Growth In Broth Culture Research Articles

The impact of physiological state and environmental stress on bacterial load estimation methodologies for Mycobacterium tuberculosis

When processed in solid or liquid medium, tuberculosis patient samples yield different proportions of a heterogenous bacterial community over the duration of treatment. We aimed to derive a relationship between methodologies for bacterial load determination and assess the effect of the growth phase of the parent culture and its exposure to stress on the results. Mycobacterium tuberculosis H37Rv was grown with and without antibiotic (isoniazid or rifampicin) and sampled on day 0, 3, 11 and 21 of growth in broth culture. The bacterial load was estimated by colony counts and the BD BACTEC MGIT system. Linear and nonlinear mixed-effects models were used to describe the relationship between time-to-positivity (TTP) and time-to-growth (TTG) versus colony forming units (CFU), and growth units (GU) versus incubation time in MGIT. For samples with the same CFU, antibiotic-treated and stationary phase cells had a shorter TTP than antibiotic-free controls and early-logarithmic phase cells, respectively. Similarly, stationary phase samples reached higher GUs and had shorter TTG than early-log phase ones. This suggests that there is a population of bacterial cells that can be differentially recovered in liquid medium, giving us insight into the physiological states of the original culture, aiding the interpretation of clinical trial outputs.

In silico virtual screening for the identification of novel inhibitors against dihydrodipicolinate reductase (DapB) of Mycobacterium tuberculosis, a key enzyme of diaminopimelate pathway.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) is one of the most devastating infectious diseases afflicting a large number of human lives. In spite of the availability of several drugs for treating TB, emergence of drug-resistant strains of the pathogen has made treatment and eradication of TB a challenging task. Hence, there is an imperative need for new intervention strategies to target M. tb. The enzymes of the diaminopimelate pathway, involved in lysine biosynthesis as well as in cell wall biosynthesis, have been considered as important anti-TB drug targets. One such enzyme is dihydrodipicolinate reductase (DapB), catalyzing the reduction of 2,3-dihydrodipicolinic acid to 2,3,4,5-tetrahydro-dipicolinic acid by utilizing NADH/NADPH as its cofactor. Here, we have generated an antisense knockdown mutant strain of dapB and demonstrated a crucial role of dapB in the in vitro and intracellular growth of M. tb. Further, in silico virtual screening was performed by employing a library of ~95,000 compounds for the identification of inhibitory molecules against DapB. The molecules with high docking scores were screened against the enzymatic activity of DapB to determine their IC50 values. Further, hit molecules that inhibited DapB were screened against the M. tb growth in broth culture and inside macrophages. The molecules exhibiting M. tb inhibition were also evaluated for their cytotoxicity against various mammalian cell lines. In summary, we have identified a lead molecule, B59, with an IC50 value of 11 µg/mL and MIC99 value of 20 µg/mL, which can be further optimized to develop potent inhibitory compounds against M. tb DapB. IMPORTANCE Non-compliance to lengthy antituberculosis (TB) treatment regimen, associated side effects, and emergence of drug-resistant strains of Mycobacterium tuberculosis (M. tb) emphasize the need to develop more effective anti-TB drugs. Here, we have evaluated the role of M. tb dihydrodipicolinate reductase (DapB), a component of the diaminopimelate pathway, which is involved in the biosynthesis of both lysine and mycobacterial cell wall. We showed that DapB is essential for the in vitro as well as intracellular growth of M. tb. We further utilized M. tb DapB, as a target for identification of inhibitors by employing in silico virtual screening, and conducted various in vitro screening assays to identify inhibitors with potential to inhibit DapB activity and in vitro and intracellular growth of M. tb with no significant cytotoxicity against various mammalian cell lines. Altogether, M. tb DapB serves as an important drug target and a hit molecule, namely, 4-(3-Phenylazoquinoxalin-2-yl) butanoic acid methyl ester has been identified as an antimycobacterial molecule in our study.

Formation <i>in vitro</i> of colistin resistance in carbapenem-resistant Gram-negative bacteria and its biological cost

Introduction. The spread of resistance to carbapenems among gram-negative bacteria have led to an increase in the consumption of polymyxins and the emergence of certain strains resistant to them. Polymyxin resistance is mainly associated with mutations in chromosomal genes. The development of mutational resistance to antibiotics can lead to a decrease in the viability of bacteria; which is manifested by an increase in the duration of the cell cycle; a decrease in virulence and competitive fitness.The purpose of the study was to assess in vitro the intensity of the formation of colistin resistance in carbapenemresistant clinical isolates of gram-negative bacteria; the stability of the formed emerged resistance and its biological cost.Materials and methods. For 46 strains of Klebsiella pneumoniae; 77 strains of Pseudomonas aeruginosa and 42 strains of Acinetobacter baumannii; real time polymerase chain reaction (PCR) was used to detect the genes of carbapenemases; the minimum inhibitory concentrations (MIC) of meropenem and colistin were determined by broth microdilution method. The selection of resistant subpopulations on Muller–Hinton agar with the addition of 16 mg/l colistin was carried out. For colistin-resistant mutants and their isogenic sensitive strains; the kinetic parameters of growth in broth culture were determined. Incubation and result recording were performed on an Infinite M200 microplate reader for 18.5 hours at 35°C with measurement of light scatter in the wells every 15 minutes.Results. The production of carbapenemases MBL VIM in P. aeruginosa; MBL NDM; KPC and OXA-48 in K. pneumoniae; OXA-23 and OXA-40 in A. baumannii was observed. All strains were sensitive to colistin (MIC varied from 0.062 to 2 mg/l). The colony growth on a selective medium with16 mg/l colistin was observed for 97.8% of K. pneumoniae strains; 16.9% of P. aeruginosa strains; and 61.9% of A. baumannii strains. The mutational nature of colistin resistance was confirmed for 21.7% of K. pneumoniae strains. For colistin-resistant mutants of K. pneumoniae; a significant increase in the duration of the lag phase (Tlag) was observed: 225.6 ± 7.037 min in the wild-type susceptible strains and 245.5 ± 8.726 in resistant mutants; p = 0.037. The indicators of the doubling time of the number of microbial cells in the exponential growth phase (Tdoubling) and the area under the bacterial growth curve did not differ significantly.Conclusion. A high frequency of formation of colistin resistance in vitro in carbapenemase-producing strains of K. pneumoniae was observed. The absence of significant changes in the kinetics of microbial growth in resistant strains makes it possible to predict the further spread of mutational resistance to colistin; as well as its preservation in microbial populations of K. pneumoniae even in the case of limiting the use of this antibiotic.

New Rabbit Erythrocyte Specific Mycelial Lectins from Fusarium sp. with Complex Saccharide Specificity

Eight Fusarium sp. namely, F. acutatum, F. globosum, F. graminearum, F. lactis, F. nivale, F. proliferatum, F. pseudoanthophilum and F. robustum were screened for the presence of lectins by hemagglutination activity using human ABO, porcine, ovine, goat and rabbit erythrocytes. Mycelial extracts of all the fungal cultures except F. graminearum displayed unique lectin activity with only rabbit erythrocytes. Enzymatic treatment of rabbit erythrocytes with neuraminidase has significantly enhanced the titre of all the lectin positive extracts of fungal cultures. In contrast, most of the lectins showed a decline in lectin activity with protease treated rabbit erythrocytes. Saccharide specificity studies have shown that majority of the lectins were inhibitory towards O-acetyl sialic acids. None of the lectins from Fusarium sp. were inhibited by dextran, meso-inositol and N-acetyl-D-glucosamine. Most of the fungal cultures displayed highest hemagglutination activity during 10th day of growth in broth cultures. The unique saccharide specificity of Fusarium sp. lectins can be used for elucidating their clinical role in glycobiology research.

Ginger and its component shogaol inhibit Vibrio biofilm formation in vitro and orally protect shrimp against acute hepatopancreatic necrosis disease (AHPND)

Specific isolates of Vibrio parahaemolyticus bacteria that cause acute hepatopancreatic necrosis disease (AHPND) colonize and form biofilms on the chitin lining of the shrimp stomach where they release toxins that cause necrosis in the adjacent hepatopancreas. One potential strategy to prevent AHPND is to inhibit biofilm formation in the stomach using feed additives. Thus, we tested ethanolic extracts of ginger (0.2 mg/ml and 2 mg/ml) for their ability to inhibit biofilm formation by an AHPND-causing isolate of V. parahaemolyticus (3HP) using chitosan coated 96-well polystyrene plastic plates to mimic the shrimp stomach lining. The extract significantly (p < .05) inhibited biofilm formation in those plates but did not inhibit cell growth in broth culture in parallel uncoated plates. Subsequent in vivo immersion-challenge experiments using 3HP (106 CFU/ml) showed that use of extract-supplemented feed (0.2 mg/g and 2 mg/g) for 7 days prior to challenge protected whiteleg shrimp (Penaeus vannamei) against mortality (40%–60% higher survival) when compared to control shrimp fed un-supplemented feed. Extract supplementation had no apparent negative effect on feed palatability or on shrimp growth. Three potentially bioactive compounds known to occur in ginger extracts (6-gingerol, 8-gingerol, and 6-shogaol) were also tested for efficacy in biofilm inhibition in vitro, and 6-shogaol was found to be the most potent followed by the others in descending order. None were found to affect bacterial growth in broth. The results of this study suggested that the use of ginger-based or other feed additives that inhibit biofilm formation may constitute a practical approach to reduce the negative impact of AHPND in shrimp aquaculture.

3D Printed Antibiotic and Chemotherapeutic Eluting Catheters for Potential Use in Interventional Radiology: In Vitro Proof of Concept Study

Additive manufacturing may be used as a form of personalized medicine in interventional radiology by allowing for the creation of customized bioactive constructs such as catheters that can act as a form of localized drug delivery. The purpose of the present in vitro study was to use three-dimensional (3D) printing to construct bioactive-laden bioabsorbable catheters impregnated with antibiotics and chemotherapeutics. Polylactic acid bioplastic pellets were coated with the powdered bioactive compounds gentamicin sulfate (GS) or methotrexate (MTX) to incorporate these drugs into the 3D printed constructs. The pellets were then extruded into drug-impregnated filament for fused deposition modeling 3D printing. Computer-aided design files were generated in the shapes of 14-F catheters. Scanning electron microscope imaging was used to visualize the presence of the additive powders on the surface of the printed constructs. Elution profiles were run on the antibiotic-laden catheter and MTX-laden catheters. Antibiotic-laden catheters were tested on bacterial broth and plate cultures. Both GS and MTX catheter constructs had sustained drug release up to the 5-day limit of testing. The 3D printed GS-enhanced catheters inhibited all bacterial growth in broth cultures and had an average zone of inhibition of 858 ± 118 mm2 on bacterial plates, whereas control catheters had no effect. The 3D printing manufacturing method to create instruments in percutaneous procedures is feasible. Further in vivo studies will substantiate these findings.

The VirAB ABC Transporter Is Required for VirR Regulation of Listeria monocytogenes Virulence and Resistance to Nisin.

Listeria monocytogenes is a Gram-positive intracellular pathogen that causes a severe invasive disease. Upon infecting a host cell, L. monocytogenes upregulates the transcription of numerous factors necessary for productive infection. VirR is the response regulator component of a two-component regulatory system in L. monocytogenes In this report, we have identified the putative ABC transporter encoded by genes lmo1746-lmo1747 as necessary for VirR function. We have designated lmo1746-lmo1747 virAB We constructed an in-frame deletion of virAB and determined that the ΔvirAB mutant exhibited reduced transcription of VirR-regulated genes. The ΔvirAB mutant also showed defects in in vitro plaque formation and in vivo virulence that were similar to those of a ΔvirR deletion mutant. Since VirR is important for innate resistance to antimicrobial agents, we determined the MICs of nisin and bacitracin for ΔvirAB bacteria. We found that VirAB expression was necessary for nisin resistance but was dispensable for resistance to bacitracin. This result suggested a VirAB-independent mechanism of VirR regulation in response to bacitracin. Lastly, we found that the ΔvirR and ΔvirAB mutants had no deficiency in growth in broth culture, intracellular replication, or production of the ActA surface protein, which facilitates actin-based motility and cell-to-cell spread. However, the ΔvirR and ΔvirAB mutants produced shorter actin tails during intracellular infection, which suggested that these mutants have a reduced ability to move and spread via actin-based motility. These findings have demonstrated that L. monocytogenes VirAB functions in a pathway with VirR to regulate the expression of genes necessary for virulence and resistance to antimicrobial agents.

Virtual Screening, pharmacophore development and structure based similarity search to identify inhibitors against IdeR, a transcription factor of Mycobacterium tuberculosis

ideR, an essential gene of Mycobacterium tuberculosis, is an attractive drug target as its conditional knockout displayed attenuated growth phenotype in vitro and in vivo. To the best of our knowledge, no inhibitors of IdeR are identified. We carried out virtual screening of NCI database against the IdeR DNA binding domain followed by inhibition studies using EMSA. Nine compounds exhibited potent inhibition with NSC 281033 (I-20) and NSC 12453 (I-42) exhibiting IC50 values of 2 µg/ml and 1 µg/ml, respectively. We then attempted to optimize the leads firstly by structure based similarity search resulting in a class of inhibitors based on I-42 containing benzene sulfonic acid, 4-hydroxy-3-[(2-hydroxy-1-naphthalenyl) azo] scaffold with 4 molecules exhibiting IC50 ≤ 10 µg/ml. Secondly, optimization included development of energy based pharmacophore and screening of ZINC database followed by docking studies, yielding a molecule with IC50 of 60 µg/ml. More importantly, a five-point pharmacophore model provided insight into the features essential for IdeR inhibition. Five molecules with promising IC50 values also inhibited M. tuberculosis growth in broth culture with MIC90 ranging from 17.5 µg/ml to 100 µg/ml and negligible cytotoxicity in various cell lines. We believe our work opens up avenues for further optimization studies.

Studies on the Kinetics of Antibacterial Agent Production in Two Actinomycete Strains, F9 and Is5, Isolated from Soil Samples, Iran

The main purpose of this research was to search various collections of soil samples for isolating antibacterial agent-producing actinomycetes against drug-resistant pathogens and find the relationship between growth and antibacterial agent production in broth culture. Primary screening for antibacterial agent production was done by overlay agar technique. Production kinetics was assessed by minimum inhibitory concentration (MIC) determination using dilution method for 10 days and compared to growth in broth culture. The Hypothetical growth cycle in solid medium was studied using slide culture technique. Among 110 isolated strains, 17% were active against Staphylococcus aureus (ATCC 6538), 17% against Bacillus subtilis (ATCC 12711), 8% against methicillin-resistant Staphylococcus aureus (MRSA), 4.5% against vancomycin-resistant enterococci (VRE), and 0.9% against Escherichia coli (ATCC 1399). Two of the best strains, F9 and Is5, were selected for assessing production kinetics. They showed an irregular growth in liquid medium. Fluctuations are the continuous growth/death processes. Antibacterial agent production reached the highest expression level in the end and middle of the first descending phase in growth curve for strains F9 and Is5, respectively. The best MIC values of strain F9 extract were 0.97, 0.97, 1.37, and 1.1 mg/L against S. aureus, B. subtilis, MRSA, and VRE, while the best MIC value of Is5 extract was 1.92 and 1.67 mg/L against S. aureus and B. subtilis, respectively. No change was seen in growth of E. coli in the presence of nearly 1500 mg/L strain Is5 extract during 10 days. Comparing the 16S rDNA gene sequences of isolates and type strains retrieved from GenBank/EzTaxon showed that strains F9 and Is5 have 99.88 and 95.68% similarity with Streptomyces chryseus and S. anulatus, respectively. 16S rDNA gene sequence of the isolates was deposited in the NCBI GenBank database, under the accession numbers KX417085 and KX417086. Based on the results obtained in the present study, it can be concluded that the separation possibility of the antibacterial agent-producing actinomycetes against Gram-negative and drug resistance bacteria is very low. However, new places can be a promising source for isolation of strains with the ability to produce new, diverse and effective antibacterial agents.

Secretion Chaperones PrsA2 and HtrA Are Required for Listeria monocytogenes Replication following Intracellular Induction of Virulence Factor Secretion.

The Gram-positive bacterium Listeria monocytogenes transitions from an environmental organism to an intracellular pathogen following its ingestion by susceptible mammalian hosts. Bacterial replication within the cytosol of infected cells requires activation of the central virulence regulator PrfA followed by a PrfA-dependent induction of secreted virulence factors. The PrfA-induced secreted chaperone PrsA2 and the chaperone/protease HtrA contribute to the folding and stability of select proteins translocated across the bacterial membrane. L. monocytogenes strains that lack both prsA2 and htrA exhibit near-normal patterns of growth in broth culture but are severely attenuated in vivo We hypothesized that, in the absence of PrsA2 and HtrA, the increase in PrfA-dependent protein secretion that occurs following bacterial entry into the cytosol results in misfolded proteins accumulating at the bacterial membrane with a subsequent reduction in intracellular bacterial viability. Consistent with this hypothesis, the introduction of a constitutively activated allele of prfA (prfA*) into ΔprsA2 ΔhtrA strains was found to essentially inhibit bacterial growth at 37°C in broth culture. ΔprsA2 ΔhtrA strains were additionally found to be defective for cell invasion and vacuole escape in selected cell types, steps that precede full PrfA activation. These data establish the essential requirement for PrsA2 and HtrA in maintaining bacterial growth under conditions of PrfA activation. In addition, chaperone function is required for efficient bacterial invasion and rapid vacuole lysis within select host cell types, indicating roles for PrsA2/HtrA prior to cytosolic PrfA activation and the subsequent induction of virulence factor secretion.

Antibacterial Properties of Pit and Fissure Sealant Containing S-PRG filler on &amp;lt;i&amp;gt;Streptococcus mutans&amp;lt;/i&amp;gt;

The purpose of this study was to evaluate the antibacterial properties of a sealant containing S-PRG filler compared to those of two contemporary commercial sealants to determine the inhibition of bacterial growth in broth culture and biofilm formation using the CDC Biofilm Reactor. The BeautiSealant containing S-PRG filler, the fluoride releasing ClinproTM sealant, which are known to have higher antibacterial effects, and the non-fluoride releasing Concise TM sealant were selected for this study. A Streptococcus mutans culture in BHI broth without sealant served as a negative control in the planktonic growth inhibition test. As a result, bacterial growth was inhibited in all three sealant groups compared to that in the control. The ClinproTM sealant showed a significantly reduced number of CFUs compared to those of the BeautiSealant and Concise TM sealants. However, no significant difference was detected between the BeautiSealant and Concise TM sealants. The Clinpro TM sealant significantly decreased biofilm formation compared to that by the BeautiSealant and Concise TM sealants. No significant difference was observed between the BeautiSealant and Concise TM sealants. In conclusion, the sealant containing S-PRG filler had a less potent anti-bacterial property and increased biofilm formation capacity compared to those of the fluoride releasing Clinpro TM sealant.

Antibacterial effect of doxycycline-coated dental abutment surfaces

Biofilm formation on dental abutment may lead to peri-implant mucositis and subsequent peri-implantitis. These cases are clinically treated with antibiotics such as doxycycline (Doxy). Here we used an electrochemical method of cathodic polarization to coat Doxy onto the outer surface of a dental abutment material. The Doxy-coated surface showed a burst release in phosphate-buffered saline during the first 24 h. However, a significant amount of Doxy remained on the surface for at least 2 weeks especially on a 5 mA–3 h sample with a higher Doxy amount, suggesting both an initial and a long-term bacteriostatic potential of the coated surface. Surface chemistry was analyzed by x-ray photoelectron spectroscopy and secondary ion mass spectrometry. Surface topography was evaluated by field emission scanning electron microscopy and blue-light profilometry. Longer polarization time from 1 h to 5 h and higher current density from 1 to 15 mA cm−2 resulted in a higher amount of Doxy on the surface. The surface was covered by a layer of Doxy less than 100 nm without significant changes in surface topography. The antibacterial property of the Doxy-coated surface was analyzed by biofilm and planktonic growth assays using Staphylococcus epidermidis. Doxy-coated samples reduced both biofilm accumulation and planktonic growth in broth culture, and also inhibited bacterial growth on agar plates. The antibacterial effect was stronger for samples of 5 mA–3 h coated with a higher amount of Doxy compared to that of 1 mA–1 h. Accordingly, an abutment surface coated with Doxy has potential for preventing bacterial colonization when exposed to the oral cavity. Doxy-coating could be a viable way to control peri-implant mucositis and prevent its progression into peri-implantitis.

MavN is a Legionella pneumophila vacuole-associated protein required for efficient iron acquisition during intracellular growth

Iron is essential for the growth and virulence of most intravacuolar pathogens. The mechanisms by which microbes bypass host iron restriction to gain access to this metal across the host vacuolar membrane are poorly characterized. In this work, we identify a unique intracellular iron acquisition strategy used by Legionella pneumophila. The bacterial Icm/Dot (intracellular multiplication/defect in organelle trafficking) type IV secretion system targets the bacterial-derived MavN (more regions allowing vacuolar colocalization N) protein to the surface of the Legionella-containing vacuole where this putative transmembrane protein facilitates intravacuolar iron acquisition. The ΔmavN mutant exhibits a transcriptional iron-starvation signature before its growth is arrested during the very early stages of macrophage infection. This intracellular growth defect is rescued only by the addition of excess exogenous iron to the culture medium and not a variety of other metals. Consistent with MavN being a translocated substrate that plays an exclusive role during intracellular growth, the mutant shows no defect for growth in broth culture, even under severe iron-limiting conditions. Putative iron-binding residues within the MavN protein were identified, and point mutations in these residues resulted in defects specific for intracellular growth that are indistinguishable from the ΔmavN mutant. This model of a bacterial protein inserting into host membranes to mediate iron transport provides a paradigm for how intravacuolar pathogens can use virulence-associated secretion systems to manipulate and acquire host iron.

Identification of Conserved and Species-Specific Functions of the Listeria monocytogenes PrsA2 Secretion Chaperone.

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen that relies on the regulated secretion and activity of a variety of proteins that sustain life within diverse environments. PrsA2 has recently been identified as a secreted peptidyl-prolyl cis/trans isomerase and chaperone that is dispensable for bacterial growth in broth culture but essential for L. monocytogenes virulence. Following host infection, PrsA2 contributes to the proper folding and activity of secreted proteins that are required for bacterial replication within the host cytosol and for bacterial spread to adjacent cells. PrsA2 is one member of a family of Gram-positive secretion chaperones that appear to play important roles in bacterial physiology; however, it is not known how these proteins recognize their substrate proteins or the degree to which their function is conserved across diverse Gram-positive species. We therefore examined PrsA proteins encoded by a variety of Gram-positive bacteria for functional complementation of L. monocytogenes mutants lacking prsA2. PrsA homologues encoded by Bacillus subtilis, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus mutans, Staphylococcus aureus, and Lactococcus lactis were examined for functional complementation of a variety of L. monocytogenes PrsA2-associated phenotypes central to L. monocytogenes pathogenesis and bacterial cell physiology. Our results indicate that while selected aspects of PrsA2 function are broadly conserved among diverse Gram-positive bacteria, PrsA2 exhibits unique specificity for L. monocytogenes target proteins required for pathogenesis. The L. monocytogenes PrsA2 chaperone thus appears evolutionarily optimized for virulence factor secretion within the host cell cytosol while still maintaining aspects of activity relevant to more general features of Gram-positive protein translocation.

Mycobacterium tuberculosis Response Regulators, DevR and NarL, Interact in Vivo and Co-regulate Gene Expression during Aerobic Nitrate Metabolism

Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M. tuberculosis NarL/NarS as a functional two-component system and identified His(241) and Asp(61) as conserved phosphorylation sites in NarS and NarL, respectively. Transcriptional profiling between M. tuberculosis H37Rv and a ΔnarL mutant strain during exponential growth in broth cultures with or without nitrate defined an ∼30-gene NarL regulon that exhibited significant overlap with DevR-regulated genes, thereby implicating a role for the DevR response regulator in the regulation of nitrate metabolism. Notably, expression analysis of a subset of genes common to NarL and DevR regulons in M. tuberculosis ΔdevR, ΔdevSΔdosT, and ΔnarL mutant strains revealed that in response to nitrite produced during aerobic nitrate metabolism, the DevRS/DosT regulatory system plays a primary role that is augmented by NarL. Specifically, NarL itself was unable to bind to the narK2, acg, and Rv3130c promoters in phosphorylated or unphosphorylated form; however, its interaction with DevR∼P resulted in cooperative binding, thereby enabling co-regulation of these genes. These findings support the role of physiologically derived nitrite as a metabolic signal in mycobacteria. We propose NarL-DevR binding, possibly as a heterodimer, as a novel mechanism for co-regulation of gene expression by the DevRS/DosT and NarL/NarS regulatory systems.

Antimycobacterial activity of selected medicinal plants traditionally used in Sudan to treat infectious diseases

Ethnopharmacological relevanceThe emergence of multidrug-resistant strains of Mycobacterium tuberculosis underscores the need for continuous development of new and efficient methods to determine the susceptibility of isolates of Mycobacterium tuberculosis in the search for novel antimycobacterial agents. Natural products constitute an important source of new drugs, and design and implementation of antimycobacterial susceptibility testing methods are necessary to evaluate the different extracts and compounds. In this study we have explored the antimycobacterial properties of 50 ethanolic extracts from different parts of 46 selected medicinal plants traditionally used in Sudan to treat infectious diseases. Materials and methodsPlants were harvested and ethanolic extracts were prepared. For selected extracts, fractionation with hydrophilic and hydrophobic solvents was undertaken. A luminometry-based assay was used for determination of mycobacterial growth in broth cultures and inside primary human macrophages in the presence or absence of plant extracts and fractions of extracts. Cytotoxicity was also assessed for active fractions of plant extracts. ResultsOf the tested extracts, three exhibited a significant inhibitory effect on an avirulent strain of Mycobacterium tubercluosis (H37Ra) at the initial screening doses (125 and 6.25µg/ml). These were bark and leaf extracts of Khaya senegalensis and the leaf extract of Rosmarinus officinalis L. Further fractions of these plant extracts were prepared with n-hexane, chloroform, ethyl acetate, n-butanol, ethanol and water, and the activity of these extracts was retained in hydrophobic fractions. Cytotoxicity assays revealed that the chloroform fraction of Khaya senegalensis bark was non-toxic to human monocyte-derived macrophages and other cell types at the concentrations used and hence, further analysis, including assessment of IC50 and intracellular activity was done with this fraction. ConclusionThese results encourage further investigations to identify the active compound(s) within the chloroform fraction of Khaya senegalensis bark.

Growth inhibition of lactic acid bacteria in ham by nisin: A model approach

Lactic acid bacteria (LAB) have been described as spoilage organisms in vacuum-packed cooked ham. A Fractional Factorial Design was performed to investigate the relative merits of sodium chloride, sodium lactate, sodium tripolyphosphate, sodium erythorbate, nisin and pediocin, in limiting the Lactobacillus sakei growth in broth culture. This allowed rejection of sodium chloride, sodium lactate and sodium erythorbate (no significant effects on growth), and a Central Composite Rotatable Design broth culture study was performed comparing the effects of nisin and pediocin. From this study, nisin was identified as a more important variable for inclusion into a cooked ham model (significant effects on growth parameters: logarithmic increase in the population, exponential microbial growth rate and lag phase extension). The validation of this outcome in a model formulation of vacuum-packed sliced cooked ham (0.001%, 0.007% and 0.013% of nisin) stored for 60days, confirmed the inhibitory effect of nisin on total LAB growth.

The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis

The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis.

Potent antimycobacterial activity of the pyridoxal isonicotinoyl hydrazone analog 2-pyridylcarboxaldehyde isonicotinoyl hydrazone: a lipophilic transport vehicle for isonicotinic acid hydrazide.

The rise in drug-resistant strains of Mycobacterium tuberculosis is a major threat to human health and highlights the need for new therapeutic strategies. In this study, we have assessed whether high-affinity iron chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class can restrict the growth of clinically significant mycobacteria. Screening a library of PIH derivatives revealed that one compound, namely, 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH), exhibited nanomolar in vitro activity against Mycobacterium bovis bacille Calmette-Guérin and virulent M. tuberculosis. Interestingly, PCIH is derived from the condensation of 2-pyridylcarboxaldehyde with the first-line antituberculosis drug isoniazid [i.e., isonicotinic acid hydrazide (INH)]. PCIH displayed minimal host cell toxicity and was effective at inhibiting growth of M. tuberculosis within cultured macrophages and also in vivo in mice. Further, PCIH restricted mycobacterial growth at high bacterial loads in culture, a property not observed with INH, which shares the isonicotinoyl hydrazide moiety with PCIH. When tested against Mycobacterium avium, PCIH was more effective than INH at inhibiting bacterial growth in broth culture and in macrophages, and also reduced bacterial loads in vivo. Complexation of PCIH with iron decreased its effectiveness, suggesting that iron chelation may play some role in its antimycobacterial efficacy. However, this could not totally account for its potent efficacy, and structure-activity relationship studies suggest that PCIH acts as a lipophilic vehicle for the transport of its intact INH moiety into the mammalian cell and the mycobacterium. These results demonstrate that iron-chelating agents such as PCIH may be of benefit in the treatment and control of mycobacterial infection.

Disruption of Mycobactin Biosynthesis Leads to Attenuation of Mycobacterium tuberculosis for Growth and Virulence

Low iron availability in the host upregulates the mbt gene cluster of Mycobacterium tuberculosis, which is responsible for mycobactin biosynthesis. However, the biological significance of mycobactins in the growth of this pathogen and in disease progression has not been elucidated. We have disrupted the mbtE gene (Rv2380c) in the mbt cluster to evaluate the importance of mycobactin biosynthesis in the growth and virulence of M. tuberculosis. The mbtE mutant (MtbΔmbtE) was unable to synthesize mycobactins, displayed an altered colony morphology, and was attenuated for growth in broth culture and in macrophages. Transmission electron microscopy revealed that MtbΔmbtE displayed an altered cell wall permeability. The growth characteristics and colony morphology of MtbΔmbtE were similar to wild type when the medium was supplemented with mycobactins or when MtbΔmbtE was genetically complemented with the mbtE gene. Moreover, guinea pigs infected with MtbΔmbtE exhibited a significantly reduced bacillary load and histopathological damage in the organs, in comparison to M. tuberculosis-infected animals. This study highlights the importance of mycobactins in the growth and virulence of M. tuberculosis and establishes the enzymes of mycobactin biosynthesis as novel targets for the development of therapeutic interventions against tuberculosis.