Rapid identification and antibiotic susceptibility testing of Yersinia pestis using bioluminescent reporter phage

The rapid identification and antibiotic susceptibility testing of Yersinia pestis is paramount for a positive prognosis. We previously engineered a Y. pestis-specific 'bioluminescent' reporter phage for the identification of Y. pestis. In this study, we generated an improved reporter phage and evaluated the ability of this phage to provide direct and rapid susceptibility testing. Compared to the first generation reporter, the second generation reporter exhibited a 100-fold increase in signal strength, leading to a 10-fold increase in assay sensitivity. Y. pestis antimicrobial testing in the presence of the reporter elicited bioluminescent signals that were drug concentration-dependent, and produced susceptibility profiles that mirrored the standard CLSI method. The phage-generated susceptibility profiles, however, were obtained within hours in contrast to days with the conventional method.     

Differentiation of Klebsiella-Enterobacter (Aerobacter)-Serratia by biochemical tests and antibiotic susceptibility

Studies were undertaken for the differentiation of members of the Klebsiella-Enterobacter (Aerobacter)-Serratia division by biochemical tests and antibiotic susceptibility. A total of 67 cultures were tested. Strain identification was readily accomplished with the use of motility tests and arabinose fermentation. In addition, a practical schema, based on sensitivity pattern, proved valuable in the classification of the different strains. Most (if not all) Klebsiella strains were susceptible to cephalothin, and all were sensitive to colistin. Enterobacter strains were resistant to cephalothin but susceptible to colistin. In contrast to those other members of the group, all Serratia strains were resistant to both cephalothin and colistin. The combination of a limited number of biochemical reactions and single-disc sensitivity tests appears to be a logical approach for the tentative identification of Klebsiella-Enterobacter (Aerobacter)-Serratia strains.     

Parentage testing in alpacas (Vicugna pacos) using semi-automated fluorescent multiplex PCRs with 10 microsatellite markers

The aim of this study was to assess and apply a microsatellite multiplex system for parentage determination in alpacas. An approach for parentage testing based on 10 microsatellites was evaluated in a population of 329 unrelated alpacas from different geographical zones in Perú. All microsatellite markers, which amplified in two multiplex reactions, were highly polymorphic with a mean of 14.5 alleles per locus (six to 28 alleles per locus) and an average expected heterozygosity ( H _E) of 0.8185 (range of 0.698–0.946). The total parentage exclusion probability was 0.999456 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.999991 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. In a case test of parentage assignment, the microsatellite panel assigned 38 (from 45 cases) offspring parentage to 10 sires with LOD scores ranging from 2.19 × 10⁺¹³ to 1.34 × 10⁺¹⁵ and Δ values ranging from 2.80 × 10⁺¹² to 1.34 × 10⁺¹⁵ with an estimated pedigree error rate of 15.5%. The performance of this multiplex panel of markers suggests that it will be useful in parentage testing of alpacas.     

Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.     

62株阴沟肠杆菌的生化特性和药敏结果

目的探讨临床分离阴沟肠杆菌的实用鉴定方法.方法对VITEK-32微生物分析仪鉴定所得的62株阴沟肠杆菌的生化反应和药敏结果作回顾性分析.结果 62株阴沟肠杆菌用鉴定卡鉴定的可信度在94%～99%,其中51株为99%、占82.3%,4株为95%、占6.5%,其他7株、占11.2%.阴沟肠杆菌对除外亚胺培南的20种抗生素均有不同程度的耐药.结论脲酶、侧金盏花醇、精氨酸水解和赖氨酸脱羧等试验对肠杆菌属中5个常见菌种(阴沟、坂崎、产气、聚团和格高菲肠杆菌)的鉴别有重要意义.     

Mixed-infection of antibiotic susceptible and resistant Helicobacter pylori isolates in a single patient and underestimation of antimicrobial susceptibility testing

Antibiotic resistance among Helicobacter pylori has been increasing worldwide and has begun to affect the overall efficacy of current antibiotic regimens adversely. We examined 220 pairs of H. pylori isolates obtained from both the antrum and corpus of separate patients; 109 (50%) harbored antibiotic-resistant H. pylori: amoxicillin (0.5%), clarithromycin (5.9%), furazolidone (1.4%), metronidazole (45.5%), nitrofurantoin (1.4%), and tetracycline (6.8%). Heteroresistance among the two biopsy sites from each patient was present in 41 of the 109 patients (38%) with antibiotic resistant H. pylori (e.g. 34% with resistant strains would be misclassified as susceptible if a biopsy of the antrum alone used for antimicrobial susceptibility testing). DNA fingerprinting genotype analysis was carried out on the 41 pairs of isolates with heteroresistance. While different patients had different fingerprinting patterns, each pair of isolates showed identical or similar fingerprinting patterns. These results suggest that antibiotic-resistant H. pylori typically develop from pre-existing susceptible strain rather than coinfection with a different strain. The minor differences in genotype (degeneration of genotype) seen reflect one of the processes for development of genetic diversity in H. pylori. No biopsy single site can be considered representative for antimicrobial susceptibility testing.     

基于幼虫粪便的小菜蛾肠道细菌分离鉴定及其抗生素敏感性分析

【目的】昆虫肠道微生物对于其食物消化、生长发育以及环境适应性等方面都具有重要作用,本研究旨在探究小菜蛾 Plutella xylostella (L.)幼虫肠道可培养细菌的菌群结构及抗生素敏感性。【方法】对小菜蛾3龄幼虫粪便进行了细菌分离培养和16S rDNA分子鉴定,并采用纸片扩散法对之进行了药敏试验。【结果】小菜蛾3龄幼虫肠道有5属6种细菌,即蒙氏肠球菌 Enterococcus mundtii、欧文氏菌属 Erwinia 2种、成团泛菌 Pantoea agglomerans 、类芽孢杆菌Paenibacillus sp.和栖稻假单胞菌 Pseudomonas oryzihabitans,均与小菜蛾中肠细菌已知种不同,其中蒙氏肠球菌数量最多;蒙氏肠球菌、桃色欧文氏菌和成团泛菌对青霉素、氨苄青霉素、麦迪霉素、克拉霉素和洁霉素均不敏感,而对磷霉素、万古霉素和强力霉素等表现出相似的敏感性。【结论】小菜蛾幼虫肠道细菌菌群组成具有多样性,且存在天然耐药性现象。本研究为进一步开展小菜蛾幼虫肠道细菌微生物区系及功能细菌的研究提供了菌株材料和研究基础。     

Comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of Helicobacter cinaedi

The aim of this study was to establish a broth microdilution method for antimicrobial susceptibility testing of Helicobacter cinaedi and to assess the prevalence and mechanisms of fluoroquinolone resistance in Japanese clinical isolates. A broth microdilution method using modified Levinthal broth was developed and compared with the agar dilution method for testing susceptibility to ampicillin, gentamicin, tetracycline and ciprofloxacin. The minimum inhibitory concentrations obtained by these two methods were almost the same for all the antibiotics tested, demonstrating the broth microdilution method to be a suitable and reliable technique for antimicrobial susceptibility testing. A broth microdilution method for antimicrobial susceptibility test for H. cinaedi was established. This method is expected to help improve treatment.     

Quicolor: A novel system for rapid antibacterial susceptibility testing

Early determination of antibacterial susceptibility increases the success of therapy, decreases unnecessary use of antibacterials and side effects and lowers the overall healthcare costs. We have evaluated a rapid antibacterial susceptibility test, Quicolor (Salubris Inc., Massachusetts, USA), which is based on a rapid culture medium that indicates growth early by changing its colour. Quicolor proved to be a reliable rapid test for determining antibacterial susceptibility, having an overall agreement of 97.6% with the conventional CLSI sisk diffusion susceptibility test results. Between two methods overall agreement was 96.7% for Enterobacteriaceae, 96.8% for staphylococci and 94.2% for non-fermentative bacteria. There was only 0.6% major discrepancy in Enterobacteriaceae, 1.7% in staphylococci and 0.9% in non-fermentative bacteria. Since the test provides results in 3.5–6 h, it can provide the means to choose the right treatment regimen the same day the infectious agent is isolated.     

Cord blood volume reduction using an automated system (Sepax) vs. a semi-automated system (Optipress II) and a manual method (hydroxyethyl starch sedimentation) for routine cord blood banking: a comparative study

Background With the development of cord blood banking, solutions have to be found to solve the storage space problem, by reducing the volume of cord blood units (CBU). Methods We compared total nucleated cell (TNC) and CD34(+) cell counts before and after processing with three different CBU volume reduction methods used consecutively in our bank: a manual method based on hydroxyethyl starch sedimentation (HES) (n=447), a top-and-bottom (TB) semi-automated method (n=181) using Optipress II, and the Sepax automated method (n=213). Statistical analysis was done using t-tests, linear regression and Spearman correlation coefficients. Adjusted variables included TNC, CD34(+) cell counts, CD34(+) cell percentage and CB volume before processing. Results TNC recovery was higher with Sepax (80.3+/-7.7%) than with HES (76.8+/-9.1%) and TB (60.7+/-13.5%) (P<0.0001, both). It was higher with HES than with TB (P<0.0001). CD34(+) cell recovery was higher with Sepax (86+/-11.6%) than with HES (81.5+/-12.5%) and TB (82.0+/-17.7%) (P<0.008 and <0.0001, respectively) and results with HES and TB were not significantly different (P=0.7). Interestingly, with Sepax, TNC and CD34(+) cell recoveries were not correlated with pre-processing values (P=0.8 and 0.4, respectively). Discussion In conclusion, the Sepax volume reduction method allows higher TNC and CD34(+) cell recoveries.     

Detection of methicillin-resistance (mec-A) gene inStaphylococcus aureus strains by PCR and determination of antibiotic susceptibility

Methicillin-ResistantStaphylococcus aureus (MSRA) has become a frequent cause of serious infections. Extended hospitalization and antibiotic therapy have been identified as additional risk factors for MRSA carrier and infection. The aim of this study was to determine the incidence of MRSA infections in the hospitals affiliated to Hamedan University of Medical Sciences. SeventyS. aureus clinical strains were isolated from patients from June 2005 to June 2006 and examined by PCR and conventional microbiological tests. Then, the antibiotic susceptibility to methicillin/oxacillin and other antibiotics were performed by Disc Diffusion Agar (DDA). The results of this study showed that methicillin resistance gene was detected in 35 (50%) and 22 (31.4%) cases by PCR and DDA, respectively. The results of antibiotic susceptibility assays also showed there were high resistance MRSA strains to penicilin (100%), cloxacillin (91.4%), tetracycline (74.2%), cotrimoxazole (68.5%), erythromycin (68.5%) and less resistance to rifampin (11.4). Two MRSA also had decreased susceptibility to vancomycin. But the strains of Methicillin-SensitiveS. aureus (MSSA) showed high sensitivity to all antibiotics profiles except to penicillin (complete resistance). As a conclusion, the resistance to methicillin/oxacillin ofS. aureus in Hamedan hospitals has reached to 50% and they show multidrug resistance.     

Characterization of Renibacterium salmoninarum with reduced susceptibility to macrolide antibiotics by a standardized antibiotic susceptibility test

Three cohorts of juvenile and subadult Chinook salmon Oncorhynchus tshawytscha received multiple treatments with macrolide antibiotics for bacterial kidney disease (BKD) during rearing in a captive broodstock program. A total of 77 mortalities among the cohorts were screened for Renibacterium salmoninarum, the etiologic agent of BKD, by agar culture from kidney, and isolates from 7 fish were suitable for growth testing in the presence of macrolide antibiotics. The minimum inhibitory concentration (MIC) of erythromycin and azithromycin was determined by a modification of the standardized broth assay using defined medium. The American Type Culture Collection (ATCC) type strain 33209 exhibited a MIC of 0.008 microg m(-1) to either erythromycin or azithromycin. Isolates from 3 fish displayed MICs identical to the MICs for the ATCC type strain 33209. In contrast, isolates from 4 fish exhibited higher MICs, ranging between 0.125 and 0.250 microg ml(-1) for erythromycin and between 0.016 and 0.031 microg ml(-1) for azithromycin. Sequence analysis of the mutational hotspots for macrolide resistance in the 23S rDNA gene and the open reading frames of ribosomal proteins L4 and L22 found identical sequences among all isolates, indicating that the phenotype was not due to mutations associated with the drug-binding site of 23S rRNA. These results are the first report of R. salmoninarum with reduced susceptibility to macrolide antibiotics isolated from fish receiving multiple antibiotic treatments.     

Method of testing insecticide susceptibility of Stenotus rubrovittatus (Hemiptera: Miridae) by topical application

Appropriate droplet size and the preferred sex and age of insects for insecticides used in topical application were investigated to standardize the method of testing the susceptibility of Stenotus rubrovittatus (Matsumura). The overflow of acetone droplets applied on the notum of the thorax of S. rubrovittatus adults occurred when the droplet size exceeded 0.20 μl per insect, and the overflow occurred more frequently in males than in females. No effect of acetone solution on mortality was observed at less than 0.20 μl droplet size per insect within 72 h after treatment. The LD₅₀ values and relative potencies of fenitrothion 24 and 48 h after treatment were almost the same in each sex. The fenitrothion susceptibility of males was twice higher than that of females. The fenitrothion susceptibility increased with age after emergence. However, there were small differences in LD₅₀ values and relative potencies between 4–7 and 8–11 days after emergence because the females 4–11 days after emergence had a small difference in body weight. These results led us to conclude that the recommended size of acetone droplets per insect is less than 0.20 μl and that it is suitable to use S. rubrovittatus females 4–11 days after emergence for topical application.     

CycleTrak: a novel system for the semi-automated analysis of cell cycle dynamics

Cell proliferation is crucial to tissue growth and form during embryogenesis, yet dynamic tracking of cell cycle progression and cell position presents a challenging roadblock. We have developed a fluorescent cell cycle indicator and single cell analysis method, called CycleTrak, which allows for better spatiotemporal resolution and quantification of cell cycle phase and cell position than current methods. Our method was developed on the basis of the existing Fucci method. CycleTrak uses a single lentiviral vector that integrates mKO2-hCdt1 (30/120), and a nuclear-localized eGFP reporter. The single vector and nuclear localized fluorescence signals simplify delivery into cells and allow for rapid, automated cell tracking and cell cycle phase readout in single and subpopulations of cells. We validated CycleTrak performance in metastatic melanoma cells and identified novel cell cycle dynamics in vitro and in vivo after transplantation and 3D confocal time-lapse imaging in a living chick embryo.     

Changes in antibiotic susceptibility in staphylococci habituated to sub-lethal concentrations of tea tree oil (Melaleuca alternifolia)

Aims: To investigate the effect of sub‐lethal challenge with tea tree oil (TTO) on the antibiotic resistance profiles of staphylococci. Methods and Results: Isolates of methicillin‐resistant/‐sensitive Staphylococcus aureus (MRSA and MSSA) and coagulase‐negative staphylococci (CoNS) were habituated to sub‐lethal concentrations of TTO (72 h). Following habituation, the minimum inhibitory concentrations (MIC) of antibiotics and TTO were determined. Habituated MRSA/MSSA cultures had higher (P < 0·05) MIC values than control cultures for the examined antibiotics. Habituated MRSA/MSSA cultures also displayed decreased susceptibility to TTO. Although the MIC of habituated MRSA/MSSA for the examined antibiotics reverted to control values after subsequent culture in the absence of TTO, the increased MIC against TTO were maintained. When compared with control cultures, habituated CoNS cultures had higher (P < 0.05) MIC values against three‐fifths of the antibiotics examined; no changes in TTO MIC were observed. Conclusions: TTO habituation ‘stress‐hardens’ MRSA and MSSA, evidenced by transient decreased antibiotic susceptibility and stable decreased TTO susceptibility. Although TTO habituation did not decrease susceptibility of CoNS to TTO, such cultures showed transient decreased antibiotic susceptibility. Significance and Impact of the Study: Application of TTO at sub‐lethal concentrations may reduce the efficacy of topical antibiotics used with TTO in combination therapies.     

Evaluation of new broth media for microdilution antibiotic susceptibility testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria

Nine pure or mixed broth media were evaluated for their suitabilities to determine MICs in a microdilution test of 19 antibacterial agents for lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Lactococcus, and Bifidobacterium. A mixed formulation of Iso-Sensitest broth (90%) and deMan-Rogosa-Sharpe broth (10%) with or without supplementation with L-cysteine, referred to as the LAB susceptibility test medium, provided the most optimal medium basis in terms of growth support of nonenterococcal LAB and correct indication of MICs of international control strains.