Staphylococcus epidermidis strain (ATCC 12228)

Staphylococcus epidermidis strains are diverse in their pathogenicity; some are invasive and cause serious nosocomial infections, whereas others are non-pathogenic commensal organisms. To analyse the implications of different virulence factors in Staphylococcus epidermidis infections, the complete genome of Staphylococcus epidermidis strain ATCC 12228, a non-biofilm forming, non-infection associated strain used for detection of residual antibiotics in food products, was sequenced. This strain showed low virulence by mouse and rat experimental infections. The genome consists of a single 2499 279 bp chromosome and six plasmids. The chromosomal G + C content is 32.1% and 2419 protein coding sequences (CDS) are predicted, among which 230 are putative novel genes. Compared to the virulence factors in Staphylococcus aureus, aside from delta-haemolysin and beta-haemolysin, other toxin genes were not found. In contrast, the majority of adhesin genes are intact in ATCC 12228. Most strikingly, the ica operon coding for the enzymes synthesizing interbacterial cellular polysaccharide is missing in ATCC 12228 and rearrangements of adjacent genes are shown. No mec genes, IS256, IS257, were found in ATCC 12228. It is suggested that the absence of the ica operon is a genetic marker in commensal Staphylococcus epidermidis strains which are less likely to become invasive.     

High-resolution melting analysis (HRMA): a highly sensitive inexpensive genotyping alternative for population studies

High-resolution melting analysis (HRMA) is a highly sensitive closed-tube genotyping method used primarily in clinical studies. As the method is rapid, inexpensive and amenable to high throughput, we decided to investigate its applicability to population studies. Small amplicons and unlabelled probes were used to genotype the nuclear genes, lactate dehydrogenase-A (ldh-A), myosin light chain-2 (mlc-2), acidic ribosomal phosphoprotein P0 (ARP) and calmodulin (CaM) in populations of swordfish, Xiphias gladius. Results indicate that HRMA is a powerful genotyping tool to study wild populations.     

Capillary isoelectric focusing--useful tool for detection of the biofilm formation in Staphylococcus epidermidis

The biofilm formation is an important factor of S. epidermidis virulence. Biofilm-positive strains might be clinically more important than biofilm-negative ones. Unlike biofilm-negative staphylococci, biofilm-positive staphylococci are surrounded with an extracellular polysaccharide substance. The presence of this substance on the surface can affect physico-chemical properties of the bacterial cell, including surface charge. 73 S. epidermidis strains were examined for the presence of ica operon, for the ability to form biofilm by Christensen test tube method and for the production of slime by Congo red agar method. Isoelectric points (pI) of these strains were determined by means of Capillary Isoelectric Focusing. The biofilm negative strains focused near pI value 2.3, while the pI values of the biofilm positive strains were near 2.6. Isoelectric point is a useful criterion for the differentiation between biofilm-positive and biofilm-negative S. epidermidis strains.     

High-resolution melting analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine

The differentiation of Diplodia pinea from closely related species, such as Diplodia scrobiculata and Diplodia seriata, and its detection in plant tissue, represented a critical issue for a long time. Molecular screening tools have recently been developed to address this topic. In this study we applied one of the most sensitive and rapid diagnostic screening method so far developed, called High-Resolution Melting Analysis (HRMA), to detect D. pinea in Austrian pine (Pinus nigra). HRMA exploits differences in the melting behaviour of PCR products to rapidly identify DNA sequence variants without the need for cumbersome post-PCR methods. We developed a HRMA method to detect specific fungal sequences in the mitochondrial small subunit ribosome gene (mt SSU rDNA). The reliability of this technique was firstly assessed on DNA extracted from pure cultures of D. pinea and closely related species. Amplicon differences were screened by HRMA and the results confirmed by direct DNA sequencing. Subsequently, HRMA was tested on DNA from symptomatic and symptomless pine shoots, and the presence of the fungus was also confirmed by both conventional and molecular quantitative approaches. The HRMA allowed the distinction of D.?pinea from closely related species, showing specific melting profiles for the each pathogen. This new molecular technique, here tested in a plant-fungus pathosystem for the first time, was very reliable in both symptomatic and symptomless shoots. HRMA is therefore a highly effective and accurate technique that permits the rapid screening of pathogens in the host.     

表皮葡萄球菌PhoU同源物(SERP0316)的抗体制备及免疫鉴定

表皮葡萄球菌可在植入性医疗材料表面形成生物膜,对多种抗生素耐受,而持留菌的形成是导致细菌生物膜耐药的原因之一。研究发现,大肠埃希菌中PhoU可影响持留菌的形成,与细菌对多种抗生素及压力条件的耐受相关,但表皮葡萄球菌中PhoU的功能仍不清楚。生物信息学分析显示,在表皮葡萄球菌中存在2个phoU同源基因,其中serp0956位于pst操纵子中,命名为phoU1;另一个编码功能未知蛋白的phoU基因同源物(serp0316)命名为phoU2。前期研究显示,敲除phoU1并不影响表皮葡萄球菌生长、生物膜形成等生物学特性,为此本研究对PhoU2进行了初步探讨。利用实时荧光定量反转录-聚合酶链反应(RT-PCR)检测phoU2基因在表皮葡萄球菌SE1457不同生长时期的转录水平,发现其在细菌生长不同时间点持续表达,其中对数期(6h)表达量相对较高。为进一步研究表皮葡萄球菌中PhoU2的表达情况,对PhoU2蛋白进行原核表达和纯化,并用纯化的重组PhoU2蛋白免疫小鼠制备PhoU2多克隆抗体。在此基础上,利用蛋白免疫印迹法检测PhoU2在SE1457菌株不同生长时间点的表达水平,结果显示PhoU2在细菌生长对数期和平台期均有表达。另外,在不同的表皮葡萄球菌临床分离株中均检测到PhoU2蛋白表达,表明PhoU2具有一定的保守性。以上研究提示,PhoU2可能作为PhoU的功能同源物发挥一定的作用,为进一步研究表皮葡萄球菌PhoU2的生物学功能及其在生物膜和持留菌形成中的作用奠定了基础。     

Glucose-6-phosphate dehydrogenase (G6PD) gene mutations detection by improved high-resolution DNA melting assay

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide including southern China. G6PD gene mutations cause deficiency of the enzyme and a large spectrum of diseases. High-resolution DNA melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. But there was a shortcoming of this method that hemizygous and homozygous genotypes were not easily distinguished from wild-types. Here we used improved HRM method for a small-scale screening of G6PD-deficient variants among people of Meizhou region. Then all amplicons were ascertained by direct DNA sequencing. These results indicated that HRM method was a major technical advance for G6PD mutations screening.     

High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.)

Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic polymorphism in plant genomes. SNP markers are valuable tools for genetic analysis of complex traits of agronomic importance, linkage and association mapping, genome-wide selection, map-based cloning, and marker-assisted selection. Current challenges for SNP genotyping in polyploid outcrossing species include multiple alleles per loci and lack of high-throughput methods suitable for variant detection. In this study, we report on a high-resolution melting (HRM) analysis system for SNP genotyping and mapping in outcrossing tetraploid genotypes. The sensitivity and utility of this technology is demonstrated by identification of the parental genotypes and segregating progeny in six alfalfa populations based on unique melting curve profiles due to differences in allelic composition at one or multiple loci. HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes. Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs.     

Kinetic analysis of rhodamines efflux mediated by the multidrug resistance protein (MRP1)

Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, k(a) = V(M)/k(m), was very similar for the four rhodamine analogs but approximately 10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes.     

Rapid detection of resistance in Staphylococcus aureus by using Quicolor ES

Traditional microbiological methods are dependent on the growth of microorganisms, and hence require prolonged periods. The methods used to detect resistance in Staphylococcus aureus should have high sensitivity and specificity, yet provide results in a timely manner. The aim of this study was to evaluate the use of Quicolor (QC) ES^® agar for the rapid detection of resistance in S. aureus. We evaluated 100 clinical S. aureus isolates. Resistance detection was performed using traditional microbiological methods. Methicillin resistance detection was evaluated using traditional and molecular microbiological methods. Traditional antibiotic susceptibility testing methods, such as disc diffusion, were conducted using QC ES and Mueller–Hinton (MH) media. The plates were incubated at 36 °C for 5, 6 and 24 h. Rapid results obtained using QC ES agar after 5 h of incubation were consistent with those using the overnight procedure with MH agar for 83 of the 100 S. aureus (including methicillin-susceptible S. aureus) strains. However, the correlation for oxacillin between MH (24 h) and QC ES (5 h) was not satisfactory (r = 0.770). The total agreement between QC ES and MH agar was 83 % after 5 h, 89 % after 6 h, and 94 % after 24 h. The accurate and rapid detection of resistance in S. aureus is critical due to the associated therapeutic problems and infection control measures. We believe that the use of QC ES for S. aureus will reduce the delay in resistance detection, thus providing physicians and infection control practitioners with early information for better management.     

利奈唑胺治疗儿童社区获得性金黄色葡萄球菌肺炎的临床疗效分析

目的:分析儿童社区获得性金葡菌肺炎应用利奈唑胺的临床疗效.方法:选择2008-1-1至2009-3-1在青岛大学医学院附属医院普通儿科及青岛市儿童医院诊断社区获得性金葡菌肺炎患儿22人为研究对象,分析其体温、咳嗽、肺部罗音、住院时间等临床资料.结果:患儿的平均住院时间为13.0±2.26天,有效率100%,不良反应发生率4.55%.结论:利奈唑胺治疗儿童社区获得性金葡菌肺炎有较高的临床治愈率,可快速改善症状.缩短住院时间.