Enumeration Of Colony Forming Units Research Articles

Antimicrobial activity of aqueous and organic extracts of a Saudi medicinal plant: Rumex nervosus

Objective:The antimicrobial effect of aerial part of Rumex nervosus obtained from the Southern region of Saudi Arabia was evaluated on bacterial strains Staphylococcus aureus, methicillin resistant S. aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas Aeruginosa, and fungal strain Candida albicans.Materials and Methods:The solvents used for the extraction were aqueous, hexane, and methanol. The in vitro antimicrobial activity was performed by agar diffusion and disk diffusion methods and the confirmation of this activity was done by the enumeration of colony forming units (CFU).Results:The aqueous extract showed the growth inhibitory effect on Gram-positive bacteria while the Gram-negative P. aeruginosa was the most sensitive microorganism as determined by the agar diffusion technique. Surprisingly, the extract showed little antibacterial activity on other Gram-negative bacteria (E. coli) by this technique. Ethanolic extract was also found to be inhibitory to the growth of microorganisms. Hexane extract was relatively low in antimicrobial activity on Gram-negative E. coli and P. aeruginosa, while both the organic extracts were inhibitory to the growth of the fungus, C. albicans. Hexane gave no conclusive results with agar or disk diffusion methods, but showed the microbial growth inhibition in CFU enumeration. The antibacterial activity of active extracts was compared with vancomycin while antifungal activity of was compared with amphotericin B.Conclusion:The results obtained in the present study suggest that R. nervosus showed a marked antimicrobial activity with the test organisms.

Endothelial progenitor cells proliferated via MEK-dependent p42 MAPK signaling pathway.

Endothelial progenitor cells (EPCs) clinical applications have been well reported. However, due to low number of EPCs that could be isolated, EPCs expansion study became one of the main focuses. Some optimized mediums to culture EPCs were currently available. However, the proliferation signaling pathway is not clearly disclosed yet. Peripheral blood was collected from eight healthy subjects, followed by mononuclear cells (MNCs) isolation. MNCs were then prepared and cultured for 2 days. After that, non-adherent cells were harvested and further cultured for 3 days. Resulted colony-forming unit (CFU)-Hill colonies were documented and enumerated under an inverted light microscope. To detect membrane markers, immunofluorescence was performed to detect CD34, VEGFR-2, and CD133. Cell documentation was conducted under a fluorescence microscope. To check cell proliferation, XTT Cell Proliferation Assay Kit was used according to kit insert. To detect possible activation of p44/42 MAPK, western blot was performed to detect p44/42 MAPK and phosphorylated p44/42 MAPK. All visualized bands were captured and quantified. Our results showed that EPCs markers (CD34, CD133 and VEGFR-2) were detected in 3 days culture. From XTT cell proliferation assay and CFU enumeration results, we found that EPCs proliferated significantly (p = 0.012) with addition of supplement. Phosphorylated-p42 MAPK expression of EPCs treated with supplement was significantly higher than the one of EPCs without treatment. Significant inhibition of p42 MAPK phosphorylation by U0126 was observed (p = 0.012). By pretreatment of U0126, number of viable cells and CFUs treated with supplement was significantly decreased (p = 0.012). Our results showed that MEK-dependent p42 MAPK pathway might play an important role in EPCs proliferation.

Study of exogenous oxidative stress response in Escherichia coli, Pseudomonas spp., Bacillus spp., and Salmonella spp.

With a previous observation of Escherichia coli growth cessation with the supplementation of 3 mM hydrogen peroxide (H2O2) at the late log phase, the current study further demonstrated the consequences of the addition of an increased concentration (6 mM) of H2O2 and further extended the investigation on such an oxidant's impact on the growth of Salmonella spp., Pseudomonas spp., and Bacillus spp. Cell culturability was measured through the enumeration of colony-forming units (CFUs) on agar plates for up to 72 h. Subsequent changes in cell morphology and arrangements were monitored, and the cell viability was simultaneously retraced by spot tests. A sharp decline in the culturable cells of E. coli was observed after 48 h with a large mass of cell aggregates upon addition of H2O2, while Pseudomonas spp. lost viability after 36 h. Impaired morphology of such stressed cells was comparable to those of the untreated cells. Notably, Pseudomonas cells were more prone to oxidative damage compared to E. coli. In contrast, the impact of H2O2 was insignificant in the case of Salmonella spp. and Bacillus spp., suggestive of a stringent defense mechanism against oxidative stress.

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Antifungal activity of silver nanoparticles in combination with nystatin and chlorhexidine digluconate against Candida albicans and Candida glabrata biofilms

Although silver nanoparticles (SN) have been investigated as an alternative to conventional antifungal drugs in the control of Candida-associated denture stomatitis, the antifungal activity of SN in combination with antifungal drugs against Candida biofilms remains unknown. Therefore, the aim of this study was to evaluate the antifungal efficacy of SN in combination with nystatin (NYT) or chlorhexidine digluconate (CHG) against Candida albicans and Candida glabrata biofilms. The drugs alone or combined with SN were applied on mature Candida biofilms (48 h), and after 24 h of treatment their antibiofilm activities were assessed by total biomass quantification (by crystal violet staining) and colony forming units enumeration. The structure of Candida biofilms was analysed by scanning electron microscopy (SEM) images. The data indicated that SN combined with either NYT or CHG demonstrated synergistic antibiofilm activity, and this activity was dependent on the species and on the drug concentrations used. SEM images showed that some drug combinations were able to disrupt Candida biofilms. The results of this study suggest that the combination of SN with NYT or CHG may have clinical implications in the treatment of denture stomatitis. However, further studies are needed before recommending the use of these drugs safely in clinical situations.

Effects of feminine hygiene products on the vaginal mucosal biome

BackgroundOver-the-counter (OTC) feminine hygiene products come with little warning about possible side effects. This study evaluates in-vitro their effects on Lactobacillus crispatus, which is dominant in the normal vaginal microbiota and helps maintain a healthy mucosal barrier essential for normal reproductive function and prevention of sexually transmitted infections and gynecologic cancer.MethodsA feminine moisturizer (Vagisil), personal lubricant, and douche were purchased OTC. A topical spermicide (nonoxynol-9) known to alter the vaginal immune barrier was used as a control. L. crispatus was incubated with each product for 2 and 24h and then seeded on agar for colony forming units (CFU). Human vaginal epithelial cells were exposed to products in the presence or absence of L. crispatus for 24h, followed by epithelium-associated CFU enumeration. Interleukin-8 was immunoassayed and ANOVA was used for statistical evaluation.ResultsNonoxynol-9 and Vagisil suppressed Lactobacillus growth at 2h and killed all bacteria at 24h. The lubricant decreased bacterial growth insignificantly at 2h but killed all at 24h. The douche did not have a significant effect. At full strength, all products suppressed epithelial viability and all, except the douche, suppressed epithelial-associated CFU. When applied at non-toxic dose in the absence of bacteria, the douche and moisturizer induced an increase of IL-8, suggesting a potential to initiate inflammatory reaction. In the presence of L. crispatus, the proinflammatory effects of the douche and moisturizer were countered, and IL-8 production was inhibited in the presence of the other products.ConclusionSome OTC vaginal products may be harmful to L. crispatus and alter the vaginal immune environment. Illustrated through these results, L. crispatus is essential in the preservation of the function of vaginal epithelial cells in the presence of some feminine hygiene products. More research should be invested toward these products before they are placed on the market.

In Vivo Germination of Bacillus anthracis Spores During Murine Cutaneous Infection

Germination is a key step for successful Bacillus anthracis colonization and systemic dissemination. Few data are available on spore germination in vivo, and the necessity of spore and host cell interactions to initiate germination is unclear. To investigate the early interactions between B. anthracis spores and cutaneous tissue, spores were inoculated in an intraperitoneal cell-free device in guinea pigs or into the pinna of mice. Germination and bacterial growth were analyzed through colony-forming unit enumeration and electron microscopy. In the guinea pig model, germination occurred in vivo in the absence of cell contact. Similarly, in the mouse ear, germination started within 15 minutes after inoculation, and germinating spores were found in the absence of surrounding cells. Germination was not observed in macrophage-rich draining lymph nodes, liver, and spleen. Edema and lethal toxin production were not required for germination, as a toxin-deficient strain was as effective as a Sterne-like strain. B. anthracis growth was locally controlled for 6 hours. Spore germination involving no cell interactions can occur in vivo, suggesting that diffusible germinants or other signals appear sufficient. Different host tissues display drastic differences in germination-triggering capacity. Initial control of bacterial growth suggests a therapeutic means to exploit host innate defenses to hinder B. anthracis colonization.

Rapid quantification of pathogenic fungi by Cellometer image-based cytometry

The objective of this study was to develop an image-based cytometric methodology for the quantification of viable pathogenic yeasts, which can offer increased sensitivity and efficiency when compared to the traditional colony forming unit (CFU) assay. Live/dead yeast quantification by flow cytometry has been previously demonstrated, however, adoption of flow cytometric detection of pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome. Here, we quantitatively assess the growth of H. capsulatum yeasts within RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with Cellometer image-based cytometry; this method faithfully recapitulates growth trends as measured by traditional CFU enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of bone marrow-derived macrophages with a GFP-expressing strain of C. albicans. To demonstrate that image-based cytometry can be used as a tool to assess the susceptibility of fungi to antifungal drugs, we perform dose response experiments with the antifungal drugs amphotericin B and itraconazole and show that image-based cytometry allows rapid assessment of the kinetics of cytotoxicity induced by these antifungals. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens, either alone or in association with host cells.

Silver nanoparticles: influence of stabilizing agent and diameter on antifungal activity against Candida albicans and Candida glabrata biofilms

The purpose of this work was to evaluate the size-dependent antifungal activity of different silver nanoparticles (SN) colloidal suspensions against Candida albicans and Candida glabrata mature biofilms. The research presented herein used SN of three different average sizes (5, 10 and 60 nm), which were synthesized by the reduction of silver nitrate through sodium citrate and which were stabilized with ammonia or polyvinylpyrrolidone. Minimal inhibitory concentration (MIC) assays were performed using the microdilution methodology. The antibiofilm activity of SN was determined by total biomass quantification (by crystal violet staining) and colony forming units enumeration. MIC results showed that all SN colloidal suspensions were fungicidal against the tested strains at very low concentrations (0·4-3·3 μg ml(-1) ). With regard to biomass quantification, SN colloidal suspensions were very effective only against C. glabrata biofilms, achieving biomass reductions around 90% at a silver concentration of 108 μg ml(-1) . In general, all SN suspensions promoted significant log(10) reduction of the mean number of cultivable biofilm cells after exposure to silver concentrations at or higher than 108 μg ml(-1) . Moreover, the results showed that the particle size and the type of stabilizing agent used did not interfere in the antifungal activity of SN against Candida biofilms. This study suggests that SN have antifungal therapeutic potential, but further studies are still required namely regarding formulation and delivery means. SN may contribute to the development of new strategies for the improvement of oral health and quality of life particularly of the complete denture wearers.

Anticandidal Efficacy of Cinnamon Oil Against Planktonic and Biofilm Cultures of Candida parapsilosis and Candida orthopsilosis

Candida parapsilosis is yeast capable of forming biofilms on medical devices. Novel approaches for the prevention and eradication of the biofilms are desired. This study investigated the anticandidal activity of sixteen essential oils on planktonic and biofilm cultures of C. parapsilosis complex. We used molecular tools, enumeration of colony-forming units, the colourimetric MTT assay, scanning electron microscopy (SEM) and a chequerboard assay coupled with software analyses to evaluate the growth kinetics, architecture, inhibition and reduction in biofilms formed from environmental isolates of the Candida parapsilosis complex; further, we also evaluated whether essential oils would interact synergistically with amphotericin B to increase their anticandidal activities. Of the environmental C. parapsilosis isolates examined, C. parapsilosis and C. orthopsilosis were identified. Biofilm growth on polystyrene substrates peaked within 48 h, after which growth remained relatively stable up to 72 h, when it began to decline. Details of the architectural analysis assessed by SEM showed that C. parapsilosis complex formed less complex biofilms compared with C. albicans biofilms. The most active essential oil was cinnamon oil (CO), which showed anticandidal activity against C. orthopsilosis and C. parapsilosis in both suspension (minimum inhibitory concentration-MIC-250 and 500 μg/ml) and biofilm (minimum biofilm reduction concentration-MBRC-1,000 and 2,000 μg/ml) cultures. CO also inhibited biofilm formation (MBIC) at concentrations above 250 μg/ml for both species tested. However, synergism with amphotericin B was not observed. Thus, CO is a natural anticandidal agent that can be effectively utilised for the control of the yeasts tested.

Factors affecting survival of Listeria monocytogenes and Listeria innocua in soil samples

We investigated the ability of several strains of L. monocytogenes and Listeria innocua strains to survive in local soil samples in vitro. Survival of three L. monocytogenes strains, EGDe, CD83, and CD1038, and three L. innocua strains, CLIP, FH2117, FH2152, was monitored in soil samples by direct enumeration of colony-forming units on selective agar. The study did not demonstrate any species-specific difference in soil survival, and all Listeria strains exhibited a marked decline in numbers over time. Bioluminescence imaging approaches to detect lux-tagged strains in soil proved largely ineffective, most likely due to the reduced metabolic activity of strains in this environment. We investigated the influence of specific factors including the presence of a background microbiota, growth temperature, moisture and strain motility upon persistence in this environment. A sequenced L. monocytogenes strain, EGDe, was capable of active growth in sterile soil yet exhibited a decline in the presence of the normal soil microbiota. Furthermore, greater survival was seen at lower incubation temperatures in normal soil. Finally, we demonstrated a direct correlation between motility and survival of L. monocytogenes in soil with highly motile L. monocytogenes strains exhibiting greater soil survival than non-motile mutants.

Enumeration of Colony forming Units of Trichoderma in formulations-Precautions to be Taken to Avoid Errors during Serial Dilution

Traditionally serial dilution method is used for the enumeration of viable propagules in a microbial formulation. Conidia of some fungal agents like Trichoderma spp. are hydrophobic in nature. They repel water and are not dispersed uniformly in water. To enhance uniform distribution, dispersing agents like Tween 20 have to be used. In many articles in the published literature, high CFUs (>10 11 CFUs g -1 ) have been either been claimed or reported to be present in formulations. Further considering the volume (4/3 × 3.14 × r 3 ) occupied by each conidium (minimum 2 micron diameter), realistically we cannot expect high Colony Forming Units (CFUs) reported or claimed in many research papers. In the present report an attempt was made to estimate the possible 'number of conidia per cc' and 'number of conidia per g' of Trichoderma harzianum spores (NBAII Th10-MTCC 5584). The results showed that even at higher dilutions (>10 -18 to 10 -20 ) colonies of Trichoderma appeared on plates which is not theoretically possible. It is proposed that while enumerating the viable propagules in a formulation the serial dilution has to be done with Tween 20 and the tips should be changed for each dilution without fail. The dilution should be restricted to 10 9 or 10 10 . The limitations of the serial dilution beyond 10 10 and precautions to be taken are discussed.

Influence of Bacteria on Spinal Implant-Centered Infection

an in vitro and in vivo experimental study. this study was undertaken to evaluate differences in the capability of inducing an implant-centered infection between Staphylococcus aureus and Mycobacterium tuberculosis. bacterial infection is still one of the most serious and devastating complications after orthopedic implant surgery despite the advent of new antibiotics and treatment methods. S. aureus and M. tuberculosis were separately cultured with titanium plates. The bacteria colonized on the plates were isolated and cultured on culture medium. They were evaluated and compared by colony-forming units enumeration. Scanning electron microscopy was used to evaluate the difference in the colonization features of the 2 pathogens. In the in vivo experiment, 22 dogs were used to assess the susceptibility to infection after a local bacterial challenge with either S. aureus or M. tuberculosis. S. aureus showed heavy adhesion and multiplication on the surface of titanium plates in vitro, whereas M. tuberculosis rarely adhered to the surface of the plates. Under scanning electron microscopy, S. aureus colonization was observed: the coccoid was widespread on the surface of the plates but only a few M. tuberculosis cells scattered on the surface of the plates. In in vivo test, the infection rateforthe S. aureus inoculation was higher than that for the M. tuberculosis challenge. The infection rate for the entire test population (n = 44 sites) was 39.58% (19/48). The infection rates were 54.17% (13/24) for the S. aureus challenge and 25% (6/24) for the M. tuberculosis challenge, respectively (P < 0.05). it is less likely for M. tuberculosis to adhere and form a biofilm on an implant surface than S. aureus. Under otherwise identical conditions, M. tuberculosis contamination following instrumented spine surgery might lead to less occurrence of infection than S. aureus contamination.

Comparison of methods for measuring viable E. coli cells during cultivation: Great differences in the early and late exponential growth phases

Four methods, namely enumeration of colony-forming units (CFU), aerobic respiration, MTT reduction capacity and succinate dehydrogenase activity were compared to determine the viability of E. coli cells at the early and late exponential growth phases. Our results revealed that great differences in cell viability existed between these methods and that the choice of method to determine cell viability must be made with caution.

Colony Forming Unit (CFU) Assay with High Resolution Digital Imaging (HRDI): a Reliable, Standardized Technology for Evaluation of Cord Blood Potency Before and After Cryopreservation.

Abstract 2160Poster Board II-137 Background:The Total Nucleated Cell dose (TNC) is the graft characteristic most commonly used as indicator of engraftment potential for CB unit selection. Even thought well standardized, TNC does not reflect the functional state of hematopoietic progenitor cells, an important determinant of their engraftment ability, and therefore, of the CB unit quality. In contrast, CFU is a better graft characteristic since it measures the functional state, repopulation capacity and number of hematopoietic progenitor cells of a CB graft. However, it has not yet been standardized, so that it can be used for comparison of CB unit quality among different CB banks, or for evaluation of cryopreserved units prior to release for transplantation. There is therefore, an urgent need of a potency assay that could allow transplant physicians to select the highest quality unit for their patients.We sought to evaluate whether our new technology that combines the CFU assay with High Resolution Digital Imaging and colony staining (CFU-HRDI) [ASH 2008;2306], would allow for a better standardization of the traditional CFU assay and the testing of a large number of CB samples in a ”High Throughput” approach. Further, we examined whether the CFU-HRDI can be a reliable method for determination of CBU quality before release for transplantation. Methods and Results:CFU growth was assessed in a total of 7,447 CBU over a period of 14 months, by using the HRDI technology. Samples were obtained after CB processing (AXP® system) and all were evaluated in duplicates. The HRDI system achieves a resolution of 7.6 μM per pixel allowing a clear view of all colonies in the dish with their barcoded IDs. A short one-step colony staining procedure provides an even better definition of CFU-GM/E and CFU-GM by bestowing a specific color on each type. A good correlation was observed after comparison of the CFU-HRDI against traditional microscopy enumeration (R2= 0.95; p<0.001). The number of CFU (average: 3109 10×3 +/- 2100 CFU/CBU) correlated strongly with total mononuclear cell count-MNC (R2=0.66, p<0.001); TNC (R2=0.64, p<0.001), nucleated red blood cells-NRBC (R2=0.44, p<0.001) and especially with CD34+ cell content (R2=0.81, p<0.001) of the CBU.To evaluate the reproducibility obtained by the use of HRDI, CB samples (n=3) were plated multiple times each (n=24 times) and five operators with different levels of expertise counted and classified CFU in all dishes. CFU-HRDI allowed excellent reproducibility for counting and classification (CFU-GM and CFU-Mixed-Red; R2≥0.9; p<0.001; n=72). Importantly, correlation was good even when HRDI showed CFU growth of several sizes and shapes for different CB units, a feature that increases the complexity of CFU classification and enumeration. Furthermore, to evaluate the effect of cryopreservation on CB quality, a total of 186 attached segments were assessed after thawing for CFU, CD34 and viability (7-AAD). Among those units, n=43 were split in two bags after processing and thus, the respective segments could be compared (R2=0.70, p<0.001). All segments showed good viability (95 +/- 5%; range 71-100). Additionally, the CFU and CD34 cell content of the unit pre-cryopreservation were well correlated when compared to the results of their respective segments (R2= 0.57, p<0.001 and R2= 0.59, p<0.001 respectively; n=58). Summary:Our new strategy (CFU-HRDI) shows reliability and reproducibility and importantly, supports high-throughput implementation as shown by the assessment of more than 7,000 CBU/year. CFU can be objectively classified and enumerated and thus CFU-HRDI can be standardized to be used as a potency determinant of CBU. Digital images can be stored for future review or further classification of CFU colonies if needed for their association with transplant engraftment. The feasibility of CFU growth testing from thawed segments and its correlation with post-processing CFU counts strengthens its use as a practical quality control measure for the cryopreservation process prior to a CBU release for transplantation. Disclosures:No relevant conflicts of interest to declare.

Replication or death: distinct fates of pathogenic Leptospira strain Lai within macrophages of human or mouse origin

Pathogenic leptospires evoke severe diseases in humans but only cause mild chronic or asymptomatic infection in many host animals. The reasons for this diversity of infection remain unclear. Here, we demonstrated that Leptospira interrogans serovar Lai strain Lai had a similar ability to adhere to and enter primary and immortal (THP-1 and J774A.1) macrophages from human and mouse, but its intracellular fate in human macrophages differed markedly from that in mouse. The leptospires resided within membrane-bound vacuoles in the murine macrophages, but occurred free in the cytosol of human macrophages, with no surrounding vesicular membrane. Most leptospires in murine macrophages co-localized with the late-endosomal/lysosomal marker LAMP-1 and then were killed by lysosomal hydrolases, while most leptospires in human macrophages did not co-localize with this marker and survived. Enumeration of colony-forming units plus quantitative fluorimetry showed that in human, but not in murine, macrophages, the amounts of leptospires increased with incubation time. The infected human macrophages differed from mouse macrophages by displaying gradually enhanced apoptosis, in parallel with the increase in number of leptospires. These data strongly suggest that the outcome for intracellular leptospires depends on differences among host macrophages, which may account for some of the differences in the severity of leptospirosis in humans and animals.

Existence of functional lingual antimicrobial peptide in bovine milk

The lingual antimicrobial peptide (LAP) belongs to the β-defensin family in cattle and is localized in epithelial cells of alveoli in mammary glands. The aim was to investigate whether LAP is secreted into milk and whether the secreted LAP has antimicrobial activity. Decaseinated bovine skim milk was applied to sample extraction cartridges, and the eluate was used for competitive enzyme immunoassay and Western blotting to test for the presence of LAP in milk. After tricine-SDS PAGE, the gel was stained using the periodic acid-Schiff reaction to examine the possibility of glycosylation of LAP. The eluate obtained from the sample extraction cartridges was subjected to a LAP antibody-coupled affinity column, after which the antimicrobial activity of its eluate against Escherichia coli was investigated with radial diffusion plate assay and colony-forming unit enumeration following the culture of bacteria with the sample. The immunoreactive LAP was detected in the eluate by competitive enzyme immunoassay (optical density=0.437±0.012 vs. 0.468±0.016). In the Western blotting analysis, immunoreactive bands were seen around 8, 14, and 17 kDa. The bands at 14 and 17 kDa, but not 8 kDa, were periodic acid-Schiff reaction-positive. The eluate of LAP antibody-coupled affinity column had antimicrobial activity against E. coli (cfu/control=0.17±0.18). These results suggest that bovine milk contains functional LAP-like substances that exert antimicrobial activity.

On the Enhanced Antibacterial Activity of Antibiotics Mixed with Gold Nanoparticles.

The bacterial action of gentamicin and that of a mixture of gentamicin and 15-nm colloidal-gold particles onEscherichia coliK12 was examined by the agar-well-diffusion method, enumeration of colony-forming units, and turbidimetry. Addition of gentamicin to colloidal gold changed the gold color and extinction spectrum. Within the experimental errors, there were no significant differences in antibacterial activity between pure gentamicin and its mixture with gold nanoparticles (NPs). Atomic absorption spectroscopy showed that upon application of the gentamicin-particle mixture, there were no gold NPs in the zone of bacterial-growth suppression in agar. Yet, free NPs diffused into the agar. These facts are in conflict with the earlier findings indicating an enhancement of the bacterial activity of similar gentamicin–gold nanoparticle mixtures. The possible causes for these discrepancies are discussed, and the suggestion is made that a necessary condition for enhancement of antibacterial activity is the preparation of stable conjugates of NPs coated with the antibiotic molecules.

Quantification of enterococci and bifidobacteria in Georgia estuaries using conventional and molecular methods

Fecal pollution is a serious threat to the estuarine environment along the Georgia coast. Culture-dependant and molecular methodologies were utilized to compare and evaluate the abundance of fecal indicator bacteria in four Georgia estuaries (Darien River, Frederica River, Gulley Hole Creek, and St. Marys River). The functionality of enterococci and bifidobacteria as indicator organisms in marine environments was assessed, as well as Bifidobacterium adolescentis densities. At each study site, enterococci were enumerated as colony forming units (CFU) on mEI agar. For quantitative polymerase chain reaction (qPCR), genus- and species-specific primer sets were used to quantify bifidobacteria and B. adolescentis as 16S rRNA gene copies and enterococci as tuf gene copies. A high correlation ( r = 0.925) was observed between CFU and qPCR enumeration of enterococci. Enterococci densities in the estuarine rivers ranged from 3–449 CFU/100 ml on mEI plates and 4.58–5.39 Log 10 gene copies/100 ml by qPCR. Bifidobacteria densities ranged from 3.62–4.14 Log 10 gene copies/100 ml and suggested the Frederica River as least affected by fecal bacteria and the Darien River as most affected by fecal pollution. A correlation of 0.46 was observed among qPCR densities of enterococci and bifidobacteria at all sample sites. Quantitative polymerase chain reaction detection of B. adolescentis was a rapid (i.e., less than 2 h) indicator of presumptive human fecal pollution and suggested that Gulley Hole Creek, the Darien River, and the St. Marys River were affected by fecal bacteria derived from a human source. Gulley Hole Creek and the Darien River had the highest levels of fecal pollution detected in the studied estuaries. Molecular quantification of bifidobacteria may be a more accurate method of determining immediate health risks associated with fecal pollution in estuarine water than traditional and contemporary assessments of enterococci.

Modulation of Mycobacterium avium growth in vivo by cytokines: involvement of tumour necrosis factor in resistance to atypical mycobacteria

The protective mechanisms associated with resistance to atypical mycobacteria infections are not clear. In an effort to broaden our understanding of the mechanisms involved, susceptible mice were infected with a virulent strain of M. avium and various treatments were applied so as to modify the course of the disease. Treatment with an antiserum against tumour necrosis factor-alpha (TNF-alpha) significantly enhanced the experimental infection, as judged by enumeration of colony-forming units (CFU) in the spleens and livers of infected mice, suggesting a role for TNF-alpha in resistance to M. avium. In other sets of experiments, recombinant cytokines were directly infused into infected mice. Infusion of recombinant interferon-gamma (IFN-gamma) did not modify the experimental infection significantly, and infusion of interleukin-2 was also without effect. Injection of TNF-alpha enhanced resistance in susceptible animals, as seen by a reduction in the viable bacilli recovered from the spleens and livers. In a final set of experiments, we demonstrate that combinations of cytokines may induce strong resistance against M. avium, namely injection of 1 micrograms of interleukin-1 alpha and 1 micrograms of TNF-alpha at 5-day intervals which was seen to eradicate M. avium in both spleens and livers of susceptible BALB/c mice. Overall, our results suggest that induction of protection against M. avium by treatment with cytokines may be feasible, and that TNF-alpha may be a pivotal molecule in resistance to M. avium.