Sensitivity of Chinese Isolates of Plasmopara viticola to Metalaxyl and Dimethomorph

During 2007 and 2008, 392 isolates of Plasmopara viticola were collected from 11 regions in seven provinces in China, and their sensitivities to metalaxyl and dimethomorph were determined by the floating leaf disk technique. Among all isolates, 13% were classified as sensitive, 26% as low‐level resistant, and 61% as resistant to metalaxyl. Of the 392, 85 were from vineyards never treated with carboxylic acid amide fungicides; these isolates were used to determine the baseline sensitivity to dimethomorph, and their EC₅₀ values ranged from 0.01 to 0.21 (mean ± SD, 0.11 ± 0.04) μg/ml. The other 307 isolates were completely inhibited by a single discriminatory dose of 1.6 μg/ml of dimethomorph.     

Variation in the sensitivity to metalaxyl of Pythium spp. isolated from carrot and other sources

Over a 3-yr period 261 isolates of 17 species of Pythium were tested for sensitivity to metalaxyl at concentrations of 5, 50 or 100 μ/ml. A wide range of responses was observed, from isolates where growth ceased at 5 μg/ml to those where growth at 100 μg/ml was similar to that of the untreated controls. In further tests isolates of 11 different species had ED50's < 1 μg/ml. A lower sensitivity was detected in isolates of six Pythium spp. where values in the range 1–10 μg/ml were obtained. This lower sensitivity was not related to previous known use of metalaxyl. Three isolates of Pythium dissotocum from sites where the fungicide had been used repeatedly had ED50's > 100 μg/ml and were considered resistant. The resistance was stable over a 2-yr period and isolates were cross-resistant to furalaxyl, benalaxyl, ofurace, cyprofuram and oxadixyl. Increasing concentrations of metalaxyl reduced or prevented the production of zoospores by four species of Pythium, although when zoospores were produced, this was followed by the normal processes of encystment and germination. Culturing P. dissotocum on different sub-lethal concentrations of metalaxyl for 18 wk did not induce a high level of resistance to the fungicide.     

海南省潜伏香蕉果实的炭疽菌对特克多的抗药性

对来自海南省13个地区的67个Colletotrichum musae（Berk. & Curt.）Arx菌株进行抗药性检测,所测菌株均表现出敏感,即未产生特克多抗药性。室内采用高浓度特克多和90～95%致死剂量紫外光进行抗性诱导,获得的抗性菌株均能在1000g/ml特克多的PDA培养基上生长,但EC50值相差很大,最高达130g/ml,而最低为0.87g/ml。抗性菌株对多菌灵和甲基托布津均表现正交互抗药性。连续无毒培养10代后,所有菌株仍可在5g/ml特克多的培养基上生长,表现抗性遗传稳定。抗性菌株的产孢能力和致病力与敏感菌相比并没有下降,表现很高的适应能力。     

Comparative Resistance Patterns of Fludioxonil and Vinclozolin in Botryotinia fuckeliana

Fludioxonil is a new non-systemic phenylpyrrole fungicide. It is a derivative of the antibiotic pyrrolnitrin and is highly active against Ascomycetes. Basidiomycetes and Deuteromycetes. In the present study we determined the baseline sensitivity of B. fuckeliana for fludioxonil in comparison to the dicarboximide vinclozolin. Subsequently these baselines were compared with the sensitivity of dicarboximide-resistant field isolates and with resistant laboratory strains selected on media amendedwith vinclozotin or fludioxonil, respectively. Additionally, sensitivities of genetically homogeneous ascospore progenies of sexual crosses of sensitive isolates with dicarboximide-resistant field isolates or resistant laboratory strains were compared to, the baselines. Baseline ECst, values for the inhibition of germination ranged from 0.07 to 0.20 μg ml^-1 of vinclozolin and for mycelial growth from 0.003 to 0.016 μg ml’ of fludioxonil and from 1.7 to 2.0 μg ml^-1 of vinclozolin and for mycelial growth from 0.003 to 0.016 μg ml^-1 of fludioxonil and from 0.13 to 0.27 μg ml^-1 of vinclozolin. Sensitivity to fludioxonil of all dicarboximide-resistant field isolates did not differ from their baseline. However, resistant laboratory strains that were selected either on media amended with fludioxonil or vinclozolin showed decreased sensitivities to both active ingredients. All sexual progenies of the crosses described above were of the parental phenotypes. We found no independent segregation of the resistance markers.     

Assessing the Risk That Phytophthora melonis Can Develop a Point Mutation (V1109L) in CesA3 Conferring Resistance to Carboxylic Acid Amide Fungicides

The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC(50) values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) μg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10(-7) and 1×10(-6), respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI = mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC(50) values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations.     

Double resistance by citrus green mould Penicillium digitatum to the fungicides guazatine and benomyl

In vitro dosage response data with different isolates of Penicillium digitatum and the fungicide guazatine indicated an approximate 10-fold shift in tolerance when compared with wild type strains. ED₅₀ values for resistant strains were approximately 0.5 μg/ml compared to approximately 0.05, μg/ml for the wild type strains. Colony growth of guazatine resistant isolates on selective media containing carbendazim showed that they were also resistant to the benzimidazole group of fungicides. In vivo tests in inoculated oranges with strains previously characterised by in vitro tests confirmed resistance to guazatine and benomyl. A combined treatment of these fungicides at 400 /μ/ml and 500 μg/ml respectively, which normally gives protection against decay, also failed to provide adequate mould control. Growth and pathogenicity of the resistant strains in these tests in oranges were indistinguishable from that of wild type strains.     

Natural antibiotic resistance of bacteria isolated from larvae of the oil fly, Helaeomyia petrolei

Helaeomyia petrolei (oil fly) larvae inhabit the asphalt seeps of Rancho La Brea in Los Angeles, Calif. The culturable microbial gut contents of larvae collected from the viscous oil were recently examined, and the majority (9 of 14) of the strains were identified as Providencia spp. Subsequently, 12 of the bacterial strains isolated were tested for their resistance or sensitivity to 23 commonly used antibiotics. All nine strains classified as Providencia rettgeri exhibited dramatic resistance to tetracycline, vancomycin, bacitracin, erythromycin, novobiocin, polymyxin, colistin, and nitrofurantoin. Eight of nine Providencia strains showed resistance to spectinomycin, six of nine showed resistance to chloramphenicol, and five of nine showed resistance to neomycin. All 12 isolates were sensitive to nalidixic acid, streptomycin, norfloxacin, aztreonam, cipericillin, pipericillin, and cefotaxime, and all but OF008 (Morganella morganii) were sensitive to ampicillin and cefoxitin. The oil fly bacteria were not resistant to multiple antibiotics due to an elevated mutation rate. For each bacterium, the number of resistant mutants per 10(8) cells was determined separately on rifampin, nalidixic acid, and spectinomycin. In each case, the average frequencies of resistant colonies were at least 50-fold lower than those established for known mutator strain ECOR 48. In addition, the oil fly bacteria do not appear to excrete antimicrobial agents. When tested, none of the oil fly bacteria produced detectable zones of inhibition on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, or Candida albicans cultures. Furthermore, the resistance properties of oil fly bacteria extended to organic solvents as well as antibiotics. When pre-exposed to 20 microg of tetracycline per ml, seven of nine oil fly bacteria tolerated overlays of 100% cyclohexane, six of nine tolerated 10% xylene, benzene, or toluene (10:90 in cyclohexane), and three of nine (OF007, OF010, and OF011) tolerated overlays of 50% xylene-50% cyclohexane. The observed correlation between antibiotic resistance and organic solvent tolerance is likely explained by an active efflux pump that is maintained in oil fly bacteria by the constant selective pressure of La Brea's solvent-rich environment. We suggest that the oil fly bacteria and their genes for solvent tolerance may provide a microbial reservoir of antibiotic resistance genes.     

长沙地区临床分离金黄色葡萄球菌的耐药监测

目的了解长沙地区临床分离金黄色葡萄球菌（以下简称金葡菌）对常用抗菌药物的耐药现状,探讨金黄色葡萄球菌对甲氧西林的耐药水平。方法收集长沙地区11家医院2009年11月至2010年11月临床分离的非重复金葡菌279株,应用Vitek-2全自动微生物分析系统进行鉴定,K-B法检测金葡菌对24种药物的敏感性,产色头孢菌素试验检测β-内酰胺酶以及D试验检测诱导型克林霉素耐药。应用头孢西丁和苯唑西林纸片扩散法筛查耐甲氧西林的金葡菌（MRSA）,琼脂稀释法检测头孢西丁和苯唑西林的最低抑菌浓度（MIC）。结果在被检测的24种药物中,敏感率〉50%的药物为9种,未发现对万古霉素、替考拉宁和利奈唑胺耐药菌株;耐药率〉50%的抗菌药物有11种,其中以青霉素和氨苄西林的耐药率最高（均为97.1%）。MRSA的分离率达54.5%,且对常用的16种抗菌药物的耐药率均显著高于甲氧西林敏感金黄色葡萄球菌（MSSA）。279株金葡菌中,β-内酰胺酶阳性250株（89.6%）;红霉素耐药而克林霉素敏感或中介的30株中,D试验阳性22株（73.3%）。苯唑西林（OXA）和头孢西丁（FOX）MIC范围分别为0.125～〉256μg/mL和2～〉256μg/mL,苯唑西林的MIC50和MIC90分别为128μg/mL和256μg/mL,头孢西丁的MIC50和MIC90分别为64μg/mL和256μg/mL。结论长沙地区临床分离金葡菌对常用抗菌药物呈多重耐药;MRSA不仅分离率高,而且对甲氧西林呈高水平耐药。     

Occurrence of fenhexamid resistance in Botrytis cinerea from greenhouse strawberries in China

This study assessed the fenhexamid sensitivity of 143 Botrytis cinerea isolates collected from greenhouse strawberries in five regions of China between 2012 and 2013, and identified four isolates with moderate levels of resistance: two from the Xinjiang Uygur Autonomous Region and two from Hebei Province. The baseline fenhexamid sensitivity of B. cinerea exhibited a unimodal distribution with a mean EC₅₀ value of 0.20 ± 0.10 μg/ml (SD). The EC₅₀ values of the fenhexamid‐resistant isolates ranged from 0.05 to 0.40 μg/ml. Molecular analysis of the fenhexamid target gene erg27 revealed that the resistant isolates collected from Xinjiang (163‐6 and 163‐22) contained three mutations that led to amino acid changes (V365A, E368D and A378T) known to be associated with fenhexamid resistance, but that the isolates from Hebei lacked any mutations, indicating that an alternative mechanism could be responsible for their resistance. Most of the biological characteristics of the fenhexamid‐resistant isolates, such as mycelial growth, sclerotia production and pathogenicity, did not significantly differ from those of the sensitive ones (p ≤ .05), but it was noted that some of the resistant isolates exhibited reduced rates of sporulation and spore germination. In addition, the resistant isolates exhibited lower osmotic sensitivity than the sensitive ones. The study found no evidence of cross‐resistance with other fungicides, but that there was negative cross‐resistance with procymidone, iprodione, carbendazim and pyraclostrobin, which indicates that the inclusion of these fungicides within an integrated pest management (IPM) programme could help to minimize the risk of fenhexamid resistance developing in B. cinerea.     

Sensitivity to metalaxyl of Phytophthora infestans populations in potato crops in south-west England in 1980 and 1981

A laboratory test was developed to assess the sensitivity of field populations of Phytophthora infestans to metalaxyl. Discs of potato leaf tissue were floated upon solutions of the fungicide at different concentrations and inoculated with spores. The extent of symptom development was noted after incubation under standard conditions for 5–6 days. In preliminary experiments growth of isolates of P. infestans obtained from culture collections was severely inhibited in discs treated at 2 μg/ml. By contrast the development of an isolate obtained from a crop in Eire in which blight control with metalaxyl had failed, and known to be markedly less sensitive in vitro, was unaffected in discs treated at 100 μg/ml. During the summer of 1980, 234 samples of P. infestans were obtained from 20 sites in south-west England, 10 of which had received sprays containing metalaxyl and 10 of which had not. All samples were sensitive to metalaxyl applied at 2 μg/ml. In 1981, 35 sites within the same area, 30 of which had received sprays containing either metalaxyl or ofurace (a related fungicide), were similarly surveyed. Most of the 79 samples of P. infestans examined proved sensitive and at all sites the amount of blight was small. However, at three sites, including one not treated with acylalanine fungicides, strains were found which were unaffected by 100 μg/ml metalaxyl in leaf disc tests. These findings are discussed in relation to the development of resistant blight in other areas and to the use of fungicide mixtures.     

油菜菌核病菌对速克灵的抗药性风险研究

在室内条件下通过菌丝生长速率法测定了分离自安徽省10个县市的油菜菌核病菌（Sclerotinia scleroti-orum）对速克灵的敏感性。结果表明,速克灵对各供试菌株的EC50值分布范围为0.0899-0.4966μg/mL,平均为0.2541μg/mL,且供试菌株在含速克灵质量浓度为10 000μg/mL的PDA平板上菌丝生长几乎完全被抑制。表明各供试菌株对速克灵十分敏感,但其敏感程度地区间存在较大差异。通过室内药剂直接诱变法,获得了抗速克灵突变株。抗性突变株抗性测定结果表明,某些地区的抗性菌株抗性消失,有些地区的抗性菌株抗性继续保持。结果显示安徽省油菜菌核病菌对速克灵具有潜在的抗药性风险。     

Leishmania donovani: isolation and characterization of sodium stibogluconate (Pentostam)-resistant cell lines

Leishmania donovani promastigotes were generated by virtue of their resistance to incrementing concentrations of sodium stibogluconate (Pentostam) under completely defined growth conditions. The PENT0400 and PENT03200 cell lines were isolated after prolonged exposure to 0.4 mg/ml and 3.2 mg/ml Pentostam (Sb concentration), respectively. Whereas the effective concentration of Pentostam which inhibited the growth of wild type cells by 50% (EC50 value) was 0.1-0.15 mg/ml, the EC50 values for the PENT0400 and PENT03200 cells were approximately 1 and 4 mg/ml, respectively. The decreased sensitivities of both PENT0400 and PENT03200 cells to Pentostam were maintained after 6 months of continuous culture in the absence of selective pressure, indicating that the Pentostam resistance in the mutant organisms was a stable genetic trait. Interestingly, wild type and PENT03200 cells were equally sensitive to growth inhibition and cytotoxicity caused by SbCl5 and SbCl3, as well as to a variety of other cations such as Cd, Zn, and As. Wild type and PENT03200 cells also displayed equivalent growth sensitivities to a spectrum of other antiprotozoal agents, including antimony potassium tartrate, melarsoprol, pyrimethamine, pentamidine, formycin B, and difluoromethylornithine. These results illustrate a potentially useful model system to study Pentostam resistance in Leishmania and suggest that Pentostam resistance in vitro may be independent of antimony toxicity.     

Baseline sensitivities to azoxystrobin and tebuconazole in Sclerotinia sclerotiorum isolates from sunflower in Iran related to sensitivities to carbendazim and iprodione

In this study, sensitivities of 156 Sclerotinia sclerotiorum isolates collected from sunflower fields of West Azarbaijan province, Iran, were assessed to carbendazim and iprodione, and the baseline sensitivities were established for azoxystrobin and tebuconazole. Resistance to carbendazim and iprodione was observed in 53.85% and 4.49% of the isolates, respectively. The 50% effective concentration (EC₅₀) values of azoxystrobin for the isolates ranged from 0.017 to 3.515 μg/ml with a mean of 0.330 μg/ml, and 8.97% of the strains showed low levels of resistance to the fungicide. However, in the presence of salicylhydroxamic acid, all isolates were sensitive to azoxystrobin and EC₅₀ values ranged from 0.015 to 0.263 μg/ml with a mean of 0.086 μg/ml. All isolates were found to be sensitive to tebuconazole, and EC₅₀ values ranged from 0.003 to 0.177 μg/ml with a mean of 0.036 μg/ml. Among the multiple-resistant isolates, the strains exhibiting resistance to both carbendazim and iprodione were detected in the highest frequency (4.49%). No correlation was observed between mycelial growth and aggressiveness with fungicide sensitivity of the isolates suggesting the absence of fitness cost associated with resistance to the studied fungicides. The results indicated that iprodione, azoxystrobin and tebuconazole could be effectively used in rotation or mixture in spray programmes to manage S. sclerotiorum in the region. The baselines established for azoxystrobin and tebuconazole would be useful in monitoring the fungal populations in the province to assess possible shifts in fungicide sensitivity of S. sclerotiorum isolates in the future.     

A spreading colony formed method for rapid evaluation of dicarboximide fungicides resistance level of field tobacco brown spot disease

A new method (spreading colony formed method) for rapidly identifying and evaluating the dicarboximide fungicides resistance level of field tobacco spot brown disease caused byAlternaria longipes was developed. Two typical colonies with distinct differences in colony morphology on media containing 5 μg/ml dicarboximide fungicides dimethachlon (CAS registration number: 24096-53-5) were discovered by using this method. The two typical colonies were named spreading colony and dense pad colony, respectively. Isolates (250) ofA. longipes were quickly separated by this method, and their growth properties (including the sensitivity to dimethachlon, the cross-resistance to phenylpyrroles fludioxonil and hyphal development) were examined. Our results indicated that (1) monospore isolates from spreading colonies and dense pad colonies were respectively resistant and sensitive to dimethachlon; (2) resistant and sensitive isolates formed respectively spreading colonies and dense pad colonies on dimethachlon media. Furthermore, molecular experiments confirmed the spreading colony formed method reliable. In conclusion, field resistant isolates and resistant situation in population level of field tobacco spot brown disease could be exactly and timely determined and evaluated by spreading colony formed method.     

Gonococcal Antibiotic Resistance in Los Angeles

One hundred and thirty-five gonococcal isolates collected from Los Angeles in 1972 were studied for antibiotic susceptibility to penicillin, ampicillin, carbenicillin, tetracycline, minocycline, doxycycline and spectinomycin. Only 12 percent of the isolates were sensitive to 0.05 μg per ml of penicillin while 35 percent required at least 0.5 μg per ml for inhibition of growth. The results were slightly better with ampicillin and nearly the same with carbenicillin. Nineteen percent of the isolates required at least 1.0 μg per ml of tetracycline for inhibition of growth and the results were similar with either minocycline or doxycycline. Forty-nine percent were sensitive to 2.0 μg per ml spectinomycin, but 37 percent required at least 8.0 μg per ml for inhibition of growth.In this study nine of eleven isolates resistant to 1.0 μg per ml of tetracycline were also resistant to both penicillin and spectinomycin. Six came from endocervical sites of female patients who contributed only 37 percent of the total number of isolates studied.Correlation between the agar dilution and disc diffusion methods was satisfactory with penicillin but not with ether tetracycline or spectinomycin.     

Resistance of Botrytis cinerea to dicarboximide fungicides in protected crops

Dicarboximide fungicides have been used for the control of grey mould in protected crops in Crete since 1977. During the 1980 growing season a decline of their efficacy was observed. In successive surveys carried out in May 1980, February 1981 and May 1981 in 28, 10 and 13 plastic houses repectively, a considerable proportion of resistant strains was found. From each of the plastic houses sampled mostly either only resistant or only sensitive strains were isolated. In three of the plastic houses with resistant strains there was an acute disease control problem. The ED₅₀ of 15 resistant strains studied was in the area of 3·5 μg/ml vinclozolin as compared with 0·2 μg/ml for the wild type strains. The vinclozolin-resistant strains were also resistant to procymidone, iprodione, and dicloran. In most of the cases strains resistant to vinclozolin were also resistant to benomyl and strains sensitive to vinclozolin were also sensitive to benomyl. In the absence of fungicide, resistant strains grew more slowly on PDA than sensitive ones, but spores germinated equally well. Vinclozolin (0·75 mg a.i./ml) did not protect eggplant seedlings against resistant strains but gave satisfactory control of sensitive ones.     

Isolation and characterization of DNA-dependent RNA polymerase A, B and C from rat brain nuclei

Abstract— DNA-dependent RNA polymerase activities were solubilized from the brain nuclei of young rats. Six forms of RNA polymerases were distinguished on DEAE-Sephadex A-25 chromatography and designated A, BI, BII, CI, CII, and Oil by their sensitivities to α-amanitin. CII enzyme was shown to derive from CIII enzyme by serine-protease digestion. CI enzyme was also suggested to be a product of a proteolytic process. Using a DNA template, enzyme A was completely resistant to α-amanitin; BI and BII enzymes were equally sensitive to this toxin (50% inhibition at 0.006 μg/ml); while C enzymes showed intermediate sensitivity (50% inhibition at 30 μg/ml). When poly[d(A-T)] was used as a template, α-amanitin sensitivities were altered in A, CI, CII, and CIII enzymes without any change in the BII enzyme. CI, CII and CIII enzymes were greatly stimulated by poly[d(A-T)], whereas A and BII enzymes were only slightly stimulated. All six forms of RNA polymerases were extensively characterized with respect to their ammonium sulphate optima, effects of divalent metal ions, template requirements and pH optima, using DNA and poly[d(A-T)] as templates. The results show new findings in several properties and supply basic data for discussion and future studies on RNA metabolism of the brain.     

大肠埃希菌高产AmpC酶的检测及耐药性分析

目的了解深圳市人民医院高产AmpC酶大肠埃希菌的存在现状及其耐药性。方法采用Tris-ED-TA纸片裂菌法检测148株大肠埃希菌的AmpC酶。用琼脂稀释法测定产酶株对11种抗生素的最低抑菌浓度（MIC）。结果 148株大肠埃希菌中6株检出AmpC酶,检出率为4.1%。所有产AmpC酶大肠埃希菌对亚胺培南敏感,MIC为0.12～0.25μg/ml;对头孢吡肟的MIC值也较低,为0.5～32.0μg/ml,仅1株耐头孢吡肟。所有产AmpC酶大肠埃希菌对头孢西丁耐药,MIC为32～128μg/ml;对氨苄西林/舒巴坦、阿莫西林/克拉维酸和哌拉西林/他唑巴坦等加酶抑制剂复合物耐药性较强。结论深圳市人民医院大肠埃希菌高产AmpC酶检出率为4.1%。产酶株对多种抗生素耐药性较高。     

Detection and molecular characterization of carbendazim-resistant Colletotrichum truncatum Isolates causing anthracnose of soybean in Thailand

A loss of fungicide efficacy, particularly for carbendazim, was noted in soybean fields in Thailand and was considered to be due to the development of Colletotrichum truncatum resistance. The carbendazim sensitivity of C. truncatum populations isolated from various soybean fields in Thailand was thus evaluated with in vitro sensitivity assays and molecular characterization of mutations in the sequences of the ß₂-tubulin (TUB2) gene that confer carbendazim resistance in the pathogen. Among 52 isolates, 46 isolates were classified as highly resistant (HR) to carbendazim (EC₅₀ > 1,000 µg/ml). All HR isolates grew on PDA amended with carbendazim at 1,000 µg/ml. Six isolates were classified as carbendazim sensitive (S) (EC₅₀ < 1 µg/ml). Mycelial growth on PDA amended with 1 µg/ml carbendazim was inhibited by over 50% compared with growth on PDA alone. When a partial TUB2 gene from the isolates was amplified and analysed using predicted amino acid sequences, an alteration from glutamic acid to alanine at codon 198 (E198A) was found in 45 HR isolates for which the EC₅₀ was higher than 2000 µg/ml. This mutation resulted from a nucleotide substitution from adenine to cytosine (GA G → GC G). The other HR isolate, CtPhS_1, with EC₅₀ of 1,127 µg/ml, had an alteration at codon 200 (F200Y) (TT C → TA C).     

Impact of reduced vancomycin susceptibility on the therapeutic outcome of MRSA bloodstream infections

Background

Resistance to anti-tuberculosis drugs is a serious public health problem. Multi-drug resistant tuberculosis (MDR-TB), defined as resistance to at least rifampicin and isoniazid, has been reported in all regions of the world. Current phenotypic methods of assessing drug susceptibility of M. tuberculosis are slow. Rapid molecular methods to detect resistance to rifampicin have been developed but they are not affordable in some high prevalence countries such as those in sub Saharan Africa. A simple multi-well plate assay using mycobacteriophage D29 has been developed to test M. tuberculosis isolates for resistance to rifampicin. The purpose of this study was to investigate the performance of this technology in Kampala, Uganda.

Methods

In a blinded study 149 M. tuberculosis isolates were tested for resistance to rifampicin by the phage assay and results compared to those from routine phenotypic testing in BACTEC 460. Three concentrations of drug were used 2, 4 and 10 μg/ml. Isolates found resistant by either assay were subjected to sequence analysis of a 81 bp fragment of the rpoB gene to identify mutations predictive of resistance. Four isolates with discrepant phage and BACTEC results were tested in a second phenotypic assay to determine minimal inhibitory concentrations.

Results

Initial analysis suggested a sensitivity and specificity of 100% and 96.5% respectively for the phage assay used at 4 and 10 μg/ml when compared to the BACTEC 460. However, further analysis revealed 4 false negative results from the BACTEC 460 and the phage assay proved the more sensitive and specific of the two tests. Of the 39 isolates found resistant by the phage assay 38 (97.4%) were found to have mutations predictive of resistance in the 81 bp region of the rpoB gene. When used at 2 μg/ml false resistant results were observed from the phage assay. The cost of reagents for testing each isolate was estimated to be 1.3US$ when testing a batch of 20 isolates on a single 96 well plate. Results were obtained in 48 hours.

Conclusion

The phage assay can be used for screening of isolates for resistance to rifampicin, with high sensitivity and specificity in Uganda. The test may be useful in poorly resourced laboratories as a rapid screen to differentiate between rifampicin susceptible and potential MDR-TB cases.