Automated Colony Counter Research Articles

Antibacterial Effect of Nitric Oxide on the Causative Agents of Hospital-Acquired Pneumonia (Experimental Study)

The aim of the study was to evaluate the antimicrobial effect of single and repeated nitric oxide (NO) exposure on the major pathogens of nosocomial pneumonia isolated from the sputum of cardiac surgery patients.Materials and Methods. A 24-hour culture of microorganisms from pan-resistant isolates of Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae from the sputum of inpatient cardiac surgery patients with nosocomial pneumonia, as well as strains of P. aeruginosa and E. coli from the American Type Culture Collection (ATCC), were exposed to 200 ppm NO (experimental sample) or medical air (control sample) in a sealed chamber for 30 minutes. After a single or 4 repeated gas exposure at 4 h intervals, Petri dishes were placed in a thermostat at 37°C and the results were evaluated at 24 and 48 h or at 12, 24, 36 and 48 h, respectively. Grown colonies were counted using an automated colony counter and recorded as CFU/mL.Results. No growth of clinical isolates of P. aeruginosa and E. coli was observed 24 and 48 h after a single exposure to NO. Growth of A. baumannii was lower compared to controls at 24 h but continued at 48 h. No effect of a single exposure to 200 ppm NO on other microorganisms was observed. After 4 exposures to NO, the growth of ATCC E. coli was not detected, the growth of other experimental strains was significantly lower compared to the control (P<0.05).Conclusion. Our results provide a rationale for the use of multiple intermittent inhalation of 220 ppm NO for the treatment of patients with hospital-acquired bacterial pneumonia.

Validation studies of the spiral plating method to determine the potency of lactobacillus-containing probiotics

Scientific relevance. Viable cell counting is an important microbiological test for the quality assessment of medicinal products containing live microorganisms. To optimise labour and material costs and enhance testing precision and reproducibility, it is practical to use partially automated instrumental methods, such as the spiral plating method.Aim. The aim was to conduct a validation study of the spiral plating method for assessing the potency of biologicals containing live bacterial cells, using a lactobacillus-containing probiotic medicinal product as a case study.Materials and methods. This study used a culture of Lactiplantibacillus plantarum isolated from a probiotic medicinal product. Spiral plating on agar-based biological culture media was performed using an automatic Eddy Jet 2 plating system. The study used an IUL Flash & Go colony counter for automatic reporting of the results. The validation study was conducted according to the general chapter on the validation of microbiological testing methods (OFS.1.1.0021.18) of the State Pharmacopoeia of the Russian Federation.Results. According to the validation results for the main parameters, the spiral plating method had the range of 104–105 CFU/mL; the limit of quantification of 102 CFU/mL; and the coefficient of linear determination, R2, of 0.99. The accuracy of the spiral plating method in determining the potency of the test sample was 93%; the repeatability was 4.9%.Conclusions. The study results confirm the similarity of the spiral plating method with an automatic colony counter to Koch’s plating method, which is currently used in accordance with the State Pharmacopoeia of the Russian Federation, in terms of the validated parameters. Therefore, spiral plating can be used to evaluate the potency of lactobacillus-containing probiotics. Spiral plating can help improve the cost-effectiveness and accuracy of testing.

Evaluation of an Automated System for the Counting of Microbial Colonies.

Counting of microbial colonies is a common technique employed in research and diagnostics. To simplify this tedious and time-consuming process, automated systems have been proposed. This study aimed to elucidate the reliability of automated colony counting. We evaluated a commercially available instrument (UVP ColonyDoc-It Imaging Station) in regard to its accuracy and potential time savings. Suspensions of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus faecium, and Candida albicans (n = 20 each) were adjusted to achieve growth of approximately 1,000, 100, 10, and 1 colony per plate, respectively, after overnight incubation on different solid media. Compared with manual counting, each plate was automatically counted by the UVP ColonyDoc-It with and without visual adjustment on a computer display. For all bacterial species and concentrations automatically counted without visual correction, an overall mean difference from manual counts of 59.7%, a proportion of isolates with overestimation/underestimation of colony numbers of 29%/45%, respectively, and only a moderate relationship (R2 = 0.77) with the manual counting were shown. Applying visual correction, the overall mean difference from manual counts was 1.8%, the proportion of isolates with overestimation/underestimation of colony numbers amounted to 2%/42%, respectively, and a strong relationship (R2 = 0.99) with the manual counting was observed. The mean time needed for manual counting compared with automated counting without and with visual correction was 70 s, 30 s, and 104 s, respectively, for bacterial colonies through all concentrations tested. Generally, similar performance regarding accuracy and counting time was observed with C. albicans. In conclusion, fully automatic counting showed low accuracy, especially for plates with very high or very low colony numbers. After visual correction of the automatically generated results, the concordance with manual counts was high; however, there was no advantage in reading time. IMPORTANCE Colony counting is a widely utilized technique in the field of microbiology. The accuracy and convenience of automated colony counters are essential for research and diagnostics. However, there is only sparse evidence on performance and usefulness of such instruments. This study examined the current state of reliability and practicality of the automated colony counting with an advanced modern system. For this, we thoroughly evaluated a commercially available instrument in terms of its accuracy and counting time required. Our findings indicate that fully automatic counting resulted in low accuracy, particularly for plates with very high or very low colony numbers. Visual correction of the automated results on a computer screen improved concordance with manual counts, but there was no benefit in counting time.

Multisite Qualification of an Automated Incubator and Colony Counter for Environmental and Bioburden Applications in Pharmaceutical Microbiology.

Traditional microbiological techniques have been used for well over a century as the basis for contamination testing of pharmaceutical products and processes. With more recent focus on faster product release and concerns around the integrity of the test data, new technologies have been implemented to detect and enumerate organisms faster and provide paperless processes to minimize data integrity issues. Manual colony counting technologies, where incubation is performed in a standard incubator, and the plate is manually transferred to the colony counter for a single read at the end of incubation, have been used for many years to reduce the potential for human error; however, they pose validation challenges due to poor counting accuracy. Colony counters that automatically perform both the incubation and enumeration functions (multiple enumeration calculations through the incubation phase) have recently been implemented for quality control (QC) laboratory analytical processes, supporting a cGMP environment. This article summarizes the findings of eight companies demonstrating the qualification of an automated colony counter technology to perform the majority of microbial tests required for QC, environmental monitoring, and bioburden for in-process, bulk drug substance, and water system testing. Comparable analytical performance and time to result data generated during individual studies at all companies allows the system to be qualified and implemented for cGMP processes while reducing data integrity risks.

Application of Automatic Colony Counter in the Detection of Dairy Products

Objective: To improve the counting efficiency and realize the traceability of raw data. The applicability, accuracy, precision and repeatability of the automatic colony counter were verified by experiments, Determine the applicability of colony count in the detection of dairy products and raw and auxiliary materials. Methods: The plate pouring method and test piece method are selected for verification test, including psychrophilic bacteria, aerobic spores, thermophilic aerobic spores, lactic acid bacteria count, total number of colonies, coliform, mold, yeast, Staphylococcus aureus and other items. The manual count is compared with the automatic colony counter to determine its feasibility, applicability and accuracy. Results: The colony count of normal Petri dish without background interference was compared with that of 3M test piece. The paired t-test was carried out by visual method and instrument method, <I>P </I>= 0.17, greater than 0.05. There was no significant difference in statistical results, and the colored medium had no significant effect on automatic counting. The counting effect of the instrument is poor when there are needle shaped colonies, the color of colonies is close to that of the culture medium, the contour of colonies is not obvious, the colonies spread greatly and the edges of colonies are irregular, there are food particles and colonies in the culture medium at the same time, or the color of colonies is close to that of food particles, which can not meet the accuracy requirements. Conclusion: For samples with uniform texture, the accuracy and precision of automatic colony counting are relatively good, the work efficiency is high, the operation time of experimental personnel is saved, and the original experimental data and images can be stored in the instrument for a long time. It is a counting method worthy of popularization in the laboratory. However, for the samples and culture medium with interference in the test verification, appropriate measures can be taken to change the counting conditions and manually correct the counting results of the instrument, but the counting efficiency will be reduced, and the laboratory can choose to use it according to the situation.

A Systematic Approach for the Evaluation, Validation, and Implementation of Automated Colony Counting Systems.

For several years, automated colony counting systems have been available with varying degrees of automation. Ever more sophisticated instruments are now increasingly used in microbiological laboratories of pharmaceutical quality control. In addition to the colony counting device, the instruments are now also equipped with robotic systems performing the entire handling of the petri dishes, e.g., automated internal transportation of petri dishes from the incubator chamber to the instrument's enumeration device and back. Moreover, the subjective evaluation of microbial enumeration tests by analysts is replaced with a more accurate and precise process. This leads to significant improvements to data integrity compliance. Automated colony counting systems also often enable cost reduction in the microbiological laboratory, e.g., by not requiring a contemporaneous verification by a second analyst. They also enable direct integration of count data into an existing laboratory information management system, reducing the hands-on time, costs per test and also preventing human errors caused by manual transcription. Altogether, these instruments will lead to improved monitoring and assurance of control of biopharmaceutical processes and manufacturing environments, as well as shortened cycle times in the supply chain. Regulators are encouraging the biopharmaceutical industry to adopt these innovative systems. For example, this year a BioPhorum member company received the first health authority approvals from EU, US, CH, Canada, Australia, and China for the use of automated colony counting systems for in-process bioburden testing and the release of drug substance lots, with an incubation time reduced by about 50%. Although these approvals are for release testing of drug substance lots, the instruments can also be used for environmental monitoring, testing of water samples, etc. This article describes a systematic 9-step approach to the evaluation, equipment qualification, and deployment of automated colony counting systems, which can be applied by biopharmaceutical companies wanting to take advantage of their numerous benefits.

Design and Implementation of an Automated Colony Counter Using Artificial Vision

Design and Implementation of an Automated Colony Counter Using Artificial Vision

A clinical investigation of dental evacuation systems in reducing aerosols

BackgroundThe route of transmission of severe acute respiratory syndrome coronavirus 2 has challenged dentistry to improve the safety for patients and the dental team during various treatment procedures. The purpose of this study was to evaluate and compare the effectiveness of dental evacuation systems in reducing aerosols during oral prophylactic procedures in a large clinical setting. MethodsThis was a single-center, controlled clinical trial using a split-mouth design. A total of 93 student participants were recruited according to the inclusion and exclusion criteria. Aerosol samples were collected on blood agar plates that were placed around the clinic at 4 treatment periods: baseline, high-volume evacuation (HVE), combination (HVE and intraoral suction device), and posttreatment. Student operators were randomized to perform oral prophylaxis using ultrasonic scalers on 1 side of the mouth, using only HVE suction for the HVE treatment period and then with the addition of an intraoral suction device for the combination treatment period. Agar plates were collected after each period and incubated at 37 °C for 48 hours. Colony-forming unit (CFU) counts were determined using an automatic colony counter. ResultsThe use of a combination of devices resulted in significant reductions in CFUs compared with the use of the intraoral suction device alone (P < .001). The highest amounts of CFUs were found in the operating zone and on patients during both HVE and combination treatment periods. ConclusionsWithin limitations of this study, the authors found significant reductions in the amount of microbial aerosols when both HVE and an intraoral suction device were used. Practical ImplicationsThe combination of HVE and intraoral suction devices significantly decreases microbial aerosols during oral prophylaxis procedures.

New insights into the standard method of assessing bacterial filtration efficiency of medical face masks

Based on the current knowledge of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) transmission, wearing a mask has been recommended during the COVID-19 pandemic. Bacterial filtration efficiency (BFE) measurements enable designing and regulating medical masks to prevent bioaerosol dissemination; however, despite the simplicity of these measurements, several scientific questions remain unanswered regarding BFE tests. Here, we investigated (1) the impact of substituting 100-mm Petri dishes with 90-mm disposable Petri dishes, (2) the impact of colony-counting methods on the bioaerosol aerodynamic size, and (3) the impact of colony-counting methods on the total viable particle counts. We demonstrated that disposable 90-mm Petri dishes can be used to replace the 100-mm dishes. We also showed that an automatic high-resolution colony counter can be used to directly count viable particles on collection substrates and to measure the bioaerosol size parameters. Our results enable possible modernization of the outdated testing methods recommended in the US and European standards for BFE measurements. Specifically, use of a modernized colony counter should be clearly regulated and permitted to avoid the counting of positive holes. The median aerodynamic diameter appears to be the most relevant parameter for characterizing bioaerosol size.

In-Vitro Model of Scardovia wiggsiae Biofilm Formation and Effect of Nicotine.

Recently, Scardovia wiggsiae has been reported to be strongly associated with caries formation. This study aimed to establish an in vitro model of S. wiggsiae biofilm and to investigate the effect of nicotine on S. wiggsiae colony-forming units (CFUs) growth. S. wiggsiae biofilm was grown overnight using brain-heart infusion (BHI) broth supplemented with 5 g of yeast extract/L (BHI-YE). The overnight culture was used as an inoculum to grow S. wiggsiae biofilm on standardized enamel and dentin samples. Samples were incubated with different nicotine concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 mg/mL) for 3 days. The dissociated biofilms were diluted, spiral plated on blood agar plates, and incubated for 24 h. CFUs/mL were quantified using an automated colony counter. A two-way ANOVA was used to compare the effect of different nicotine concentrations on S. wiggsiae CFUs. This study demonstrated that S. wiggsiae biofilm could be initiated and formed in vitro. Increased CFUs was observed through 0.5-4 mg/mL and 0.5-8 mg/mL of nicotine using enamel and dentin substrates, respectively. 16 and 32 mg/mL of nicotine were determined as the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), respectively. S. wiggsiae formed greater biofilm on enamel than dentin specimens in response to the nicotine stimulus. This study demonstrated the negative effect of smoking on increasing S. wiggsiae biofilm. Establishing S. wiggsiae biofilm in vitro may allow researchers in the future to have a better understanding of caries pathogenesis and bacterial interaction.

Experimental setup and image processing method for automatic enumeration of bacterial colonies on agar plates.

Automated colony counting methods have long been known in Microbiology. Numerous methods for automated image analysis have been described and a wide range of commercial products exists. Known advantages are saving cost by reducing enumeration time, automatic documentation, reproducibility, and operator independence. Still, even today the realization of all advantages of automated image analysis makes it necessary to either invest in an expensive, high performance commercial system, or to acquire expert knowledge in image processing. This is a considerable obstacle for many laboratories, and the reason why manual colony counting is still done frequently. This article describes an easy to apply automatic colony counting system–including suggestions for sample preparation–that can be put into operation with basic knowledge of image processing and low budget.

Hand Bacterial Repopulation Dynamics Following Two Methods of Surgical Hand Preparation during Elective Orthopedic Surgeries.

Context:Antiseptic hand preparations are routine prior to surgical procedures to reduce microbial load on the operating gloved hands. Two methods of surgical hand preparations available are the antibacterial detergent hand wash and an alcohol-based hand rub.Aim:The aim of the study was to compare quantitatively, the efficacy of the two methods in hand bacterial reductions during elective orthopedic surgeries.Setting and Design:This comparative study was conducted at the orthopedic surgery department of a tertiary health facility.Subjects and Methods:One-hundred and sixty dominant hands of operating surgeons and nurses involved in forty elective orthopedic surgeries were studied. The subjects were randomly assigned to either the antibacterial detergent hand-washing (HW) or the alcohol-based hand-rubbing (HR) groups. Swab samples were obtained from the hands before and after hand preparations and at the end of surgeries following removal of the operating gloves. These samples were then subjected to culture. The bacterial counts on these were then obtained through an automated colony counter, and the results were expressed in logarithmic values (log10).Statistical Analysis Used:The analysis was done using IBM SPSS software version 20. The mean results obtained were subjected to an independent t-test analysis with the statistical significance level set at P < 0.05.Results:Both methods of hand antisepsis showed comparable efficacies in attaining surgical hand hygiene at 1-min postapplication (P = 0.73). HR group, however, showed greater sustained effects during the period of surgeries, though not statistically significant (P = 0.18).Conclusion:Scrubbing using the HR method is a viable alternative to the HW method during elective orthopedic surgery.

Abstract 4398: In vitro Tumor Treating Fields (TTFields) alter proliferation and morphology of patient-derived high-grade meningioma cell lines

Abstract Introduction: Meningiomas are the most common neoplasm of the central nervous system (CNS), accounting for 37% of primary CNS tumors. Though derived from the arachnoid cap cells within the meninges, high-grade meningiomas (WHO grades II and III) invade into the normal brain tissue, and have a high rate of recurrence. As there are no efficacious chemotherapies for these high-grade meningiomas, standard of care is currently limited to maximal surgical resection followed by radiotherapy, with a median overall survival of only 2.5-3 years. Clinical trials with TTFields therapy are under way for patients with high-grade meningiomas. In this study, in vitro experiments comparing TTFields to untreated controls were performed on two patient-derived anaplastic (WHO grade III) meningioma cell lines to determine the effects of TTFields on cell proliferation and clonogenicity. Methods: Studies utilizing patient tumor specimens were approved by the Wayne State University Institutional Review Board and written informed consent was obtained from participants. Meningioma tumor specimens were collected immediately following microsurgical resection. Single-cell suspensions from the tumor tissues were prepared by enzymatic and mechanical disruption. Cells were plated on plastic coverslips (10,000 each) in DMEM/F12 media supplemented with 10% fetal bovine serum. TTFields were applied at ~1.6 V/cm at 100, 200, 300, or 400 kHz (groups n=4-5) with one control group. Culture media were replaced every day. After 12 days of treatment, cell proliferation was assessed with the XTT assay and cells were harvested and replated for clonogenic assays (500 cells/well), which were stained with crystal violet and counted with an automated colony counter. Harvested cells were also fixed and immunostained with the diagnostic meningioma cytoplasmic marker vimentin to observe the cell morphology in control vs. TTFields-treated cells. Control vs. TTFields-treated groups were compared by one- or two-tailed t-tests. Results: The 12-day TTFields treatment significantly reduced cell proliferation, with the greatest decrease found in the 200 kHz group (0.508±0.21; mean±SEM) compared to the control (1.515±0.08; two-tailed t test p=0.0019). Similar results were found in the clonogenic assay, with control cell counts of 4219±793 compared to the 200kHz treated cells 1793±444 (one-tailed t test p=0.0279). In the vimentin-stained cells, the control cells displayed a typical meningioma spindle morphology, while the TTFields-treated cells demonstrated pleomorphic and irregular morphologies. Conclusions: Application of TTFields in vitro reduced proliferation and clonogenicity and altered morphology of patient-derived anaplastic meningioma cell lines. This is the first report on the in vitro effects of TTFields on meningioma. Citation Format: Sharon K. Michelhaugh, Sam Kiousis, Sam A. Michelhaugh, Neil V. Klinger, Sandeep Mittal. In vitro Tumor Treating Fields (TTFields) alter proliferation and morphology of patient-derived high-grade meningioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4398.

Abstract 3309: Tumor-treating fields decrease proliferation and clonogenicity of patient-derived WHO grade IV glioma cell lines

Abstract Despite decades of research, efficacious treatments for malignant glioma tumors are limited. Tumor-treating fields (TTFields) are FDA-approved for the treatment of newly-diagnosed and recurrent glioblastoma. In this study, in vitro experiments comparing TTFields to untreated controls were performed on patient-derived grade IV glioma cell lines to determine the effects of TTFields on cell proliferation and clonogenicity. Methods: Studies utilizing patient tumor specimens were approved by the Wayne State University Institutional Review Board and written informed consent was obtained from participants. Patient tumor specimens (glioblastoma and gliosarcoma) were collected immediately following microsurgical resection. Single-cell suspensions from the tumor tissues were prepared by enzymatic and mechanical disruption. Equal numbers of cells were plated on plastic coverslips in DMEM/F12 media supplemented with 10% fetal bovine serum. TTFields were applied at 200 kHz to half of the coverslips. Culture media was replaced every day. At the conclusion of the 2 week treatment, cell proliferation was assessed with the XTT assay and cells were harvested and replated for clonogenic assays (10,000 cells/well). The resulting colonies were fixed and stained with crystal violet and counted with an automated colony counter. Control vs. TTFields treated groups were compared by two-tailed t-tests. Results: The two-week TTFields treatment significantly reduced cell proliferation in both the glioblastoma (41.6 ± 11.1 % control; n=4; p&amp;lt;0.001) and in the gliosarcoma (41.6 ± 16.6 % control; n=4; p&amp;lt;0.002) as measured by XTT assay. The clonogenic assay revealed that the number of colonies generated from both cell lines was reduced by TTFields treatment. For the glioblastoma cell line, control cells yielded 847 colonies with an average diameter of 462.8 ± 5.6 µM while TTFields-treated cells yielded 561 colonies with an average diameter of 435.1 ± 7.3 µM, which was a statistically significant decrease (p=0.0022). For the gliosarcoma cell line, control cells yielded 809 colonies vs. 144 colonies from the TTFields treated cells, although the average diameter of the colonies was unchanged between groups (control 375.1 ± 176.1 µM; TTFields 362.9 ± 196.0 µM). Conclusions: In vitro application of TTFields markedly reduced cell proliferation and clonogenicity in both patient-derived WHO grade IV glioblastoma and gliosarcoma cell lines. This is the first report on the in vitro effects of TTFields on gliosarcoma. Citation Format: Sharon K. Michelhaugh, Sam Kiousis, Adrianne Wallace-Povirk, Sandeep Mittal. Tumor-treating fields decrease proliferation and clonogenicity of patient-derived WHO grade IV glioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3309. doi:10.1158/1538-7445.AM2017-3309

The effect of modified citrus pectin-probiotic on faecal lactobacilli in Balb/c mice

Abstract Intestinal bacteria utilize the constituent of ingested food for their metabolic activities making the gut microbiota a target in the modification of functional foods. This study examines the effect of modified citrus pectin and encapsulated probiotic, Lactobacillus acidophilus ATCC 4356 on colon lactobacilli microbiota in healthy Balb/c mice. Mice were orally administered once daily with modified citrus pectin alginate probiotic, alginate calcium probiotic, modified citrus pectin and water for 28 days. Faecal lactobacilli count were determined using automated colony counter Doc-It® imaging station. The number of faecal lactobacilli in the modified citrus pectin alginate probiotic-treated mice significantly increased at day 7, 14 and 28 [7.99 ± 0.22, 8.32 ± 0.08 and 8.36 ± 0.23 (log10cfu/g) respectively]. A little or no increase in the faecal lactobacilli count was noted in the modified citrus pectin-treated mice. Modified citrus pectin alginate combined with live probiotic, Lactobacillus acidophilus ATCC 4356 supplements can be used as probiotic therapy to improve the intestinal lactobacilli microbiota in healthy host.

Development of a high-throughput method to evaluate serum bactericidal activity using bacterial ATP measurement as survival readout.

Serum Bactericidal Activity (SBA) assay is the method of choice to evaluate the complement-mediated functional activity of both infection- and vaccine-induced antibodies. To perform a typical SBA assay, serial dilutions of sera are incubated with target bacterial strains and complement. The conventional SBA assay is based on plating on agar the SBA reaction mix and counting the surviving bacterial colony forming units (CFU) at each serum dilution. Even with automated colony counting, it is labor-intensive, time-consuming and not amenable for large-scale studies. Here, we have developed a luminescence-based SBA (L-SBA) method able to detect surviving bacteria by measuring their ATP. At the end of the SBA reaction, a single commercially available reagent is added to each well of the SBA plate, and the resulting luminescence signal is measured in a microplate reader. The signal obtained is proportional to the ATP present, which is directly proportional to the number of viable bacteria. Bactericidal activity is subsequently calculated. We demonstrated the applicability of L-SBA with multiple bacterial serovars, from 5 species: Citrobacter freundii, Salmonella enterica serovars Typhimurium and Enteritidis, Shigella flexneri serovars 2a and 3a, Shigella sonnei and Neisseria meningitidis. Serum bactericidal titers obtained by the luminescence readout method strongly correlate with the data obtained by the conventional agar plate-based assay, and the new assay is highly reproducible. L-SBA considerably shortens assay time, facilitates data acquisition and analysis and reduces the operator dependency, avoiding the plating and counting of CFUs. Our results demonstrate that L-SBA is a useful high-throughput bactericidal assay.

Pharmacokinetic/Pharmacodynamic (PK/PD) Evaluation of Dalbavancin Alone and in Combination with Ceftaroline against Methicillin-Resistant Strains (MRSA) of Staphylococcus aureus

BackgroundGlycopeptide antibiotics, principally, vancomycin, have been the first armamentarium against MRSA. However, Staphylococcus aureus strains have now developed reduced susceptibility to common glycopeptides. (DAL) is a novel, 2 ndgeneration lipoglycopeptide antibiotic with reported activity against Gram-positive bacterial pathogens, including MRSA. The long lipophilic lateral chain in DAL’s structure extends its half-life and promotes its anchoring to the cell membrane (vancomycin lacks this side chain). While a considerable amount of data is available on susceptibility testing for this agent, information regarding the potential for synergy with β-lactams with newer lipoglycopeptides, including dalbavancin, is lacking. Our objective was to evaluate the impact of DAL or vancomycin (VAN) in combination with ceftaroline (CPT) against MRSA.MethodsMRSA 494 was studied in six different regimens independently. All the experiments were performed in one-compartment PK/PD models in duplicate during 7 days. DAL 1,500 mg day one fCmax = 30.1 µg/mL, VAN 2 g q12hr fCmax = 36 µg/mL, CPT 600 mg every 12 hours fCmax = 17.04 µg/mL, DAL 1,500 mg day 1 plus CPT 600 mg q12hr, VAN 2 g q12hr plus Ceftaroline 600 mg q12hr and Growth Control. Antibiotic carry over was accounted for by washing and centrifugation of the samples. Model samples were plated and counted using an automated colony counter and differences in log 10CFU/mL between regimens was evaluated.ResultsCombination of DAL + CPT offered a significant reduction in log10CFU/mL amounts (more than 5 log10 CFU/mL and up to detection limits) in 24 hours compared with CPT alone. CPT alone demonstrated bactericidal activity with a reduction >3 log10CFU/mL by 32 hours although regrowth without resistance was observed after 72 hours. Mean CFU/mL for DAL models reached detection limits in 72 hours and no regrowth was detected after this time.ConclusionCombination of DAL+ ceftaroline offers encouraging results for MRSA strain 494. This combination therapy can potentially lead to optimizing patient outcomes and preserving dalbavancin therapy for serious MRSA infections through utilization of ideal combinations and dose exposures.Disclosures M. J. Rybak, Allergen: Scientific Advisor, Consulting fee.

ColonyCountJ: A User-Friendly Image J Add-on Program for Quantification of Different Colony Parameters in Clonogenic Assay

Objective: There are several commercial automatic colony counters available but they are relatively expensive and associated with several quantifications related problems. Some of the key problems in automated colony counting are clustering of colonies and edge effects. Thus the main objective of this study is to develop a user friendly program for determining colony parameters of clonogenic assay. Methods: In the present study, a watershed algorithm was employed along with other ImageJ tools to develop ImageJ based add-on program i.e to calculate various colony parameters. To test this program, we have used colonies obtained after exposure of MCF-7 cells to different doses of γ-radiation. Results: The results obtained using this program were compared with the manual counting as well as with automated counting provided by Oxford Optronix GelCount colony counter. It was found that our results corroborated well with the manual as well as with commercial program. As a whole, the performance of with regard to processing time and sensitivity was at par with the commercial colony counter. Conclusion: In conclusion, ColonyCountJ add-on program customized for ImageJ with optimized parameters is a reliable method for quantifying the colonies obtained from clonogenic assay. This program will be of great use for researchers working in the area of toxicology, radiation biology, and cancer biology.

Validation of an automated colony counting system for group A Streptococcus.

BackgroundThe practice of counting bacterial colony forming units on agar plates has long been used as a method to estimate the concentration of live bacteria in culture. However, due to the laborious and potentially error prone nature of this measurement technique, an alternative method is desirable. Recent technologic advancements have facilitated the development of automated colony counting systems, which reduce errors introduced during the manual counting process and recording of information. An additional benefit is the significant reduction in time taken to analyse colony counting data. Whilst automated counting procedures have been validated for a number of microorganisms, the process has not been successful for all bacteria due to the requirement for a relatively high contrast between bacterial colonies and growth medium. The purpose of this study was to validate an automated counting system for use with group A Streptococcus (GAS).ResultsMethods: Twenty-one different GAS strains, representative of major emm-types, were selected for assessment. In order to introduce the required contrast for automated counting, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) dye was added to Todd–Hewitt broth with yeast extract (THY) agar. Growth on THY agar with TTC was compared with growth on blood agar and THY agar to ensure the dye was not detrimental to bacterial growth. Automated colony counts using a ProtoCOL 3 instrument were compared with manual counting to confirm accuracy over the stages of the growth cycle (latent, mid-log and stationary phases) and in a number of different assays. The average percentage differences between plating and counting methods were analysed using the Bland–Altman method.ConclusionsResults: A percentage difference of ±10 % was determined as the cut-off for a critical difference between plating and counting methods. All strains measured had an average difference of less than 10 % when plated on THY agar with TTC. This consistency was also observed over all phases of the growth cycle and when plated in blood following bactericidal assays. Agreement between these methods suggest the use of an automated colony counting technique for GAS will significantly reduce time spent counting bacteria to enable a more efficient and accurate measurement of bacteria concentration in culture.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-1875-z) contains supplementary material, which is available to authorized users.

Does a surface coating inhibit bacterial adhesion on silicone hydrogel contact lenses

Event Abstract Back to Event Does a surface coating inhibit bacterial adhesion on silicone hydrogel contact lenses Kevin Mccabe1 1 Johnson & Johnson Vision Care inc, Research & Development, United States Purpose: Adhesion of bacteria to contact lens (CL) surfaces is a major contributory factor in the development of CL adverse events. This study sought to determine if surface treating silicone hydrogel (SiHy) CL offers any substantial benefits against adhesion of gram positive and negative bacteria. Method: Two surface-treated SiHy CL (lotrafilcon B; delefilcon A) & 3 uncoated SiHy CL (comfilcon A; samfilcon A; senofilcon A) from manufacturers packaging were soaked in an artificial tear mimicking solution for 15 h under rotative agitation at 35°C. Lenses (n=15) were then rinsed and challenged with either P aeruginosa ATCC 9027 & S. aureus ATCC 25923, using an inoculum of approx. 1x106 colony forming units per milliliter (CFU/mL) for 15 h. Adherent bacteria were recovered and the bacterial supernatant was enumerated for viable microorganisms by a spiral plate technique. CFU counts were determined using an automated colony counter. Results: The 2 surface-treated SiHy CL (lotrafilcon B and delefilcon A) exhibited average Log colony forming units (CFU) of 5.64 & 5.86 for P. aeruginosa and 3.74 & 3.83 for S. aureus respectively. The counts for the 3 uncoated SiHy CL (comfilcon A; samfilcon A; senofilcon A) were 5.86, 5.86 & 5.70 for P. aeruginosa and 3.89, 3.85 & 4.02 for S. aureus. No significant difference was determined between surface-treated and untreated materials (p=0.268). P. aeruginosa exhibited a higher propensity to adhere to the materials than S. aureus (p<0.001). Conclusions: In vitro bacterial adhesion was not modified by applying a surface treatment to the lens materials evaluated and all materials accumulated the gram negative bacteria to a greater extent than the gram positive organism. This suggests that the surface treatments applied to lotrafilcon B and delefilcon A does not provide a protective effect from bacterial adhesion. Further evaluation of the materials post-wear is required to validate the clinical relevance of these in vitro findings. Centre for Contact lens research at the University of Waterloo Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials for ophthalmic applications Citation: Mccabe K (2016). Does a surface coating inhibit bacterial adhesion on silicone hydrogel contact lenses. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02416 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kevin Mccabe Google Kevin Mccabe Google Scholar Kevin Mccabe PubMed Kevin Mccabe Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.