Comparison of direct antimicrobial susceptibility testing methods for rapid analysis of bronchial secretion samples in ventilator-associated pneumonia

Two hundred and fifty tracheal aspirates were subjected to direct antimicrobial susceptibility testing by disk diffusion, Etest and inoculation on antibiotic-enriched MacConkey agar plates. Results were compared with those obtained using an automated system on microorganisms recovered from standard quantitative culture. A total of 255 microorganisms were isolated from 194 positive samples by the standard quantitative procedure. A total of 85.1%, 82.5% and 72.5% agreement between direct disk diffusion, Etest and antibiotic-enriched MacConkey agar plates, respectively, and the standard procedure was observed in 64 microorganisms obtained from monomicrobial cultures that corresponded to 240 individual microorganism-antimicrobial agent combinations. Three (1.3%) and four (1.7%) very major errors for direct disk diffusion and Etest methods were observed, respectively. The antibiotic-enriched MacConkey agar plate method compared with the standard procedure demonstrated an unacceptable rate of very major (6.7%) and major errors (14.2%). Clinical evaluation of direct susceptibility tests based on the speculative impact on clinical practice by guiding patient's early treatment, if all positive cultures corresponded to infection, was correct in 79.9% for the direct disk diffusion test, 77.8% for the direct Etest method and 68.0% for antibiotic-enriched MacConkey agar plates. Direct diffusion tests (Etest or disk diffusion) applied on respiratory samples are rapid techniques that provide results comparable with standard antimicrobial susceptibility testing in <24 h.     

Setting interpretive breakpoints for antimicrobial susceptibility testing using disk diffusion

Antimicrobial susceptibility testing plays a key role in clinical microbiology. The disk diffusion test dates back to the 1940s and became standardised from the 1950s, with the International Collaborative Study (ICS) and National Committee for Clinical Laboratory Standards (NCCLS) as the two major standards. Interlaboratory variation of disk test results was recognised early but has never been dealt with in a satisfactory manner. The error-rate bounded method was described in 1974 and its role is discussed. Species-specific susceptibility interpretation was coined in 1980 for Proteus mirabilis and chloramphenicol. In the late 1970s, more extensive use of species-specific breakpoints was introduced in Lund (Sweden). At the same time, P. Mouton constructed species-specific regression lines and pointed out the difficulties with narrow ranges of minimal inhibitory concentration (MIC) values. A more general use of species-specific regression lines was made possible with single-strain regression analysis, using one well-defined strain tested in disk diffusion with a range of disk contents. This method made it possible to calibrate the disk test in an individual laboratory. Other methods to achieve such calibration are also described. A recent method, ‘MIC-coloured zone diameter histogram-technique’, has proven useful for the validation of species-specific interpretive breakpoints. The microbiological breakpoint proposed by Williams in 1990 has experienced a renaissance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off value (ECOFF). MIC and zone diameter distributions with accompanying ECOFFs for species-antimicrobial combinations are published on the EUCAST website. A method for the reconstruction of wild-type zone diameter populations, namely normalised resistance interpretation, is described. This method can produce resistance figures that are truly comparable between laboratories.     

Comparison of E-test and disk diffusion assay to evaluate resistance of Helicobacter pylori isolates to amoxicillin, clarithromycin, metronidazole and tetracycline in Costa Rica

MIC distribution and susceptibility to four antimicrobial agents were determined by E-test for 94 Helicobacter pylori isolates from Costa Rica. Disk diffusion was evaluated as an alternative method to determine susceptibility and compared with the E-test results by linear regression analysis and an error-rate bounded method. Thirty-eight (40.4%) of the isolates were resistant to metronidazole, 5.3% to clarithromycin and 5.3% to amoxicillin. No isolate was resistant to tetracycline. Multiple resistance was found in 4.3% of the isolates. H. pylori isolates were categorised as resistant to amoxicillin, clarithromycin and tetracycline when inhibition diameters were less than 25, 21 and 25 mm, respectively, in the disk diffusion assay. A breakpoint diameter for metronidazole with disk diffusion could not be firmly established.     

355株金黄色葡萄球菌药敏试验结果分析

目的：探讨金黄色葡萄球菌（S-aureus）对抗菌药物的敏感性,为临床应用抗生素提供科学依据。方法：按《全国临床检验操作规程》对S—aureus进行培养分离鉴定,采用纸片扩散法进行药敏试验,参照美国国家临床实验室标准化委员会标准（NCCLS）读取结果。结果：8年来从各类临床标本中分离出355株S—aureus,其中耐甲氧西林金黄色葡萄球菌（MRSA）为140株。金黄色葡萄球菌对万古霉素、利福平、头孢哌酮、丁胺卡那霉素、头孢唑啉、庆大霉素、环丙沙星、氟哌酸、氨苄西林、青霉素G、苯唑西林的敏感率分别为100．0％、97．8％、76．1％、76．1％、69．9％、60．3％、60．1％、54．7％、3．9％、2．1％、0．9％。结论：MRSA感染呈上升趋势,青霉素类已不能成为治疗S—aureus感染的一线药物,万古霉素、利福平可作为治疗S—aureus感染的首选药物。     

In vitro susceptibility of Achromobacter spp. isolates: comparison of disk diffusion,Etest and agar dilution methods

In this study, we analysed the antimicrobial susceptibility of 92 strains of Achromobacter spp. isolated from clinical samples to 18 antimicrobial agents. The disk diffusion method and Etest were compared with the agar dilution method, and the breakpoints of susceptibility and resistance for the disk diffusion method for the antimicrobials tested were determined. The most active antibiotics were piperacillin, piperacillin/tazobactam and the carbapenems. By applying the linear least-squares regression method, breakpoints could be established for antibiotics active against this genus such as imipenem, meropenem, ertapenem and trimethoprim/sulfamethoxazole (SXT). Other active antibiotics, such as piperacillin and minocycline, could be tested by the Etest method. The less active antibiotics such as gentamicin, doxycycline and tetracycline could be tested by the disk diffusion method. For the rest of the antimicrobial agents tested, breakpoints could not be established owing to the high percentage of errors and/or the poor linear regression coefficient obtained. Therefore, these antimicrobial agents should be tested by minimal inhibitory concentration determination. In summary, we recommend the following zone diameter breakpoints for resistant and susceptible, respectively: ≤11 mm and ≥22 mm for imipenem; ≤13 mm and ≥24 mm for meropenem; ≤17 mm and ≥24 mm for ertapenem; ≤15 mm and ≥21 mm for gentamicin; ≤27 mm and ≥28 mm for SXT; ≤20 mm and ≥29 mm for tetracycline; and ≤20 mm and ≥24 mm for doxycycline.     

Interpreting streptomycin susceptibility test results for Salmonella enterica serovar Typhimurium

Resistance or susceptibility of Salmonella enterica to streptomycin is widely used as an epidemiological marker. However, there is no clear consensus on the interpretation of streptomycin susceptibility test results. Comparison of results obtained with the Clinical and Laboratory Standards Institute (CLSI) disk diffusion method, the minimum inhibitory concentration (MIC) determined by Etest and streptomycin resistance genotype for 90 isolates of S. enterica serovar Typhimurium suggests that appropriate interpretive criteria for MIC results are susceptible at ≤8 mg/L and resistant at ≥16 mg/L. For CLSI disk diffusion, we propose susceptible at a zone diameter ≥13 mm and resistant at ≤10 mm.     

Antimicrobial susceptibility of Neisseria gonorrhoeae isolates determined by the agar dilution,disk diffusion and Etest methods: comparison of results using GC agar and chocolate agar

Although the use of GC agar for determining Neisseria gonorrhoeae antimicrobial susceptibilities is suggested by Clinical and Laboratory Standard Institute (CLSI) guidelines, chocolate agar is still used in some regions owing to its low cost and availability. To determine the differences in susceptibilities determined using GC and chocolate agars, 163 non-duplicate N. gonorrhoeae isolates were tested. Minimum inhibitory concentrations (MICs) and percent susceptibilities determined using the GC agar dilution method, respectively, were as follows: ceftriaxone, 0.004–0.125 mg/L, 100%; cefixime, 0.002 mg/L to >32 mg/L, 98.2%; and ciprofloxacin, 0.002 mg/L to >32 mg/L, 3.1%. Comparison of ceftriaxone MICs determined by the Etest using GC agar and chocolate agar showed that use of GC agar tended to result in lower MICs than GC agar dilution, whilst use of chocolate agar tended to result in higher MICs (concordance, 55.8% and 82.8%, respectively). Disk inhibition zones obtained using GC agar and chocolate agar (and their correlation coefficients) were, respectively: ceftriaxone, 35–55 mm and 25–50 mm (0.46); ciprofloxacin, 6–55 mm and 6–43 mm (0.84); and penicillin, 6–47 mm and 6–50 mm (0.93). Use of chocolate agar with the disk diffusion method for ceftriaxone was associated with a 5.5% false resistance rate. In summary, compared with GC agar, susceptibility testing using chocolate agar tends to yield higher MICs with the Etest and smaller disk inhibition zones with disk diffusion methods. Clinical microbiology laboratories should strictly adhere to CLSI recommendations by using GC agar instead of chocolate agar when performing susceptibility testing for N. gonorrhoeae.     

Neo-Sensitab纸片扩散法与NCCL SM27-A微量稀释法测定97株酵母对抗真菌药物敏感性的比较

目的比较Neo-Sensitab纸片扩散法和NCCLS微量稀释法的一致性,评价该商品化方案的稳定性。方法同时采用NCCLSM27-A微量稀释法和Neo-Sensitab纸片扩散法测定临床分离的97株酵母对两性霉素B(AMB)、氟康唑(FCZ)、5-氟胞嘧啶(5-FC)、酮康唑(KCZ)和伊曲康唑(ITC)等五种常用抗真菌药物的敏感性。结果两种方法的药敏结果一致率分别为AMB100%,FCZ94.8%,5-FC93.8%,KCZ75.3%和ITC84.5%。结论纸片扩散法和微量稀释法药敏测试结果一致率较高,以FCZ、AMB和5-FC最佳,该方法检测敏感株的准确率较高,而在区分耐药株和中介株方面有所不足。     

Evaluation of the in vitro activity of caspofungin against bloodstream isolates of Candida species from cancer patients: comparison of Etest and NCCLS reference methods

The in vitro activity of caspofungin (CAS) was compared with the activity of fluconazole, itraconazole and amphotericin B against 178 bloodstream Candida spp. from cancer patients. The activities were assessed using the reference NCCLS M-27A microdilution method and the Etest method. With both the NCCLS microdilution reference method and the Etest method, CAS was the most active agent (MIC90s 0.19–0.5 mg/l) against Candida albicans, C. glabrata and C. tropicalis. CAS showed substantial activity against azole-resistant Candida. The percentages of agreement within ±2 dilutions between the NCCLS reference microdilution method and Etest MICs ranged from 81 to 97%. CAS showed good in vitro activity against invasive azole-susceptible and azole-resistant Candida isolates. The CAS Etest MICs correlated well with the NCCLS reference MICs and may provide more choice for laboratories in assessing the activity of antifungal agents. The clinical correlation of these in vitro observations needs to be established.     

Formation of Pseudomonas aeruginosa inhibition zone during tobramycin disk diffusion is due to transition from planktonic to biofilm mode of growth

Pseudomonas aeruginosa PAO1 (tobramycin MIC?=?0.064 µg/mL) was used to perform agar diffusion tests employing tobramycin-containing tablets. Bacterial growth and formation of inhibition zones were studied by stereomicroscopy and by blotting with microscope slides and staining with methylene blue, Alcian blue and a fluorescent lectin for the P. aeruginosa PSL, which was studied by confocal laser scanning microscopy. Diffusion of tobramycin from the deposit was modelled using a 3D geometric version of Fick's second law of diffusion. The time-dependent gradual increase in the minimum biofilm eradication concentration (MBEC) was studied using a Calgary Biofilm Device. The early inhibition zone was visible after 5 h of incubation. The corresponding calculated tobramycin concentration at the border was 1.9 µg/mL, which increased to 3.2 µg/mL and 6.3 µg/mL after 7 h and 24 h, respectively. The inhibition zone increased to the stable final zone after 7 h of incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained less than ca. 64 cells and production of polysaccharide matrix including PSL had begun; thereafter, the small bacterial aggregates were killed by tobramycin. Bacteria at the border of the stable inhibition zone and beyond continued to grow to a mature biofilm and produced large amount of polysaccharide-containing matrix. Formation of the inhibition zone during agar diffusion antimicrobial susceptibility testing is due to a switch from a planktonic to biofilm mode of growth and gives clinically important information about the increased antimicrobial tolerance of biofilms.     

Antimicrobial activity and chemical composition of some essential oils

In this study the composition and antimicrobial properties of essential oils obtained from Origanum onites, Mentha piperita, Juniperus exalsa, Chrysanthemum indicum, Lavandula hybrida, Rosa damascena, Echinophora tenuifolia, Foeniculum vulgare were examined. To evaluate the in vitro antibacterial activities of these eight aromatic extracts; their in vitro antimicrobial activities were determined by disk diffusion testing, according to the NCCLS criteria. Escherichia coli (ATTC 25922), Staphylococcus aureus (ATCC 25923) and Pseudomonas aeruginosa (ATTC 27853 were used as standard test bacterial strains. Origanum onites recorded antimicrobial activity against all test bacteria, and was strongest against Staphylococcus aureus. For Rosa damascena, Mentha piperita and Lavandula hybrida antimicrobial activity was recorded only to Staphylococcus aureus. Juniperus exalsa, and Chrysanthemum indicum exhibited antibacterial activities against both Staphylococcus aureus and Escherichia coli. We also examined the in vitro antimicrobial activities of some components of the essential oils and found some components with antimicrobial activity.     

Comparison of Etest, agar dilution, broth microdilution and disk diffusion methods for testing in vitro activity of levofloxacin against Staphylococcus spp. isolated from neutropenic cancer patients

The susceptibility to levofloxacin of 194 consecutive staphylococcal (45 Staphylococcus aureus and 149 coagulase-negative staphylococci) isolates from neutropenic patients was determined by Etest and the results compared with those obtained using NCCLS-methods (broth microdilution, agar dilution and disk diffusion). Overall agreement at +/- 1log(2) dilution for Etest compared with broth microdilution and agar dilution was 99.0 and 83.5%, respectively. The Etest category agreement with broth microdilution and disk diffusion was 95.9 and 89.7%, respectively. Comparison of categories with Etest and agar dilution method gave only 67.0% absolute categorical agreement, with 29.9% minor errors and 10.7% major errors. No very major errors occurred by the four methods tested. Our results show that Etest is a valid alternative to the reference NCCLS-methods for monitoring the clinical usefulness of levofloxacin against staphylococci isolates from neutropenic patients.     

Multiple pathways towards reduced membrane potential and concomitant reduction in aminoglycoside susceptibility in Staphylococcus aureus

Staphylococcus aureus is responsible for life-threatening and difficult-to-treat infections worldwide and antimicrobial resistance is an increasing concern. Whilst acquired resistance has been widely studied, little is known of the contributions from chromosomal determinants that upon inactivation may reduce the susceptibility of S. aureus to antibiotics. The aim of this study was to identify genetic determinants that upon inactivation reduce aminoglycoside susceptibility in S. aureus. The Nebraska Transposon Mutant Library of 1920 single-gene inactivations in S. aureus strain JE2 was screened for reduced susceptibility to gentamicin. Nine mutants were confirmed by Etest to display between 2- and 16-fold reduced susceptibility to this antibiotic. All of the identified genes were associated with the electron transport chain and energy metabolism. Four mutant strains (menD, hemB, aroC and SAUSA300_0355) conferred the largest decrease in gentamicin susceptibility and three exhibited a small colony variant phenotype, whereas the remaining mutants (qoxA, qoxB, qoxC, ndh and hemX) displayed colony morphology similar to the wild-type. All of the mutants, except hemX, displayed reduced membrane potential suggesting that reduced uptake of gentamicin is the predominant mechanism leading to reduced susceptibility. The results of this study demonstrate that S. aureus possesses multiple genes that upon inactivation by mutagenesis reduce the membrane potential and thereby reduce the lethal activity of gentamicin.     

淋球菌对头孢曲松的敏感性及淋球菌多抗原测序分型研究

目的了解中国广东地区2010—2016年淋球菌对头孢曲松的敏感性以及相应菌株的淋球菌多抗原测序分型(NGMAST)基因型别。方法 2010—2016年在广东省疾病预防控制中心收集的285株淋球菌,经分离纯化及鉴定后,采用美国临床实验室标准化委员会(CLSI)推荐的琼脂稀释法测定其对头孢曲松的最小抑菌浓度(minimal inhibitory concentration,MIC);菌株培养后利用试剂盒提取DNA,并进行淋球菌多抗原测序分型(NG-MAST)。结果本次测试的285株淋球菌除1株MIC>0.25μg/mL外,其他都属于敏感菌株(CLSI敏感标准为≤0.25μg/mL),MIC≥0.06μg/mL的菌株比例为63.2%,其中2016年度MIC≥0.06μg/mL菌株比例为44.4%,2010年MIC≥0.06μg/mL菌株比例为70.6%。NG-MAST分型研究显示,285株淋球菌共有166个型别,菌株多样性较高,其中73种为已知型别,93种为新型别。测定的所有菌株中主要包括ST568(n=13),ST270(n=9),ST421(n=7),ST2288(n=5),ST1731(n=4),ST1766(n=4),ST1866(n=4),ST1870(n=4),ST1053(n=4),ST2318(n=4),ST5990(n=4),ST1614(n=4),ST1866(n=3)等。相同NG-MAST型别的菌株具有相同或相近的MIC值。结论广东地区淋球菌对头孢曲松MIC≥0.06μg/mL菌株比例较高,需要对此进行长期监测。7年间菌株的优势型别有较大变化,显示该地区性网络可能发生较大波动。NG-MAST分型可以作为分子生物学标记用于淋球菌耐药监测。     

动物及健康人肠道共生的大肠埃希菌耐药性及产生原因分析

目的了解目前动物和人肠道共生的大肠埃希菌耐药性,分析其产生的原因。方法从甘肃、湖北、北京、山东、四川等地相对封闭的养殖场及养殖场附近的健康人群采集鸡、猪、鱼、人粪便样本,分离大肠埃希菌。用AP I20E鉴定条鉴定怀疑为大肠埃希菌的菌种,采用KB纸片法检测生化鉴定为大肠埃希菌的菌株的耐药性,利用WHONET 5.3软件进行药敏试验数据分析。用脉冲场凝胶电泳技术检测具有相似耐药谱型的大肠埃希菌菌株间的同源性。结果①共收集571株大肠埃希菌,其中从海水养殖鱼类和淡水养殖鱼类分离31株,另从海水养殖鱼类和淡水养殖鱼类分离嗜水气单胞菌株57株;②来源鸡的大肠埃希菌对所有检测的抗菌药物的耐药率最高;③除β-内酰胺类抗生素和阿米卡星外,鸡、猪和人来源菌株的耐药率表现为高、中、低现象,对老的一些抗菌药物和喹诺酮类抗菌药物尤其明显;不同地区分离株的耐药性也有较大的差异;④本次调查首次在国内养鸡场发现产ESBL大肠埃希菌,并且非常多见;⑤嗜水气单胞菌和大肠埃希菌的耐药性有较大的差异,47株耐药谱型相近的菌株中发现了三组基因水平同源性菌株(相似度大于95%)。结论①从不同地区、不同种类动物分离菌株的耐药性不同,与抗生素的使用情况相关。目前我国家禽养殖业滥用喹诺酮类和β-内酰胺类尤其是三代头孢菌素类抗生素的现象较为普遍,应该加以严格控制;②不同菌种的生物学特性不同,导致耐药性不同;③同源性分析发现耐药菌株可在同一种类动物间传播,不同类动物的大肠埃希菌之间可能存在耐药基因的水平传播;④应当加强养殖动物分离的大肠埃希菌耐药性监测。     

抗菌药物纸片法敏感性试验的质量评价与改进

目的　采用实例分析 ,系统评价药敏试验的质量 ,促进药敏试验质量的持续改进。方法　利用WHONET5软件对北京、湖北两地采用纸片扩散法得到的质控资料和细菌耐药性监测数据进行分析。结果　采用不同质控菌株对同一抗生素药敏纸片进行测试 ,有助于了解、分析和确认测量值偏移原因。对质控结果和耐药性调查时 ,试验人员读取数据的人为因素会造成直方图分布偏移。有时质控试验的结果符合要求 ,但并不能保证对所有患者分离株的药敏测试都能得到准确的结果。当发现不典型或不稳定的结果时 ,应重复试验和 /或鉴定过程 ,以确保结果的准确。结论　利用 WHONET5软件 ,及时对日常细菌耐药性监测数据进行回顾性分析 ,可持续改进药敏试验的质量 ,保证试验数据的可靠性。     

Vibriocidal activity of certain medicinal plants used in Indian folklore medicine by tribals of Mahakoshal region of central India

Objectives:

Screening of the medicinal plants and determination of minimum inhibitory concentration (MIC) against Vibrio cholerae and Vibrio parahaemolyticus.

Materials and Methods:

A simple in vitro screening assay was employed for the standard strain of Vibrio cholerae, 12 isolates of Vibrio cholerae non-O1, and Vibrio parahaemolyticus. Aqueous and organic solvent extracts of different parts of the plants were investigated by using the disk diffusion method. Extracts from 16 medicinal plants were selected on account of the reported traditional uses for the treatment of cholera and gastrointestinal diseases, and they were assayed for vibriocidal activities.

Results:

The different extracts differed significantly in their vibriocidal properties with respect to different solvents. The MIC values of the plant extracts against test bacteria were found to be in the range of 2.5-20 mg/ml.

Conclusions:

The results indicated that Lawsonia inermis, Saraca indica, Syzygium cumini, Terminalia belerica, Allium sativum, and Datura stramonium served as broad-spectrum vibriocidal agents.     

Extended antimicrobial resistance screening of the dominant faecal Escherichia coli and of rare resistant clones

Fifty faecal samples from healthy adults were grown on MacConkey agar and three pink colonies were subcultured, identified to species level and their antimicrobial susceptibility determined. Forty-seven samples yielded 141 isolates of Escherichia coli that were susceptible to most antimicrobials. Resistance was noted for ampicillin (30.5%), chloramphenicol (12.1%), tetracycline (23.4%), trimethoprim (24.8%) and co-trimoxazole (22.7%). A direct faecal plating method was used for extended resistance screening with E. coli as the indicator organism. Zone breakpoints were determined using normalised resistance interpretation and gave similar susceptibility results. Eighty-eight isolates of E. coli from within the zones of inhibition revealed four times more antimicrobial resistance. Extended antimicrobial resistance screening both provides the susceptibility profile of the dominant E. coli isolate and detects greater resistance in rare isolates.     

Results of an interlaboratory test on antimicrobial susceptibility testing of bacteria from animals by broth microdilution

A standard operating procedure for the determination of minimum inhibitory concentrations (MICs) of antimicrobial agents by the broth microdilution method was developed and evaluated for its fitness for use in an interlaboratory ring trial involving 46 routine diagnostic laboratories. All laboratories tested five strains (one reference strain and four field strains) against a total of 22 different antimicrobial agents. Gram-negative strains were tested against 16 different antimicrobial agents and Gram-positive strains against 14 different antimicrobial agents. Tests were performed once a week for three consecutive weeks. At least 80% of the results determined by 35 of the 46 participating laboratories were within the expected range (mode MIC ± 1 dilution step), with the 18 participating laboratories experienced in MIC determination showing a slightly higher mean percentage of accurate results (89.3% reproducible results) than the 28 non-experienced laboratories (86.7% reproducible results). The most accurate results were obtained for the Escherichia coli field strain, whilst the results for the Streptococcus uberis field strain showed the highest error rate. Among the 22 antimicrobial agents tested, the highest variabilities in the results (mean value for all antimicrobial agents 12.3%) were recorded for ceftiofur (27.8%), penicillin G (20.8%) and cefoperazone (20.6%).