Activity Of Gram-negative Bacteria Research Articles

Glycoengineering with neuraminic acid analogs to label lipooligosaccharides and detect native sialyltransferase activity in gram-negative bacteria.

Lipooligosaccharides (LOS) are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host-microbe interactions. Certain Gram-negative pathogenic bacteria cap their LOS with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides (LOS) at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their LOS. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the LOS of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering (MOE) and Selective Exo-Enzymatic Labeling (SEEL), and that together they provide tools to modify, label, detect and visualize sialylation of bacterial LOS.

Bioactivities of black soldier fly larvae protein hydrolysate

One of the potential insects for feed and food sources is the Black Soldier Fly (BSF) (Hermetia illucens). The BSF originated in South America and has now spread to many tropical countries. The BSF larvae (BSFL) had attracted much attention due to their good nutrient content and were recently reported as having useful bioactivities. BSFL has been widely used as feed and has been found to reduce bad smells associated with the growth of pathogenic microbes. The high protein content in BSFL is advantageous in the production of bioactive peptides. This research aimed to extract and hydrolyze the protein from BSFL with protease enzyme to produce protein hydrolysate or peptides with antioxidant and antibacterial activities. The antioxidant activity was measured using DPPH and FRAP, while the antibacterial was analyzed using the agar well diffusion method. BSFL was hydrolyzed with bromelain extracted from pineapple or commercial enzyme (Enzyplex). The IC50 shown by protein hydrolysate was 9.43±0.84% (v/v) or 0.72 mg/mL. The FRAP value was 521.21±264.53 µM for BSFL protein without enzyme addition, 792.14±291.02 for BSFL with the addition of bromelain, and 5,352.114±861.99 µM for BSFL with the addition of Enzyplex. The addition of bromelain increased the FRAP value 1.2 times, while the addition of Enzyplex increased the FRAP value 10.2 times. The antibacterial activity indicated that protein hydrolysate with phosphate buffer pH 8 at 50℃ and 24 hrs incubation showed the highest antibacterial activity to the lowest are Vibrio cholerae, Bacillus cereus and Staphylococcus aureus, respectively. The results suggest that BSFL has antibacterial and antioxidant bioactivities using bromelain extracted from pineapple and Enzyplex, a commercial enzyme that contains protease, lipase and amylase. Using Enzyplex as an enzyme to hydrolyze BFSL shows a very high antioxidant capacity compared to bromelain as an enzyme. This suggests that more protein and other macromolecules contribute to the antioxidant capacity. Using bromelain as an enzyme to hydrolyze BSFL, shows that BSFL peptide has higher antibacterial activity in gramnegative bacteria.

Outer-Membrane Permeabilization, LPS Transport Inhibition: Activity, Interactions, and Structures of Thanatin Derived Antimicrobial Peptides.

Currently, viable antibiotics available to mitigate infections caused by drug-resistant Gram-negative bacteria are highly limited. Thanatin, a 21-residue-long insect-derived antimicrobial peptide (AMP), is a promising lead molecule for the potential development of novel antibiotics. Thanatin is extremely potent, particularly against the Enterobacter group of Gram-negative pathogens, e.g., E. coli and K. pneumoniae. As a mode of action, cationic thanatin efficiently permeabilizes the LPS-outer membrane and binds to the periplasmic protein LptAm to inhibit outer membrane biogenesis. Here, we have utilized N-terminal truncated 16- and 14-residue peptide fragments of thanatin and investigated structure, activity, and selectivity with correlating modes of action. A designed 16-residue peptide containing D-Lys (dk) named VF16 (V1PIIYCNRRT-dk-KCQRF16) demonstrated killing activity in Gram-negative bacteria. The VF16 peptide did not show any detectable toxicity to the HEK 293T cell line and kidney cell line Hep G2. As a mode of action, VF16 interacted with LPS, permeabilizing the outer membrane and binding to LptAm with high affinity. Atomic-resolution structures of VF16 in complex with LPS revealed cationic and aromatic surfaces involved in outer membrane interactions and permeabilization. Further, analyses of an inactive 14-residue native thanatin peptide (IM14: IIYCNRRTGKCQRM) delineated the requirement of the β-sheet structure in activity and target interactions. Taken together, this work would pave the way for the designing of short analogs of thanatin-based antimicrobials.

Bisbenzamidine and Bisbenzguanidine Ureas Act as Antibacterial Agents against Pseudomonas aeruginosa.

Due to the global rise in the number of antibiotic resistant bacterial infections over the past 20 years, there is a dire need for the development of small molecule antibiotics capable of overcoming resistance mechanisms in pathogenic bacteria. Antibiotic development against Gram-negative pathogens, such as Pseudomonas aeruginosa, is especially challenging due to their additional outer membrane which reduces antibiotic entry. Recently, it has been shown that a broad range of nitrogen functionality, including guanidines, amidines, primary amines, imidazolines, and imidazoles, promote antibiotic and adjuvant activity in Gram-negative bacteria, but few of these have been targeted towards Pseudomonas aeruginosa specifically despite this pathogen being deemed a critical threat by the United States Centers for Disease Control and Prevention. Herein, we examined a small series of known and unknown nitrogenous dimers, with guanidine, amidine, dimethyl amine, and pyridine functionality, for antibacterial activity against multidrug resistant Pseudomonas aeruginosa. We found that two, with bisbenzguanidine and bisbenzamidine functionality, are potent against clinical isolates of multidrug resistant and biofilm forming Pseudomonas aeruginosa.

Identification and structure–activity relationships for a series of N, N-disubstituted 2-aminobenzothiazoles as potent inhibitors of S. aureus

An internal collection of commercial and synthetically derived small molecule compounds was screened against several drug-resistant bacterial pathogens. Compound 1, a known N, N-disubstituted 2-aminobenzothiazole, was found to be a potent inhibitor of Staphylococcus aureus and several associated clinically relevant strains of methicillin-resistant S. aureus suggesting a possible novel mechanism of inhibition. It failed to show activity in any of the Gram-negative pathogens it was tested in. Evaluation in Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1, as well as in their respective hyperporinated and efflux pump-deletion mutants revealed that activity in Gram-negative bacteria is diminished because this benzothiazole scaffold is a substrate for bacterial efflux pumps. Several analogs of 1 were synthesized to generate basic structure–activity relationships for the scaffold which highlighted that the N-propyl imidazole moiety was critical for the observed antibacterial activity.

Antimicrobial Activity of Gram Negative Bacteria in Blood Cultures: A Study with Blood Agar in ICU of LGH, Lahore

Aim: To evaluate gram-negative rods' prevalence and antibiotic resistance, which are responsible for bloodstream infections in ICU patients. Study design: Cross-sectional study Place and duration of study: Lahore General Hospital, Lahore from 1stJuly 2022 to 31th December 2022. Methodology: Total 200 patients in ICU suspected to have bloodstream infection were enrolled in this study. Blood samples were cultured on Blood agar and then subjected to antibiotic susceptibility testing by the disk diffusion method.Statistical analysis was performed by using SPSS Software. Results: 135 were positive for Gram-negative bacterial strains and 13 show gram positive strain with 25 showing mixed growth and 27 showing no growth .In all positive cases for different bacterial isolates resistance for amikacin was 60.8%, cefepime 66.8%, ceftriaxone 79%, ceftazidime 75.6%, cotrimoxazole 57.4%, ciprofloxacin 61.4%, gentamycin 60.8%, levofloxacin 59.4%, imipenem 58.7%, meropenem 65.5%, doxycycline 51.3%, tazobactam 71.6%, and piperacillin + tazobactam74.3%. Overall gram-negative bacterial isolates exhibited a high resistance rate for cefotaxime at 79% and the lowest resistance rate for doxycyclineat 51.3%. Out of 148 positive cases, 70(35%) were male and 78(39%) were females. Most positive cases were reported in the age group of 16-25 years (33.3%). Conclusion: All the positive samples showed gram-negative rods and gram positive on gram staining. Klebsiella 37(18.5%) was the most prevalent pathogen found in blood samples of ICU patients. Keywords: Prevalence, antibiotic resistance, bloodstream infections, gram-negative rods.

Macrolide Resistance and In Vitro Potentiation by Peptidomimetics in Porcine Clinical Escherichia coli.

ABSTRACTEscherichia coli is intrinsically resistant to macrolides due to outer membrane impermeability, but may also acquire macrolide resistance genes by horizontal transfer. We evaluated the prevalence and types of acquired macrolide resistance determinants in pig clinical E. coli, and we assessed the ability of peptidomimetics to potentiate different macrolide subclasses against strains resistant to neomycin, a first-line antibiotic in the treatment of pig-enteric infections. The erythromycin MIC distribution was determined in 324 pig clinical E. coli isolates, and 62 neomycin-resistant isolates were further characterized by genome sequencing and MIC testing of azithromycin, spiramycin, tilmicosin, and tylosin. The impact on potency achieved by combining these macrolides with three selected peptidomimetic compounds was determined by checkerboard assays in six strains representing different genetic lineages and macrolide resistance gene profiles. Erythromycin MICs ranged from 16 to >1,024 μg/mL. Azithromycin showed the highest potency in wild-type strains (1 to 8 μg/mL), followed by erythromycin (16 to 128 μg/mL), tilmicosin (32 to 256 μg/mL), and spiramycin (128 to 256 μg/mL). Isolates with elevated MIC mainly carried erm(B), either alone or in combination with other acquired macrolide resistance genes, including erm(42), mef(C), mph(A), mph(B), and mph(G). All peptidomimetic-macrolide combinations exhibited synergy (fractional inhibitory concentration index [FICI] < 0.5) with a 4- to 32-fold decrease in the MICs of macrolides. Interestingly, the MICs of tilmicosin in wild-type strains were reduced to concentrations (4 to 16 μg/mL) that can be achieved in the pig intestinal tract after oral administration, indicating that peptidomimetics can potentially be employed for repurposing tilmicosin in the management of E. coli enteritis in pigs.IMPORTANCE Acquired macrolide resistance is poorly studied in Escherichia coli because of intrinsic resistance and limited antimicrobial activity in Gram-negative bacteria. This study reveals new information on the prevalence and distribution of macrolide resistance determinants in a comprehensive collection of porcine clinical E. coli from Denmark. Our results contribute to understanding the correlation between genotypic and phenotypic macrolide resistance in E. coli. From a clinical standpoint, our study provides an initial proof of concept that peptidomimetics can resensitize E. coli to macrolide concentrations that may be achieved in the pig intestinal tract after oral administration. The latter result has implications for animal health and potential applications in veterinary antimicrobial drug development in view of the high rates of antimicrobial-resistant E. coli isolated from enteric infections in pigs and the lack of viable alternatives for treating these infections.

Data-Driven Derivation of Molecular Substructures That Enhance Drug Activity in Gram-Negative Bacteria.

The complex cell envelope of Gram-negative bacteria creates a formidable barrier to antibiotic influx. Reduced drug uptake impedes drug development and contributes to a wide range of drug-resistant bacterial infections, including those caused by extremely resistant species prioritized by the World Health Organization. To develop new and efficient treatments, a better understanding of the molecular features governing Gram-negative permeability is essential. Here, we present a data-driven approach, using matched molecular pair analysis and machine learning on minimal inhibitory concentration data from Gram-positive and Gram-negative bacteria to uncover chemical features that influence Gram-negative bioactivity. We find recurring chemical moieties, of a wider range than previously known, that consistently improve activity and suggest that this insight can be used to optimize compounds for increased Gram-negative uptake. Our findings may help to expand the chemical space of broad-spectrum antibiotics and aid the search for new antibiotic compound classes.

&lt;p&gt;&lt;strong&gt;Effect of Quercetin loaded Silver nanoparticles on Gram negative and Gram positive bacteria&lt;/strong&gt;&lt;/p&gt;

Microorganisms are increasingly becoming resistant to multiple antibiotics and search for effective antibacterial agents continue to challenge researchers in the respective domain. Silver nanoparticles (AgNPs) with physicochemical properties different from their bulk counterparts and show better cellular penetration resulting in higher antimicrobial activity. Here, we studied the antimicrobial activity of AgNPs in pure state as well as when coated with quercetin, and also the mechanism of action. Quercetin is known for its antimicrobial activity, hence applied here for coating it on AgNPs. Spherical AgNPs of size ranging between 10 and 28 nm were synthesised using chemical reduction method. The AgNPs show promising antimicrobial activity in Gram negative bacteria Escherchia coli; however, AgNPs loaded with quercetin did not enhance the antimicrobial activity of AgNPs. The mechanism of antimicrobial activity of AgNPs unravelled in this study was inhibition of bacterial catalase and decrease in membrane potential (MMP) of the bacterial cell with increase in concentration of AgNPs. Another mechanistic aspect was production of reactive oxygen species (ROS) by AgNPs resulting in apoptosis. However, there was no degradation of cellular DNA which suggests that it does not contribute to antibacterial activity of AgNPs.

Compound Uptake into E.coli Can Be Facilitated by N-Alkyl Guanidiniums and Pyridiniums.

Multidrug-resistant Gram-negative bacterial infections are on the rise, and with no FDA approvals for new classes of broad-spectrum antibiotics in over 50 years, these infections constitute a major threat to human health. A significant challenge is the inability of most compounds to accumulate in Gram-negative bacteria. Recently developed predictive guidelines show that appending a primary amine to an appropriately shaped compound can enhance Gram-negative accumulation. Here, we report that other positively charged nitrogen functional groups, namely, N-alkyl guanidiniums and pyridiniums, can also facilitate compound uptake into Gram-negative bacteria. The accumulation of a set of 60 nonantibiotic compounds, consisting of 20 primary amines and their corresponding guanidiniums and pyridiniums, was assessed in Escherichia coli. We also installed these alternate functional groups onto antibiotic scaffolds and assessed their accumulation and antibacterial activity in Gram-negative bacteria. The results suggest that other positively-charged, nitrogen-containing functional groups should be considered when designing antibiotics with Gram-negative activity.

Fully bio-based coating from chitosan and phytate for fire-safety and antibacterial cotton fabrics

In this study, a fully bio-based coating was constructed by layer-by-layer deposition of chitosan (CS) and ammonium phytate (AP), to obtain fire-safety and antibacterial cotton fabrics. With about 8% weight gains of CS/AP coatings, the treated cotton fabrics self-extinguished in the vertical burning test. The data obtained from cone calorimetry showed CS/AP/cotton had much lower smoke and heat production, which indicated the fire safety of the fabrics was significantly improved for the presence of CS/AP coatings. The flame-retardant mechanism of this system was finally proposed according to the analysis of gaseous products and char residues. What is more, CS/AP coatings had higher antibacterial activity in Gram-negative bacteria and did improve the tensile strength of cotton fabrics compared with AP coating. With its ease of operation and use of non-toxic chemicals, this fully bio-based coating can further offer a feasible flame-retardant and antibacterial solution of the inflammable natural fabrics.

Addition of ethylamines to the phenols of bithionol and synthetic retinoids does not elicit activity in gram-negative bacteria

Our labs have demonstrated the activity of bithionol and synthetic retinoids against methicillin-resistant Staphylococcus aureus (MRSA), as well as their membrane-acting mechanism of action. However, the compounds lack activity in gram-negative species. Herein, we apply a known strategy for converting gram-positive agents into broad-spectrum therapies: addition of an alkylamine. By appending an alkylamine to the phenols of these known membrane disruptors, we test whether this approach is applicable to our compounds. Ultimately, biological testing in four MRSA strains and three gram-negative species showed abolished or diminished activity in all our analogs compared to their parent compounds and no gram-negative activity. Thus, we find that alkylamines would not elicit broad-spectrum activity from bithionol or CD437 derivatives.

Performance Evaluation of the MBT STAR®-Carba IVD Assay for the Detection of Carbapenemases With MALDI-TOF MS.

Objectives: The increasing rate of carbapenem resistance in Gram-negative bacteria is a major public health problem and rapid detection is essential for infection management. We evaluated the performances of the MBT STAR®-Carba IVD assay (Bruker Daltonics) to detect carbapenemase-producing organisms (CPO) from bacterial colonies and directly from positive blood culture bottles with MALDI-TOF MS.Methods: We analyzed 130 strains with a reduced susceptibility to at least one carbapenem including 109 CPO (6 KPC, 27 NDM, 21 VIM, 1 IMP, 41 OXA-48-like, 8 OXA-23, 2 OXA-24/-40, and 2 OXA-58) and 21 non-CPO. The assay on colonies was performed with all 130 strains while the assay on spiked blood cultures was performed with 45 strains. Samples were prepared with the MBT STAR®-CARBA IVD kit and imipenem hydrolysis by the potential carbapenemase was analyzed with the MBT STAR®-BL module (Bruker Daltonics) on MALDI-TOF MS.Results: Performed on colonies, the assay detected all carbapenemase-producing Enterobacteriaceae (n = 78), Pseudomonas spp. (n = 19) and Acinetobacter spp. (n = 12). All 21 tested non-CPO remained negative resulting in sensitivity and specificity of 100%. Performed on positive blood cultures, the assay detected all carbapenemase-producing Enterobacteriaceae (n = 23) and Pseudomonas spp. (n = 4) but missed 9/12 carbapenemase-producing Acinetobacter spp. However, a prolonged imipenem-incubation time of the strain pellet improved carbapenemase detection. Non-CPO from positive blood culture bottles remained negative (n = 5) with the assay with the exception of one Klebsiella pneumoniae isolate.Conclusion: The MBT STAR®-Carba IVD assay is a highly reliable method for the detection of carbapenemase activity in Gram-negative bacteria. However, time-consuming sample preparation steps and reagent costs need to be considered before implementation in a routine clinical microbiology laboratory.

Magnetically navigable 3D printed multifunctional microdevices for environmental applications

Microrobotic prototypes for water cleaning are produced combining stereolithography 3D printing and wet metallization. Different metallic layers are deposited on 3D printed parts using both electroless and electrolytic deposition to impart required functionalities. In particular, by exploiting the flexibility and versatility of electrolytic codeposition, pollutants photodegradation and bacteria killing are for the first time combined on the same device by coating it with a composite nanocoating containing titania nanoparticles in a silver matrix. The microstructure of the microrobots thus obtained is fully characterized and they are successfully actuated by applying rotating magnetic fields. From the water cleaning point of view, devices show evident photocatalytic activity towards water pollutants and antimicrobial activity for gram negative bacteria.

Effect of chemically synthesis compared to biosynthesized ZnO nanoparticles using aqueous extract of C. halicacabum and their antibacterial activity

In the present work, chemically synthesis compared to Biosynthesized ZnO NPS using aqueous extract of C. halicacabum (0.2, 0.4, and 0.6 mL) for various concentrations. Influence of the synthesized products were characterized by Ultra violet–Vis spectroscopy, Photoluminescence, Dynamic light scattering analysis, Zeta potential study, X-ray diffraction, Fourier transform infrared, Scanning electron microscopy and anti-bacterial activities, respectively, UV–visible spectrometry study revealed surface plasmon resonance at 387 nm, Dynamic light scattering analysis shows average particle size of 48 nm whereas zeta potential value confirms the stability of formed zinc oxide nanoparticles, The XRD patterns show the cubic phase and average particle sizes are 65, 62, 55, and 48 nm, The functional groups and band area of the samples were established by FT-IR spectroscopy the presence of both leaf extract and ZnO NPs. The surface morphologies analysis has been hexagonal quartzite structure confirmed from SEM. Finally antibacterial activities against Gram-positive and negative bacteria. The good antibacterial activities of Gram negative bacteria. The present Bio -ZnO NPs nanoparticles suggests that have the great potential applications on various industrial and medical fields of research.

Efflux pump inhibitor CCCP to rescue colistin susceptibility in mcr-1 plasmid-mediated colistin-resistant strains and Gram-negative bacteria.

Efflux in bacteria is a ubiquitous mechanism associated with resistance to antimicrobials agents. Efflux pump inhibitors (EPIs) have been developed to inhibit efflux mechanisms and could be a good alternative to reverse colistin resistance, but only CCCP has shown good activity. The aim of our study was to identify CCCP activity in a collection of 93 Gram-negative bacteria with known and unknown colistin resistance mechanisms including isolates with mcr-1 plasmid-mediated colistin resistance. Colistin MIC was evaluated with and without CCCP and the fold decrease of colistin MIC was calculated for each strain. In order to evaluate the effect of this combination, a time-kill study was performed on five strains carrying different colistin resistance mechanisms. Overall, CCCP was able to reverse colistin resistance for all strains tested. The effect of CCCP was significantly greater on intrinsically colistin-resistant bacteria (i.e. Proteus spp., Serratia marcescens, Morganella morganii and Providencia spp.) than on other Enterobacteriaceae (P < 0.0001). The same was true for bacteria with a heteroresistance mechanism compared to bacteria with other colistin resistance mechanisms (P < 0.0001). A time-kill study showed the combination was bacteriostatic on strains tested. These results suggest an efflux mechanism, especially on intrinsically resistant bacteria and Enterobacter spp., but further analysis is needed to identify the molecular support of this mechanism. EPIs could be an alternative for restoring colistin activity in Gram-negative bacteria. Further work is necessary to identify new EPIs that could be used in humans.

The role of porins in copper acquisition by mycobacteria

Aims and objectivesCopper poisoning in macrophages plays an important role in immunity against invading pathogens. Many bacteria, including Mycobacterium tuberculosis (Mtb), have evolved mechanisms to combat copper-derived innate immune responses. Copper homeostasis in Mtb consists of several components, including a multi-copper oxidase, copper efflux pump, cytoplasmic metallothionein and copper-sensing transcriptional regulators. However, components involved in copper uptake are unknown, which prompted this study to investigate the possible role of porins in copper uptake in mycobacteria. MethodsMycobacterium smegmatis porin mutants were created and tested for their ability to grow under copper-reduced or copper-rich conditions. The M. smegmatis porin gene mspA was expressed in Mtb, and its copper susceptibility profile was investigated in the presence of different copper concentrations. The expression level of a copper detoxifying protein, mycobacterial multi-copper oxidase (MmcO), was monitored by western blot to assess intracellular copper content. ResultsDeletion of porin genes from M. smegmatis caused a severe growth defect on trace copper medium. Copper supplementation alleviated this phenotype. The inability to acquire copper in sufficient amounts due to lack of porins can explain this phenomenon. Moreover, porin mutants showed elevated tolerance to copper at concentrations that were toxic for wild-type strains, indicating that the lack of porins protects these strains from copper poisoning. On the other hand, heterologous expression of mspA in Mtb significantly impaired growth at 2.5μM copper and eliminated growth at 15μM, while wild-type Mtb eventually reached its normal cell density at this copper concentration. Consistent with a role of porins in copper uptake, expression levels of MmcO in Mtb expressing the M. smegmatis porin mspA was above wild-type levels, indicating that cytoplasmic copper-sensing transcriptional regulators respond by derepressing the expression of copper resistance genes. Moreover, the polyamine spermine, a known inhibitor of porin activity in gram-negative bacteria, increased the tolerance of wild-type Mtb for copper suggesting that endogenous outer membrane proteins with channel-forming activity exist and contribute to copper acquisition and toxicity in Mtb. ConclusionsIt was concluded from these results that porins are involved in copper uptake in mycobacteria. Moreover, the outer membrane of Mtb was found to be an important barrier against copper intoxication so that permeabilization of this barrier (e.g., by porins) renders Mtb extremely vulnerable to copper. Consequently, copper homeostasis of Mtb provides a promising drug target for the development of a new class of anti-tuberculosis compounds that can induce a copper hypersensitivity phenotype in Mtb.

Trends and exceptions of physical properties on antibacterial activity for Gram-positive and Gram-negative pathogens.

To better understand the difficulties surrounding the identification of novel antibacterial compounds from corporate screening collections, physical properties of ∼3200 antibacterial project compounds with whole cell activity against Gram-negative or Gram-positive pathogens were profiled and compared to actives found from high throughput (HTS) screens conducted on both biochemical and phenotypic bacterial targets. The output from 23 antibacterial HTS screens illustrated that when compared to the properties of the antibacterial project compounds, the HTS actives were significantly more hydrophobic than antibacterial project compounds (typically 2-4 log units higher), and furthermore, for 14/23 HTS screens, the average clogD was higher than the screening collection average (screening collection clogD = 2.45). It was found that the consequences of this were the following: (a) lead identification programs often further gained hydrophobic character with increased biochemical potency, making the separation even larger between the physicochemical properties of known antibacterial agents and the HTS active starting point, (b) the probability of plasma protein binding and cytotoxicity are often increased, and (c) cell-based activity in Gram-negative bacteria was severely limited or, if present, demonstrated significant efflux. Our analysis illustrated that compounds least susceptible to efflux were those which were highly polar and small in MW or very large and typically zwitterionic. Hydrophobicity was often the dominant driver for HTS actives but, more often than not, precluded whole cell antibacterial activity. However, simply designing polar compounds was not sufficient for antibacterial activity and pointed to a lack of understanding of complex and specific bacterial penetration mechanisms.

Porins Increase Copper Susceptibility of Mycobacterium tuberculosis

Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 μM copper by 40% and completely suppressed growth at 15 μM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis.

Antibacterial Activity of Some New Azole Compounds

The development of antimicrobial agents to treat infections has been one of the most notable medical achievements of the past century. Despite the growing list of azoles, their clinical value has been limited by their relatively high risk of toxicity and the emergence of drug resistance. This necessitates the development of more effective broad spectrum antimicrobials with fewer side effects. We previously described the design and synthesis of some new azole derivatives in both imidazole and triazole groups. Here we selected 30 analogues of them in 4 different categories (imidazoles (1), benzimidazoles (2), triazoles (3) and benztriazoles (4)) and investigated their antibacterial activity against different species of gram positive and gram negative microorganisms. Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Bacillus subtilis as gram positive and Escherichia coli, Salmonella typhi, Shigella sonnei, Pseudomonas aeroginosa and Proteus vulgaris as gram negative bacteria were chosen. Disc plate method and serial dilution assay were used to establish the minimum inhibitory concentration of the above compounds. The results show compounds 1-dodecyl- 2-methyl-1H-imidazole (1g), 2-(1H-1-imidazolyl)-1-cyclohexanol (1a) and 2-(2-methyl-4-nitro-1H-1-imidazolyl)-1- cyclohexanol (1o) had desirable antibacterial activity on both gram positive and gram negative species. Compounds 1- trityl-1H-benzo (d) imidazole (2a) and 1-octyl-1H-1,2,4-triazole (3b) had higher effect for gram positive and gram negative bacteria respectively. Compounds 2-methyl-1- nonyl-1H-imidazole (1f) and 1-butyl-2-methyl-;4-nitro-1H-imidazole (1l) had moderate activity for gram negative bacteria. Keywords: Imidazole, triazole, benzimidazole, benztriazole, antibacterial, Medicinal Chemistry, 1-dodecyl-2-methyl-1H-imidazole, azole ring