Rapid determination of carbapenem resistance by low-cost colorimetric methods: Propidium Iodide and alamar blue staining.

Carbapenems are a class of ß-lactam antibiotics with a broad antimicrobial activity spectrum. Owing to their sturdy structures resistant to most ß-lactamases, they have been regarded as one of the last-resort antibiotics for combating multidrugresistant bacterial infections. However, the emergence of carbapenem resistance increases predominantly in nosocomial pathogens. To prevent spread of carbapenem resistance in early stages, it is imperative to develop rapid diagnostic tests that will substantially reduce the time and cost in determining carbapenem resistance. Thus, we devised a staining-based diagnostic method applicable to three different Gram-negative pathogens of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae, all with the high potential to develop carbapenem resistance. Regardless of the resistance mechanisms presented by bacterial species and strains, double staining with propidium iodide (PI) and alamar blue (AB) identified resistant bacteria with an average sensitivity of 95.35%, 7 h after imipenem treatments in 343 clinical isolates. Among the three species tested, A. baumannii showed the highest diagnostic sensitivity of 98.46%. The PI and ABmediated staining method could be a promising diagnostic method with high-throughput efficacy and low cost.

Comparative assessment of qPCR enumeration methods that discriminate between live and dead Escherichia coli O157:H7 on beef.

Quantitative Polymerase Chain Reaction (qPCR) is a molecular method commonly used to detect and quantify bacterial DNA on food but is limited by its inability to distinguish between live and dead cell DNA. To overcome this obstacle, propidium monoazide (PMA) alone or with deoxycholate (DC) was used to prevent dead cell detection in qPCR. qPCR methods were used to detect strains of Escherichia coli O157, which can cause infection in humans with an infectious dose of less than 10 cells. A 5 strain E. coli O157:H7 cocktail was inoculated onto beef steaks and treated with interventions used in meat facilities (lactic acid (5%), peroxyacetic acid (200 ppm) or hot water (80 °C for 10 s)). Treatment of PMA or PMA + DC was applied to samples followed by DNA extraction and quantification in qPCR. RNA was also quantified in addition to conventional plating. For lactic acid intervention, qPCR DNA quantification of E. coli O157:H7 yielded 6.59 ±â€¯0.21 and 6.30 ±â€¯0.11 log gene copy #/cm2 for control and lactic acid samples, respectively and after treatment with PMA or PMA + DC this was further reduced to 6.31 ± 0.21 and 5.58 ± 0.38, respectively. This trend was also observed for peroxyacetic acid and hot water interventions. In comparison, RNA quantification yielded 7.65 ± 0.13 and 7.02 ± 0.38 log reverse transcript/cm2 for rRNA control and lactic acid samples, respectively, and for plating (LB), 7.51 ±â€¯0.06 and 6.86 ±â€¯0.32 log CFU/cm2, respectively. Our research determined that treatment of PMA + DC in conjunction with qPCR prevented dead cell DNA detection. However, it also killed cells injured from intervention that may have otherwise recovered. RNA quantification was more laborious and results had higher variability. Overall, quantification with conventional plating proved to be the most robust and reliable method for live EHEC detection on beef.

Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact.

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity.

Assessment of bacteria and archaea in metalworking fluids using massive parallel 16S rRNA gene tag sequencing.

Determination of the bacterial diversity in industry-based liquid in-use water-miscible metalworking fluid (MWF) samples was targeted by massive parallel multiplex DNA sequencing, either directly or upon pretreatment with propidium monoazide (PMA) that allows differentiation between intact and physically damaged cells. As MWFs provide a suitable basis of life for micro-organisms, the majority is preserved by biocides. 'Bio-concept' fluids on the other hand are bactericide free, which intentionally leads to substantial bacterial populations. Samples from both fluid types were chosen: A median of 51 operational taxonomic units at genera level (OTUs) were detected per sample, but only 13 were present at or above 1·0% of the total population in any PMA-treated sample analysed. As both fluid types were mainly dominated by Pseudomonas spp., we resolved this genus on the species level and found the Pseudomonas oleovorans/pseudoalcaligenes group to predominate. We also looked for archaea and detected Methanobrevibacter spp., albeit in <3% of all samples analysed. SIGNIFICANCE AND IMPACT OF THE STUDY: Water-miscible metalworking fluids provide a suitable base of life for micro-organisms, mainly bacteria and fungi. Earlier publications suggested that the diversity is rather low, but these studies were largely based on heterotrophic plate counts. This might have resulted in underestimation of population density and microbial diversity as some organisms might just refuse to grow. This study used high-throughput sequencing in the absence and presence of propidium monoazide to explore bacterial and archaeal presence in metalworking fluids. We established that diversity is low and bacterial populations are dominated by the genus Pseudomonas spp.

Assessment of silibinin as a potential antifungal agent and investigation of its mechanism of action.

Silibinin, which is derived from Silybum marianum (milk thistle), has used as a traditional remedy for liver or biliary disorders and known to have superior antioxidant activity. In addition, silibinin was recently reported to have antifungal effect related to fungal apoptosis against Candida albicans and the interest in the therapeutic effect is increasing. In this study, we found another mode of antifungal action of silibinin and its antibiofilm activity on C. albicans. To investigate influence on fungal plasma membrane, propidium iodide and bis-(1, 3-dibutylbarbituric acid) trimethineoxonol [DiBAC4 (3)] assay were primarily carried out. After 5-h incubation with silibinin (50, 100, 150, or 200 µg/mL), the propidium iodide fluorescent percentages increased by 11.90%, 28.50%, 34.10%, and 44.52%, respectively, and the DiBAC4 (3) fluorescent percentages increased by 13.18%, 34.64%, 46.99%, and 57.15%, respectively. As a result, we thought that silibinin concentrations of more than 100 µg/mL have a membrane-damaging effect. Subsequently, to estimate the degree of membrane damage, we used Fluorescein isothiocyanate-labelled dextrans (FDs) of various sizes and the results indicated that silibinin allowed penetration of molecules smaller than approximately FD20 (3.3 nm). In addition, silibinin inhibited the dimorphic transition of C. albicans and resulted in the inhibition of biofilm development at an early stage. In conclusion, we found membrane-damaging effect of silibinin and its antibiofilm effect in C. albicans. © 2017 IUBMB Life, 69(8):631-637, 2017.

Quantitative assessment of viable cells of Lactobacillus plantarum strains in single, dual and multi-strain biofilms.

Biofilms of Lactobacillus plantarum are a potential source for contamination and recontamination of food products. Although biofilms have been mostly studied using single species or even single strains, it is conceivable that in a range of environmental settings including food processing areas, biofilms are composed of multiple species with each species represented by multiple strains. In this study six spoilage related L. plantarum strains FBR1-FBR6 and the model strain L. plantarum WCFS1 were characterised in single, dual and multiple strain competition models. A quantitative PCR approach was used with added propidium monoazide (PMA) enabling quantification of intact cells in the biofilm, representing the viable cell fraction that determines the food spoilage risk. Our results show that the performance of individual strains in multi-strain cultures generally correlates with their performance in pure culture, and relative strain abundance in multi-strain biofilms positively correlated with the relative strain abundance in suspended (planktonic) cultures. Performance of individual strains in dual-strain biofilms was highly influenced by the presence of the secondary strain, and in most cases no correlation between the relative contributions of viable planktonic cells and viable cells in the biofilm was noted. The total biofilm quantified by CV staining of the dual and multi-strain biofilms formed was mainly correlated to CV values of the dominant strain obtained in single strain studies. However, the combination of strain FBR5 and strain WCFS1 showed significantly higher CV values compared to the individual performances of both strains indicating that total biofilm formation was higher in this specific condition. Notably, L. plantarum FBR5 was able to outgrow all other strains and showed the highest relative abundance in dual and multi-strain biofilms. All the dual and multi-strain biofilms contained a considerable number of viable cells, representing a potential source of contamination.

A flow cytometric method for viability assessment of Staphylococcus aureus and Burkholderia cepacia in mixed culture.

Mixed bacterial communities are commonly encountered in microbial infections of humans. Knowledge on the composition of species and viability of each species in these communities allows for a detailed description of the complexity of interspecies dynamics and contributes to the assessment of the severity of infections. Several assays exist for quantification of specific species in mixed communities, including analysis of quantitative terminal restriction fragment length polymorphisms. While this method allows for species-specific cell enumeration, it cannot provide viability data. In this study, flow cytometry was applied to assess the viability of Staphylococcus aureus and Burkholderia cepacia in mixed culture by membrane integrity analysis using SYBR® Green I and propidium iodide staining. Both bacteria are relevant to pulmonary infections of cystic fibrosis patients. Fluorescence staining was optimized separately for each species in pure culture due to differences between species in cell wall structure and metabolic capabilities. To determine viability of species in mixed culture, a protocol was established as a compromise between optimum conditions determined before for pure cultures. This protocol allowed the detection of viable and dead cells of both species, exhibiting an intact and a permeabilized membrane, respectively. To discriminate between S. aureus and B. cepacia, the protocol was combined with Gram-specific fluorescent staining using wheat germ agglutinin. The established three-color staining method was successfully tested for viability determination of S. aureus and B. cepacia in mixed culture cultivations. In addition, growth of both species was monitored by quantitative terminal restriction fragment length polymorphisms. The obtained data revealed alterations in viability during cultivations for different growth phases and suggest interspecies effects in mixed culture. Overall, this method allows for rapid simultaneous Gram-differentiation and viability assessment of bacterial mixed cultures and is therefore suitable for the analysis of dynamics of mixed communities of medical, environmental, and biotechnological relevance.

Improved assessment of frozen/thawed mouse spermatozoa using fluorescence microscopy.

Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).

A 'fragile cell' sub-population revealed during cytometric assessment of Saccharomyces cerevisiae viability in lipid-limited alcoholic fermentation.

AIMS: To show that in anaerobic fermentation with limiting lipid nutrients, cell preparation impacts the viability assessment of yeast cells, and to identify the factors involved. METHODS AND RESULTS: Saccharomyces cerevisiae viability was determined using propidium iodide staining and the flow cytometry. Analyses identified intact cells, dead cells and, under certain conditions, the presence of a third subpopulation of apparently damaged cells. This intermediate population could account for up to 40% of the entire cell population. We describe, analyse and discuss the effects of different solutions for cell resuspension on the respective proportion of these three populations, in particular that of the intermediate population. We show that this intermediate cell population forms in the absence of Ca(2+)/Mg(2+). CONCLUSIONS: Cell preparation significantly impacts population viability assessment by FCM. The intermediate population, revealed under certain conditions, could be renamed as 'fragile cells'. For these cells, Ca(2+) and Mg(2+) reduce cell membrane permeability to PI. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that analyses and discusses the factors influencing the formation of an intermediate population when studying viability in yeast alcoholic fermentation. With a wider application in biological research, this study provides important support to the relatively new questioning of propidium iodide staining as a universal cell death indicator.

Modified annexin V/propidium iodide apoptosis assay for accurate assessment of cell death.

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability. The Annexin V/PI protocol is a commonly used approach for studying apoptotic cells. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).

Comparison between propidium iodide and 7-amino-actinomycin-D for viability assessment during flow cytometric analyses of the human sperm acrosome.

Evaluation of the acrosome reaction can shed light on the fertilising competence of spermatozoa. To eliminate false-positive results when evaluating the acrosome status of human sperm cells, two viability probes propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) were compared for their ability to stain nonviable cells post-fixation and permeabilisation. Both the mean fluorescence and % dead cells differed significantly with time (P < 0.0001). Unlike PI, 7-AAD did not leach from cells and fluorescence remained stable for up to 4 h. Furthermore, 7-AAD proved to be a proficient marker to exclude dead sperm cells during flow cytometric evaluation of ionophore-induced acrosome reaction.

Cytometric assessment of histone H2AX phosphorylation: a reporter of DNA damage.

DNA damage that leads to formation of DNA double-strand breaks (DSBs) induces phosphorylation of histone H2AX on Ser-139 at sites flanking the breakage. Immunocytochemical detection of phosphorylated H2AX (denoted as gammaH2AX) thus provides a marker of DSBs. The method presented in this chapter describes the detection of gammaH2AX for revealing the presence of DSBs, combined with differential staining of cellular DNA for revealing the cell cycle phase. The detection of gammaH2AX is based on indirect immunofluorescence using secondary antibody tagged with fluorescein isothiocyanate (FITC) while DNA is counterstained with propidium iodide (PI). Intensity of cellular green (FITC) and red (PI) fluorescence is measured by flow cytometry and bivariate analysis of the data is used to correlate the presence of DSBs with the cell cycle phase.

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

Assessment of equine sperm mitochondrial function using JC-1.

The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged through a Percoll gradient (PERC). Spermatozoa were resuspended to 25 x 10(6) cells/mL, samples were split, and one sample was repeatedly flash frozen (FF) in LN2 and thawed. The following gradients of PERC:FF were prepared: 100:0 (100), 75:25(75), 50:50 (50), 25:75 (25) and 0:100 (0). Samples were stained with 2.0 microM JC-1 and assessed for staining by flow cytometry and by a fluorescence microplate reader. A total of 10,000 gated events was analyzed per sample with flow cytometry. The mean percentage of cells staining orange for the 100, 75, 50, 25 and 0 treatments was 92.5, 72.8, 53.4, 27.3 and 7.3, respectively. The expected percentage of spermatozoa forming JC-1 aggregates was correlated with the actual percentage of orange labeled sperm cells determined by flow cytometry (r2=0.98). Conversely, JC-1 monomer formation was negatively correlated with expected mitochondrial membrane potential (r2=-0.98). The blank corrected orange fluorescence, assessed by microplate assay, was significantly (P<0.0001) correlated with the expected (r2=0.49) and with the flow cytometric (r2=0.50) determination of percentage of spermatozoa with mitochondria of high membrane potential. Total orange and orange:green fluorescence was also correlated with mitochondrial function. These results indicate that JC-1 staining can accurately detect changes in mitochondrial membrane potential of equine spermatozoa. The relative fluorescence of JC-1 labeling patterns of equine spermatozoa can be accurately and objectively determined by flow cytometry and by a fluorescence microplate reader assay.

Assessment of stallion spermatozoa viability by flow cytometry and light microscope analysis.

Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable, and subjectively assess only 100 to 200 spermatozoa per ejaculate. We collected two ejaculates from each of 4 stallions, and extended them to 50x10(6) sperm/mL in a nonfat dried milk solids glucose extender (EZ Mixin). Half the ejaculate was freeze-killed by immersing in liquid nitrogen for 10 min. Aliquots using appropriate volumes of live and freeze-killed spermatozoa provided the following ratios of live:dead spermatozoa: 100:0, 75:25, 50:50, 25:75, 0:100. We determined the viability of each aliquot by 1) motility; 2) eosin-nigrosin staining; and 3) dual fluorescent staining. For the latter, aliquots incubated with SYBR-14 and propidium iodide had live and dead spermatozoa quantitated by fluorescent microscope (2 x 100 sperm/sample) and flow cytometry (10,000 sperm/sample). We found a linear relationship between the ratio of live:dead spermatozoa and the percentage of spermatozoa counted as live (P<0.0001). For fresh spermatozoa, correlation coefficients of the known live:dead ratio were high for all methods (eosin-nigrosin, r>0.75; fluorescent microscope, r>0.76; flow cytometry, r>0.75; motility, r>0.76). To determine viability of cryopreserved equine spermatozoa, we froze 17 fresh ejaculates from 6 stallions in a glycine extender. Each sample was thawed, extended 1:1 with EZ Mixin and evaluated as above. Cryopreserved spermatozoa assessed by flow cytometry tended to be less well correlated (r<0.68) with the other methods, and estimates were significantly higher with eosin-nigrosin staining (P<0.001). This study shows that different methods may equally estimate viability of fresh equine spermatozoa. However, evaluation by flow cytometry appears to be less precise with cryopreserved spermatozoa.