Number Of CFU Research Articles

Effect of (-)-α-Bisabolol on the Inflammatory Response in Systemic Infection Experimental Model in C57BL/6 Mice.

(-)-α-Bisabolol (BISA) is an unsaturated monocyclic sesquiterpenes compound, mainly found in the essential oil of chamomile (Matricaria chamomilla). It has been reported that this compound has several biological activities, but there are few studies evaluating the activity of this compound in the systemic inflammatory response in infectious processes. The aim of this study was to evaluate the effect of BISA on the inflammatory response and survival rate in a systemic infection model, and in vitro neutrophils phagocytic activity. BISA at concentration of 3, 10, 30, and 90 μg/ml did not presented in vitro cytotoxicity in MTT assay, and at concentrations of 1 and 3 μg/ml the BISA treatment increased in vitro phagocytic neutrophil activity. For the inflammatory response study, we verified the BISA treatment effect in a cecal ligation and puncture (CLP)-induced systemic infection model in mice; in this model, we demonstrate that BISA at dose of 100 mg/kg reduced the leukocyte recruitment in peritoneal cavity; at dose of 200 mg/kg, the NO concentration was increased in the peritoneal cavity. The bacteria CFU number was reduced in mice blood in the BISA treatment, at doses of 100 and 200 mg/kg. The BISA treatment at doses of 50 and 100 mg/kg increased the myeloperoxidase activity and reduction NO production in lung tissue of mice in CLP model. At dose of 100 mg/kg, the BISA treatment was able to reduce the mortality rate of mice submitted to CLP-induced sepsis and observed for 7 days. The results suggest an effect of BISA on inflammatory response, with activity on leukocyte chemotactic and NO production, in addition to increasing the survival rate of animals submitted to CLP model.

Type II Secretion Promotes Bacterial Growth within the Legionella-Containing Vacuole in Infected Amoebae.

It was previously determined that the type II secretion system (T2SS) promotes the ability of Legionella pneumophila to grow in coculture with amoebae. Here, we discerned the stage of intracellular infection that is potentiated by comparing the wild-type and T2SS mutant legionellae for their capacity to parasitize Acanthamoeba castellanii Whereas the mutant behaved normally for entry into the host cells and subsequent evasion of degradative lysosomes, it was impaired in the ability to replicate, with that defect being first evident at approximately 9 h postentry. The replication defect was initially documented in three ways: by determining the numbers of CFU recovered from the lysates of the infected monolayers, by monitoring the levels of fluorescence associated with amoebal monolayers infected with green fluorescent protein (GFP)-expressing bacteria, and by utilizing flow cytometry to quantitate the amounts of GFP-expressing bacteria in individual amoebae. By employing confocal microscopy and newer imaging techniques, we further determined the progression in volume and shape of the bacterial vacuoles and found that the T2SS mutant grows at a decreased rate and does not attain maximally sized phagosomes. Overall, the entire infection cycle (i.e., entry to egress) was considerably slower for the T2SS mutant than it was for the wild-type strain, and the mutant's defect was maintained over multiple rounds of infection. Thus, the T2SS is absolutely required for L. pneumophila to grow to larger numbers in its intravacuolar niche within amoebae. Combining these results with those of our recent analysis of macrophage infection, T2SS is clearly a major component of L. pneumophila intracellular infection.

Safety Evaluation of Individual Pillboxes to Control Cross-Contamination in the Drug Circuit in Hospitals.

This study aims to evaluate the potential role of pillboxes used for the preparation and delivery of individual daily medical treatments in the drug circuit of the Military Instruction Hospital (France) as reservoirs of bacterial contaminants. Samples were obtained from 32 pillboxes after decontamination (T1), after preparation in the pharmacy (T2), after use in two different medical units (T3), and again after usual mechanical washing (T4). Qualitative (identification and antibiotic susceptibility) and quantitative (contamination rate and number of colony forming units—CFUs) bacteriological tests were performed. Susceptible and resistant strains of environmental saprophytes were identified. The pillbox contamination rate was relatively low at T1 (13%). It was significantly increased at T2 (63%, p = 0.001 vs. T1), again at T3 (88%, p < 0.05 vs. T2, p < 0.001 vs. T1), and finally decreased dramatically at T4 (31%, p < 0.001 vs. T3, p > 0.05 vs. T1). The number of CFUs was significantly increased at T2 compared with that of T1 (36.7 ± 13.4 and 5.36 ± 3.64, respectively, p < 0.001) and again at T3 (84.4 ± 19.4, p < 0.001 vs. T1 and T2) and was significantly reduced at T4 (7.0 ± 2.0 vs. T3, p < 0.001) to a level that was not significantly different from that at T1. So, the use of pillboxes to deliver individual medications to patients in the hospital is a potential risk factor for bacterial cross-contamination.

Vascular Graft Pre-Treatment with Daptomycin Prior to Implantation Prevents Graft Infection with Staphylococcus aureus in an In Vivo Model.

Background: Infection of vascular grafts is a life-threatening complication in cardiovascular surgical procedures. This experimental study tested the efficacy and possible harmful effects of daptomycin pre-treatment in vivo in prevention of vascular graft infection caused by Staphylococcus aureus. Methods: Polyethylene terephthalate (PET) patches (5 × 7 mm) were sewn on the infra-renal abdominal aorta of 32 New Zealand White rabbits. Before implantation, patches either were pre-treated for 15 min with daptomycin in one group (n = 13) or left untreated in the other group (n = 13) before contamination with 100 mcL bacterial solution (1 × 1010 colony-forming units [CFU] per mL). Six animals with uninfected patches without (n = 3) or with (n = 3) daptomycin pre-treatment served as controls. On postoperative day seven, all patches were explanted, washed with phosphate buffered saline, and sonicated to release viable adherent bacteria. The CFUs were quantified and aortic tissues were histologically examined. In addition, bacterial adherence on the patches was analyzed using scanning electron microscopy (SEM). Results: In the daptomycin pre-treatment group, significantly reduced numbers of CFUs on the patches were observed, compared with non-pre-treated patches (3.21 × 102 ± 1.02 × 103 mL-1 vs. 5.18 × 105 ± 1.05 × 106 mL-1; p < 0.001). Peri-vascular abscesses were visible in all rabbits with S. aureus infected patches, whereas no signs of inflammation were found in the daptomycin pre-treatment group or the control groups. Conclusions: Daptomycin showed excellent in vivo antibacterial activity against vascular graft infection caused by S. aureus, compared with non-pre-treated grafts, resulting in a significant reduction in bacterial infection and prevention of abscess formation. No harmful effects of the antibiotic pre-treatment could be observed.

EXPERIMENTAL SUBSTANTIATION OF THE USE OF MARAL DEER ANTLERS FOR COMBATING EXTREME PSYCHO-EMOTIONAL STRESS

This work was aimed at investigating the effect of maral antler powder on the activity of animal hematopoietic stem cells both in vivo and in vitro.For in vivo experiments based on the model of sleep deprivation, male mice of the CBA/CaLac line were used. Prior to the experiment, mice in the experimental and control groups were intragastrically administered with a water dispersion of a maral antler powder and distilled water, respectively. Subsequently, the extraction of bone marrow from the femur, cloning of erythro- and granulo-monocytopoiesis precursors and count of the number of cell colonies were performed. Experiments in vitro involved the extraction of bone marrow cells from the femur followed by their cultivation both in a culture containing a maral antler powder (experimental) and distilled water (control culture). The number of CFU was counted 7 days following the beginning of the experiment.Maral antlers are found to exhibit no noticeable modulating effect on the colony-forming activity of mouse hematopoietic stem cells in vitro. However, according to our in vivo experiments on mice, a preventive administration of an antler powder before a stressful infl uence (sleep deprivation) prevents suppression of erythropoiaesis processes, thus exhibiting a modulating effect on the activity of CFU-E and CFU-GM by increasing the number of CFU-E and reducing the number of CFU-GM by more than three times. The modulating effect of maral antlers on the activity of hematopoietic and stem cells is based on the infl uence of biologically active substances contained therein on the neuroendocrine regulation of the hematopoietic system occurring in living organisms.

Evaluation of the number of colony forming units on the skin of dogs after clipping the hair with two sizes of clipper blades.

To evaluate erythema and number of CFUs on the skin of dogs with hair clipped by use of 2 sizes of clipper blades. 67 client-owned dogs receiving an epidural. Hair was clipped with a No. 10 blade (approx hair length, 1.5 mm) on one half and a No. 40 blade (approx hair length, 0.25 mm) on the other half of each epidural site. Skin was surgically scrubbed with 2% chlorhexidine gluconate and 70% isopropyl alcohol. Samples were obtained immediately after clipping, after skin was scrubbed, and again 24 hours after clipping. Number of CFUs for both sides of the clipped areas, types of microorganisms, and growth on MacConkey agar were evaluated every 24 hours for 72 hours. Colonies were evaluated for bacterial morphology and Gram stain characteristics. Sites were evaluated 24 hours after clipping for evidence of erythema. 24 hours after hair was clipped, there was a significantly higher incidence of erythema and higher number of Micrococcaceae bacteria for the side clipped with the No. 40 blade than the side clipped with the No. 10 blade. Number of CFUs did not differ significantly between size of clipper blades. Clipping hair with a No. 40 blade resulted in a significant increase in the incidence of erythema and higher number of Micrococcaceae bacteria, compared with results for clipping with a No. 10 blade. These results supported use of a No. 10 clipper blade to prevent erythema and reduce variation in the skin microbiome.

Growth of Microorganisms in Propofol and Mixture of Propofol, Lidocaine and Fentanyl.

To determine the growth of microorganisms in propofol when combined with fentanyl and lidocaine in different temperatures and times in order to find out whether there is any improvement in antimicrobial effect to lengthen the safe duration of time for application of propofol. Cross-sectional study. Istanbul Aydin University Laboratory, Istanbul, Turkey, from June to September 2018. The studied drugs and thier combination was used to determine their effect on bacterial growth of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and Acinetobacter baumanni. Bacterial solutions were prepared at 0.5 MacFarland in sterile 0.9% physiological saline and diluted at 1:100 concentration. Colony numbers were measured as colony forming units mL-1 at 0, 8, and 24 hours and at 4oC, 22oC and 37oC. In general, propofol supported the growth of microorganisms. Fentanyl with propofol also promoted the growth, especially in room and body temperature at 8th and 24th hours but when combined with lidocaine, the number of CFUs was reduced significantly compared with propofol + fentanyl group. Lidocaine inhibited the growth of microorganisms in all the solutions except for candida albicans. Lidocaine was shown to have antibacterial effect which carries advantage for inhibiting infections due to propofol; but aseptic technique is essential during preparation of propofol infusions. Fentanyl like propofol also promoted the growth at room and body temperatures.

Estimation of the POD Function and the LOD of a Binary Microbiological Measurement Method from an Interlaboratory Experiment

Abstract Background: We deal with interlaboratory experiments (collaborative studies) in which k participating laboratories, selected randomly from a population of laboratories, use samples from one and the same material or matrix. They perform binary microbiological measurements for which the measurement results are either “0” (target microorganisms not detected) or “1” (target microorganisms detected). The performance of such a measurement method is described by its probability of detection (POD) function, i.e., the POD as a function of the contamination of the sample (CFU per gram or CFU per milliliter), or by the level of detection (LODp), i.e., the contamination level of the sample that is detected (measurement result “1”) with a specified probability p. Objective: We derive an approximate statistical analysis that is simple enough to be implemented in a spreadsheet application. Methods: Under the assumption of a Poisson distribution of the number of CFU in the samples, we estimate the mean POD function of the laboratories and the SD of the laboratory effect based on a complementary log-log model, a special case of the Generalized Linear Model in the special situation in which the contamination level is known by means other than the POD. The estimates are obtained by maximization of the Laplace approximation of the likelihood function. By simulation, a bias correction factor for the estimate of the SD is obtained. With the estimated POD function, LODs can be estimated. The model can also be used to evaluate the relative LOD of an alternative method with repect to a reference method. Results: The EXCEL program PODLOD-interlab_ver1.xls for this method of statistical analysis can be downloaded from http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich. Highlights: A simple approximate statistical method for the estimation of the POD and LOD is derived. The method also allows the estimation of the RLOD of an alternative method with respect to reference method. The method is implemented in an EXCEL program that can be downloaded from http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich.

The influence of substrate surface conditioning and biofilm age on the composition of Enterococcus faecalis biofilms.

To investigate the null hypothesis that neither the surface conditioning (collagen, serum, saliva) of hydroxyapatite (HA) discs, nor the biofilm age (3days vs. 21days) has a significant effect on the cellular and matrix composition of biofilms, using Enterococcus faecalis as the model organism. Sterile HA discs were conditioned with collagen, saliva or serum, and inoculated with E.faecalis to form 3-day and 21-day-old biofilms. Unconditioned discs served as controls. The biofilms were analysed using culture-dependent and independent (confocal microscopy and biochemical analysis) methods, to determine the colony-forming units and the biofilm matrix composition (polysaccharides and proteins), respectively. Statistical analyses were performed using appropriate parametric and nonparametric tests (P=0.05). Collagen conditioning significantly increased the number of CFUs in the 21-day biofilms, compared to the 3-day biofilms (P<0.05). Although the biochemical analysis revealed that surface conditioning had no significant effect on the total carbohydrate content in the 21-day biofilms, confocal microscopic analysis revealed that collagen and saliva conditioning selectively increased the polysaccharide content of 21-day biofilms, compared to the 3-day biofilms (P<0.05). The results of this study raise an important methodological concern that the substrate conditioning substances and biofilm age differentially influence the cellular and extracellular matrix components of E.faecalis biofilms.

Photothermal inactivation of methicillin-resistant Staphylococcus aureus: anti-biofilm mediated by a polypyrrole-carbon nanocomposite.

Widespread resistance to antibiotics amongst pathogens has become a tremendous challenge of high morbidity and mortality rates which increases the needs to exploring novel methods of treatment. An efficient antimicrobial procedure to root out pathogenic bacteria is photothermal therapy. In this study, antimicrobial effects of a polypyrrole-carbon nanocomposite (PPy-C) upon laser irradiation in order to destroy the pathogenic gram-positive bacterium, methicillin-resistant Staphylococcus aureus (MRSA) were assessed. The bacterial cells were incubated with 500, 750 and 1000 μg ml-1 concentrations of PPy-C and irradiated with an 808-nm laser at a power density of 1.0 W cm-2. To indicate the biocompatibility and toxic effect of the nanocomposite without and with laser irradiation, the authors counted the number of CFUs and compared it to an untreated sample. Antibacterial mechanisms of PPy-C were assessed through temperature increment, reactive oxygen species production, and protein and DNA leakages. Photothermal heating assay showed that 26°C temperature increases in the presence of 1000 µg ml-1 PPy-C led to >98% killing of MRSA. Furthermore, 20 min radiation of near-infrared light to PPy-C in different concentrations indicated destruction and reduction in the MRSA biofilm formation. Therefore, PPy-C was introduced as a photothermal absorber with a bactericidal effect in MRSA.

FBXW11 VARIANTS DECREASED HSCS QUIESCENCE AND MYELOID-BIASED DIFFERENTIATION IN VIVO

Hematopoietic stem cells (HSCs) maintain their balance of self-renewal and differentiation under the regulation of sophisticated signal transduction networks. However, the intrinsic molecular mechanism that regulates this transformative property remains elusive. Fbxw11, as a constituent of the SCF (Skp1-Cul1-F box) ubiquitin ligase complex, can target for degradation several important transcription factors. So, Fbxw11 may play a pivotal role in many aspects of hematopoiesis through regulating various signal transduction pathways. In this study, we found that the expression of Fbxw11were lower in long term hematopoietic stem cell (LT‐HSCs), and higher in hematopoietic stem and progenitor cells (LSK) and various hematopoietic progenitor cells compared with BM cells. Competitive repopulation assays were performed and we observed that Fbxw11-overexpressed HSCs impaired their ability to repopulation. Colony-forming cell assay (CFC) showed that the number of CFU-GEMM, CFU-GM, CFU-G and the total number of CFU were lower in LSK with overexpression of Fbxw11 variants when compared with LSK control. Hoechst 33342 and Ki67 staining assay showed that the percentage of LSK cells in G0 phase was decreased when Fbxw11 overexpression. Fbxw11-overexpressed HSCs expressed lower level of self-renewal associated transcription factors, such as HoxA5, Hoxbl3, Myc, Sox2 and Tim1, suggesting that Fbxw11 decreased the self-renewal ability of HSCs. Fbxw11 also led to a progressive enlargement of myeloid population and reduce of lymphocyte population. Fbxw11-overexpressed HSCs expressed higher level of myeloid differentiation associated Csf1r, CSF2r and Runx1. Our results, therefore, demonstrate a critical and unique role for Fbxw11 in regulating self-renewal of HSCs and myeloid differentiation.

The BtaF Adhesin Is Necessary for Full Virulence During Respiratory Infection by Brucella suis and Is a Novel Immunogen for Nasal Vaccination Against Brucella Infection.

Brucella enters their hosts mostly through mucosae from where it spreads systemically. Adhesion to extracellular matrix (ECM) components or to host cells is important for the infectious process, and is mediated by several adhesins, including the BtaF trimeric autotransporter. Although Th1 responses and gamma interferon (IFN-γ) are important for protection, antibodies able to block adhesions might also contribute to prevent Brucella infection. We evaluated the importance of BtaF for respiratory Brucella infection, and characterized the immune response and protection from mucosal challenge induced by nasal vaccination with recombinant BtaF. While lung CFU numbers did not differ at day 1 p.i. between mice intratracheally inoculated with B. suis M1330 (wild type) and those receiving a ΔbtaF mutant, they were reduced in the latter group at 7 and 30 days p.i. For vaccination studies the BtaF passenger domain was engineered and expressed as a soluble trimeric protein. Mice were immunized by the nasal route with BtaF or saline (control group) plus the mucosal adjuvant c-di-AMP. Specific anti-BtaF antibodies (IgG and IgA) were increased in serum, including a mixed IgG2a/IgG1 response. In vitro, these antibodies reduced bacterial adhesion to A549 alveolar epithelial cells. Specific IgA antibodies were also increased in several mucosae. Spleen cells from BtaF immunized mice significantly increased their IL-2, IL-5, IL-17, and IFN-γ secretion upon antigen stimulation. In cervical draining lymph nodes, antigen-experienced CD4+ T cells were maintained mainly as central memory cells. A BtaF-specific delayed-type hypersensitivity response was detected in BtaF immunized mice. Lung cells from the latter produced high levels of IFN-γ upon antigen stimulation. Although nasal immunization with BtaF did not protect mice against B. suis respiratory challenge, it conferred significant protection from intragastric challenge; the splenic load of B. suis was reduced by 3.28 log CFU in immunized mice. This study shows that nasal vaccination with BtaF+c-di-AMP protects against intragastric challenge with B. suis by inducing local and systemic antibody responses, central memory CD4+ T cells and strong Th1 responses. Therefore, although BtaF vaccination did not protect from B. suis respiratory infection, this adhesin constitutes a promising immunogen against mucosal B. suis infection.

SOCS1 Antagonist-Expressing Recombinant Bacillus Calmette-Guérin Enhances Antituberculosis Protection in a Mouse Model.

Suppressor of cytokine signaling 1 (SOCS1) plays a key role in the negative regulation of JAK/STAT signaling, which is involved in innate immunity and subsequent adaptive immunity. Bacillus Calmette-Guérin (BCG) induces upregulation of SOCS1 expression in host cells, which may lead to the suppression of immune responses by BCG via inhibition of the JAK/STAT signaling pathway. This might cause A reduction in the protective effect of a BCG vaccine. In the current study, we assessed the immune responses to and the protective efficacy of a recombinant BCG secreting a dominant negative mutant of the SOCS1 molecule (rBCG-SOCS1DN). C57BL/6 mice were immunized with rBCG-SOCS1DN or parental BCG Tokyo vaccine strain harboring an empty plasmid vector (rBCG-pSO). rBCG-SOCS1DN enhanced the activation of bone marrow-derived dendritic cells and the activation of T cells compared with those with rBCG-pSO. The amounts of IFN-γ, TNF-α, and IL-6 produced by splenocytes of rBCG-SOCS1DN-immunized mice were larger than those produced by splenocytes of rBCG-pSO-immunized mice. Moreover, the rBCG-SOCS1DN-immunized mice showed a substantial reduction in the number of CFU of Mycobacterium tuberculosis in the lungs and spleens compared with that in control BCG-immunized mice when the immunized mice were infected with a highly pathogenic M. tuberculosis strain by inhalation. These findings provide evidence for the possibility of rBCG-SOCS1DN being an effective M. tuberculosis vaccine with a novel concept of rBCG as a tool for immunomodulation in host cells.

Effect of Inhibition of Bacterial Proliferation by Neutral Electrolytic Water inDental Unit Waterlines

Background: Microbial contamination in dental unit waterlines (DUWLs) has recently become an important issue in the field of dental infection control. Using neutral electrolytic water as a new disinfecting method has attracted considerable attention. However, long term data about the effect of using neutral electrolytic water in clinical settings are scarce. This is the first study to evaluate the long term effectiveness of inhibition of bacterial proliferation using the purification system to supply neutral electrolytic water to refine the waterworks in DUWLs. Methods: Before the study, we investigated the actual levels of bacterial contamination in DUWLs. Then we did thorough cleaning of DUWLs and water samples were collected 6 dental units. Three dental units assigned as Group A had purification systems that used neutral electrolytic water, and the other 3 units were the Control group. Water samples were collected from the gargle water, high-speed handpieces and the three-way syringe. We utilized the equipment in Group A, and both groups were maintained for daily clinic work for 14 months. We counted the bacterial colony forming units (cfu) for each sample and identified the pathogenic bacterial species. Results: 3 and 14 months later, no microbes were detected during the study period in Group A whereas numbers of cfu which grew from the Control group increased and glucose non-fermenting gram-negative rod of possible pathogenic organisms to human were identified in the control groups. Conclusions: The water purification system using neutral electrolytic water was effective to control the proliferation of bacteria and could maintain a hygienic environment in DUWLs.

Improving the aseptic transfer procedures in hospital pharmacies part A: methods for the determination of the surface bioburden on ampoules and vials

ObjectivesTo develop methods for surface bioburden determination of ampoules and vials to be used in the validation of the disinfection procedures and in routine monitoring of ampoules and vials.MethodsThe surface...

Estimation of the POD Function and the LOD of a Binary Microbiological Measurement Method from an Interlaboratory Experiment.

Background: We deal with interlaboratory experiments (collaborative studies) in which k participating laboratories, selected randomly from a population of laboratories, use samples from one and the same material or matrix. They perform binary microbiological measurements for which the measurement results are either "0" (target microorganisms not detected) or "1" (target microorganisms detected). The performance of such a measurement method is described by its probability of detection (POD) function, i.e., the POD as a function of the contamination of the sample (CFU per gram or CFU per milliliter), or by the level of detection (LODp), i.e., the contamination level of the sample that is detected (measurement result "1") with a specified probability p. Objective: We derive an approximate statistical analysis that is simple enough to be implemented in a spreadsheet application. Methods: Under the assumption of a Poisson distribution of the number of CFU in the samples, we estimate the mean POD function of the laboratories and the SD of the laboratory effect based on a complementary log-log model, a special case of the Generalized Linear Model in the special situation in which the contamination level is known by means other than the POD. The estimates are obtained by maximization of the Laplace approximation of the likelihood function. By simulation, a bias correction factor for the estimate of the SD is obtained. With the estimated POD function, LODs can be estimated. The model can also be used to evaluate the relative LOD of an alternative method with repect to a reference method. Results: The EXCEL program PODLOD-interlab_ver1.xls for this method of statistical analysis can be downloaded from <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich">http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich</ext-link>. Highlights: A simple approximate statistical method for the estimation of the POD and LOD is derived. The method also allows the estimation of the RLOD of an alternative method with respect to reference method. The method is implemented in an EXCEL program that can be downloaded from <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="url" xlink:href="http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich">http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich</ext-link>.

Modification of the Francis Medium for the Conversion of Histoplasma capsulatum to the Yeast-Like Growth Phase.

We propose a modification of Francis agar used for identification of the causative agent of histoplasmosis by in vitro conversion of the mycelial culture to the yeast-like growth phase. For improving of the growth characteristics of the medium, we used FT-agar with glucose-vitamin additives developed for culturing of the tularemia causative agent. The modified Francis medium is characterized by significantly higher growth properties and allowed 10-fold increasing the number of CFU of yeast-like cells of both American and African histoplasmosis causative agent.

Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System.

Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.

Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci.

Precise enumeration of living intracellular bacteria is the key step to estimate the invasion potential of pathogens and host immune responses to understand the mechanism and kinetics of bacterial pathogenesis. Therefore, quantitative assessment of host-pathogen interactions is essential for development of novel antibacterial therapeutics for infectious disease. The gentamicin protection assay (GPA) is the most widely used method for these estimations by counting the CFU of intracellular living pathogens. Here, we assess the longstanding drawbacks of the GPA by employing an antistaphylococcal endopeptidase as a bactericidal agent to kill extracellular Staphylococcus aureus We found that the difference between the two methods for the recovery of intracellular CFU of S. aureus was about 5 times. We prove that the accurate number of intracellular CFU could not be precisely determined by the GPA due to the internalization of gentamicin into host cells during extracellular bacterial killing. We further demonstrate that lysostaphin-mediated extracellular bacterial clearance has advantages for measuring the kinetics of bacterial internalization on a minute time scale due to the fast and tunable activity and the inability of protein to permeate the host cell membrane. From these results, we propose that accurate quantification of intracellular bacteria and measurement of internalization kinetics can be achieved by employing enzyme-mediated killing of extracellular bacteria (enzyme protection assay [EPA]) rather than the host-permeative drug gentamicin, which is known to alter host physiology.

Effect of various drugs on differentially detectable persisters of Mycobacterium tuberculosis generated by long-term lipid diet

Persisters of Mycobacterium tuberculosis (Mtb) that fail to form colonies on agar media when de-stressed are termed as differentially detectable (DD) persisters. Since in the host, Mtb primarily survives by utilizing lipids, we used a long-term lipid diet model to induce DD persisters of M. tuberculosis. Persisters were induced by replacing the dextrose-containing medium with one containing fatty acids instead of dextrose (FAM). After 2, 4 or 6 weeks, CFU and most probable number assays were performed; the difference between the two gave an estimate of DD persisters. Since rifampicin has been shown to induce formation of DD persisters in vitro, one set of FAM cultures were also given short-term rifampicin stress after 2, 4 or 6 weeks. Fraction of DD persisters increased with time and rifampicin treatment enhanced the effect of fatty acids, at 2 and 4 weeks. At six weeks, even in the absence of rifampicin, ∼95% population were DD persisters. The DD persisters were vulnerable to drugs interfering with bacterial respiration such as thioridazine, bedaquiline and clofazimine. The study indicates potential formation of DD persisters of Mtb in a lipid-rich microenvironment in the host even before antibiotic therapy.