High Intrinsic Resistance Research Articles

Advanced strategies for combating bacterial biofilms.

Biofilms are communities of microorganisms that are formed on and attached to living or nonliving surfaces and are surrounded by an extracellular polymeric material. Biofilm formation enjoys several advantages over the pathogens in the colonization process of medical devices and patients' organs. Unlike planktonic cells, biofilms have high intrinsic resistance to antibiotics and sanitizers, and overcoming them is a significant problematic challenge in the medical and food industries. There are no approved treatments to specifically target biofilms. Thus, it is required to study and present innovative and effective methods to combat a bacterial biofilm. In this review, several strategies have been discussed for combating bacterial biofilms to improve healthcare, food safety, and industrial process.

Multilocus sequence analysis, biofilm production, antibiotic susceptibility and synergy tests of Burkholderia species in patients with and without cystic fibrosis

Burkholderia spp. emerged as important pathogens in the airways of immunocompromised humans, especially those with cystic fibrosis (CF). Failure of identification with conventional techniques, high intrinsic resistance to most antibiotics and biofilm formation can cause difficulties in the treatment of these infections. The aim of this study was to identify Burkholderia spp. strains isolated from CF and non-CF patients with with routine microbiological methods, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multilocus sequence analysis (MLSA), to determine of the antibiotic susceptibility and synergies, and to evaluate biofilm formation of these isolates. A total of 38 Burkholderia spp. (25 CF, 13 non-CF) from 26 patients were identified by biochemical, phenotypical and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and sequence types were revealed by multilocus sequence analysis (MLSA). Sequence types of isolates were identified using the PubMLST database. Characteristics of biofilm formation of clinical isolates were evaluated by microplate method. Antibiotic susceptibilities of ceftazidime, meropenem, trimethoprim-sulfamethoxazole (TMP-SXT) and levofloxacin were determined by broth microdilution method according to CLSI (2017) guidelines. Synergy tests were performed by checkerboard method. Clinical isolates were identified as Burkholderia cenocepacia (n= 16), Burkholderia contaminans (n= 11), Burkholderia gladioli (n= 4), Burkholderia dolosa (n= 4), Burkholderia multivorans (n= 2) and Burkholderia seminalis (n= 1). Sequence types of these isolates were determined as ST19, ST72, ST102, ST180, ST482, ST602, ST629, ST740, ST839 and ST1392. The correct identification at the species-level with MALDI-TOF MS was 94-100% for all isolates except B.contaminans. Biofilm formation among the identified species in the study was determined as 53% (n= 20). There was no statistical difference when the biofilm production was evaluated separately among Burkholderia species and biofilm production rates between CF (56%, 14/25) and non-CF (46%, 6/13) Burkholderia isolates (p> 0.05). Overall rates of resistance to ceftazidime, meropenem, TMP-SXT, and levofloxacin of the isolates were 35%, 66%, 50% and 40%, respectively. The antibiotic resistance against Burkholderia spp., isolates obtained from CF patients were more susceptible to ceftazidime, but no significant difference was found for other antibiotics. Synergy was determined between meropenem and TMP-SXT in two isolates. Antagonism was detected in 15 isolates, 12 of them were between meropenem and ceftazidime, three of them were between ceftazidime and TMP-SXT. Numerous resistance mechanisms may lead to higher resistance in this bacteria, whereas the antagonism between meropenem and ceftazidime in this study might be attributed to the expression of beta-lactamases. In this study, the distinctness of sequence types between Burkholderia spp. isolated from CF and non-CF patient, provided a better understanding about the importance of biofilm formation for the infections with these bacteria and emphasized that the management of therapy should be driven by the antibiotic test results.

Multilocus sequence analysis and synergy tests of Burkholderia species isolated from cystic fibrosis and non-cystic fibrosis patients

Background: Burkholderia spp. are problematic infectious agents for cystic fibrosis (CF) or chronic granulomatous disease patients. Due to the failure of identification with conventional techniques and high intrinsic resistance to most antibiotics, these infections very difficult to treat and can cause a marked reduction in life expectancy. The aim of this study was to identify Burkholderia spp. strains isolated from CF and non-CF patients with multilocus sequence analysis (MLSA). Also, the antibiotic resistance and synergies of antibiotics were evaluated. Methods & Materials: A total of 38 Burkholderia spp. (25 CF, 13 non-CF) isolates were analyzed. All the Burkholderia spp. were identified by MLSA. Antibiotic susceptibilities of ceftazidime, meropenem, trimethoprim/sulfamethoxazole (TMP-SXT) and levofloxacin were determined by broth microdilution method according to CLSI guidelines. Synergy tests are performed by checkerboard method. Results: Multilocus sequence analysis identified clinical isolates as B. cenocepacia (n = 16), B. contaminans (n = 11), B. gladioli (n = 4), B. dolosa (n = 4), B. multivorans (n = 2) and B. seminalis (n = 1). Among the 38 Burkholderia spp. isolates, overall rates of resistance to ceftazidime, meropenem, TMP-SXT, and levofloxacin was 34.2%, 65.8%, 50% and 39.5%, respectively. No significant difference in antibiotic resistance between cystic fibrosis and non-cystic fibrosis patients were detected. Synergy has shown between meropenem and TMP-SXT at two isolates. Antagonism has detected at 15 isolates (between meropenem and ceftazidime (n = 12); between ceftazidime and TMP-SXT (n = 3)) Conclusion: A variety of molecular approaches have been developed for the identification of closely related species and MLSA has successfully provided a reliable advanced discrimination method for Burkholderia isolates. Expression of several resistance mechanisms in species may lead to higher resistance, whereas antagonism between meropenem and ceftazidime might be attributed to the expression of the beta-lactamases.

Competition mechanisms of fatigue crack growth behavior in lath martensitic steel

AbstractThe fatigue crack growth behavior in 20CrMnTi lath martensitic steel with a hierarchical structure was investigated at room temperature. The microstructure was quantitatively characterized by optical microscopy, scanning electron microscopy, electron backscattering diffraction, and transmission electron microscopy. Crack growth tests were performed at a stress ratio of 0.1 under the control of a stress intensity factor range using compact tension specimens. The results show that the lath martensite with medium grain size quenched in ice salt water possessed the highest fatigue crack propagation resistance, which was attributed to the relatively high intrinsic propagation resistance and extrinsic propagation resistance. Meanwhile, the results indicate that the intrinsic propagation resistance increased with the grain size, while the extrinsic propagation resistance showed an inverse tendency. In addition, the effect of substructure on the fatigue propagation properties is discussed. The deflection angle of block interfaces in the crack growth path indicated the block unit had a significant effect on the fatigue crack propagation behavior of lath martensitic steel. Based on the Taylor model, the block size (db) was considered to be the effective grain size for controlling the crack growth threshold value (∆Kth).

The Small RNA ncS35 Regulates Growth in Burkholderia cenocepacia J2315.

Burkholderia cenocepacia J2315 is a member of the B.cepacia complex. It has a large genome with three replicons and one plasmid; 7,261 genes code for annotated proteins, while 113 code for functional RNAs. Small regulatory RNAs of B.cenocepacia have not yet been functionally characterized. We investigated a small regulatory RNA, designated ncS35, that was discovered by differential RNA sequencing. Its expression under various conditions was quantified, and a deletion mutant, ΔncS35, was constructed. Compared to planktonic growth in a rich medium, the expression of ncS35 was elevated when B.cenocepacia J2315 was grown in biofilms and in minimal medium. Cells of the deletion mutant showed increased aggregation, higher metabolic activity, a higher growth rate, and an increased susceptibility to tobramycin. A transcriptomic analysis revealed upregulation of the phenylacetic acid and tryptophan degradation pathways in ΔncS35. Computational target prediction indicated that ncS35 likely interacts with the first gene of the tryptophan degradation pathway. Overall, we demonstrated that small RNA ncS35 is a noncoding RNA with an attenuating effect on the metabolic rate and growth. It is possible that slower growth protects B.cenocepacia J2315 against stressors acting on fast-dividing cells and enhances survival under unfavorable conditions. IMPORTANCE Small RNAs play an important role in the survival of bacteria in diverse environments. We explored the physiological role of ncS35, a small RNA expressed in B.cenocepacia J2315, an opportunistic pathogen in cystic fibrosis patients. In cystic fibrosis patients, infections can lead to "cepacia syndrome," a rapidly progressing and often fatal pneumonia. Infections with Burkholderia spp. are difficult to threat with antibiotics because of their high intrinsic resistance and ability to form biofilms. We show that ncS35 attenuates the growth and reduces the metabolic rate of B.cenocepacia and influences biofilm structure. This demonstrates that as-yet-uncharacterized small RNAs with regulatory function can influence physiological traits of B.cenocepacia that are relevant for infection.

Regulatory genes coordinating antibiotic-induced changes in promoter activity and early transcriptional termination of the mycobacterial intrinsic resistance gene whiB7.

Diseases caused by various Mycobacterium sp., especially Mycobacterium tuberculosis, are a major burden on global health care. Due to high intrinsic antibiotic resistance, treatment options are severely limited. In mycobacteria, WhiB7 coordinates intrinsic resistance to a broad range of antibiotics. While WhiB7 has been established as an auto-regulatory transcriptional activator, the signals and genes needed to induce its expression are poorly understood. Using Mycobacterium smegmatis as a model, we coupled transposon mutagenesis and next generation sequencing with WhiB7-specific antibiotic selection to identify genes that contribute to WhiB7regulation and function. We showed that whiB7 expression was regulated by two coordinated processes: early termination of the whiB7 transcript and increased whiB7 promoter activity. Early termination was irreversibly maintained by constitutive expression of a putative aspartate aminotransferase gene, MSMEG_4060. A pair of hypothetical genes, MSMEG_3637 and MSMEG_3638, were identified as important contributors to whiB7 promoter induction on antibiotic challenge. Expansion of our understanding of the WhiB7-resistance pathway may lead to identification of inhibitors that allow the use of previously ineffective antibiotics to treat mycobacterial diseases.

PsrA controls the synthesis of the Pseudomonas aeruginosa quinolone signal via repression of the FadE homolog, PA0506.

Pseudomonas aeruginosa is a ubiquitous, Gram-negative opportunistic pathogen that can cause disease in various sites within the human body. This bacterium is a major source of nosocomial infections that are often difficult to treat due to high intrinsic antibiotic resistance and coordinated virulence factor production. P. aeruginosa utilizes three cell-to-cell signaling systems to regulate numerous genes in response to cell density. One of these systems utilizes the small molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]) as a signal that acts as a co-inducer for the transcriptional regulator PqsR. Quinolone signaling is required for virulence in multiple infection models, and PQS is produced during human infections, making this system an attractive target for potential drug development. In this study we have examined the role of a TetR-type transcriptional regulator, PsrA, in the regulation of PQS production by P. aeruginosa. Previous studies showed that PsrA regulates genes of the fatty acid β-oxidation pathway, including PA0506, which encodes a FadE homolog. In this report, we show that deletion of psrA resulted in a large decrease in PQS production and that co-deletion of PA0506 allowed PQS production to be restored to a wild type level. We also found that PQS production could be restored to the psrA mutant by the addition of oleic or octanoic acid. Taken together, our data suggest that psrA positively affects PQS production by repressing the transcription of PA0506, which leads to a decrease in the conversion of acyl-CoA compounds to enoyl-CoA compounds, thereby allowing some octanoyl-CoA to escape the ß-oxidation pathway and serve as a PQS precursor.

Mycobacterium abscessus WhiB7 Regulates a Species-Specific Repertoire of Genes To Confer Extreme Antibiotic Resistance.

Mycobacterium abscessus causes acute and chronic bronchopulmonary infection in patients with chronic lung damage, of which cystic fibrosis (CF) patients are particularly vulnerable. The major threat posed by this organism is its high intrinsic antibiotic resistance. A typical treatment regimen involves a 6- to 12-month-long combination therapy of clarithromycin and amikacin, with cure rates below 50% and multiple side effects, especially due to amikacin. In the present work, we show that M. abscessuswhiB7, a homologue of Mycobacterium tuberculosis and Mycobacterium smegmatis whiB7 with previously demonstrated effects on intrinsic antibiotic resistance, is strongly induced when exposed to clinically relevant antibiotics that target the ribosome: erythromycin, clarithromycin, amikacin, tetracycline, and spectinomycin. The deletion of M. abscessuswhiB7 results in sensitivity to all of the above-mentioned antibiotics. Further, we have defined and compared the whiB7 regulon of M. abscessus with the closely related nontuberculous mycobacterium (NTM) M. smegmatis to demonstrate the induction of a species-specific repertoire of genes. Finally, we show that one such gene, eis2, is specifically induced in M. abscessus by whiB7 and contributes to its higher levels of intrinsic amikacin resistance. This species-specific pattern of gene induction might account for the differences in drug susceptibilities to other antibiotics and between different mycobacterial species.

Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa.

For clinicians, Pseudomonas aeruginosa is a nightmare pathogen that is one of the top three causes of opportunistic human infections. Therapy of P. aeruginosa infections is complicated due to its natural high intrinsic resistance to antibiotics. Active efflux and decreased uptake of drugs due to cell wall/membrane permeability appear to be important issues in the acquired antibiotic tolerance mechanisms. Bacterial cell wall biosynthesis enzymes have been shown to be essential for pathogenicity of Gram-negative bacteria. However, the role of these targets in virulence has not been identified in P. aeruginosa. Here, we report knockout (k.o) mutants of six cell wall biosynthesis targets (murA, PA4450; murD, PA4414; murF, PA4416; ppiB, PA1793; rmlA, PA5163; waaA, PA4988) in P. aeruginosa PAO1, and characterized these in order to find out whether these genes and their products contribute to pathogenicity and virulence of P. aeruginosa. Except waaA k.o, deletion of cell wall biosynthesis targets significantly reduced growth rate in minimal medium compared to the parent strain. The k.o mutants showed exciting changes in cell morphology and colonial architectures. Remarkably, ΔmurF cells became grossly enlarged. Moreover, the mutants were also attenuated in vivo in a mouse infection model except ΔmurF and ΔwaaA and proved to be more sensitive to macrophage-mediated killing than the wild-type strain. Interestingly, the deletion of the murA gene resulted in loss of virulence activity in mice, and the virulence was restored in a plant model by unknown mechanism. This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection.

Structure, function and regulation of Pseudomonas aeruginosa porins

Pseudomonas aeruginosa is a Gram-negative bacterium belonging to the γ-proteobacteria. Like other members of the Pseudomonas genus, it is known for its metabolic versatility and its ability to colonize a wide range of ecological niches, such as rhizosphere, water environments and animal hosts, including humans where it can cause severe infections. Another particularity of P. aeruginosa is its high intrinsic resistance to antiseptics and antibiotics, which is partly due to its low outer membrane permeability. In contrast to Enterobacteria, pseudomonads do not possess general diffusion porins in their outer membrane, but rather express specific channel proteins for the uptake of different nutrients. The major outer membrane 'porin', OprF, has been extensively investigated, and displays structural, adhesion and signaling functions while its role in the diffusion of nutrients is still under discussion. Other porins include OprB and OprB2 for the diffusion of glucose, the two small outer membrane proteins OprG and OprH, and the two porins involved in phosphate/pyrophosphate uptake, OprP and OprO. The remaining nineteen porins belong to the so-called OprD (Occ) family, which is further split into two subfamilies termed OccD (8 members) and OccK (11 members). In the past years, a large amount of information concerning the structure, function and regulation of these porins has been published, justifying why an updated review is timely.

Defect Antiperovskite Compounds Hg3Q2I2 (Q = S, Se, and Te) for Room-Temperature Hard Radiation Detection.

The high Z chalcohalides Hg3Q2I2 (Q = S, Se, and Te) can be regarded as of antiperovskite structure with ordered vacancies and are demonstrated to be very promising candidates for X- and γ-ray semiconductor detectors. Depending on Q, the ordering of the Hg vacancies in these defect antiperovskites varies and yields a rich family of distinct crystal structures ranging from zero-dimensional to three-dimensional, with a dramatic effect on the properties of each compound. All three Hg3Q2I2 compounds show very suitable optical, electrical, and good mechanical properties required for radiation detection at room temperature. These compounds possess a high density (>7 g/cm3) and wide bandgaps (>1.9 eV), showing great stopping power for hard radiation and high intrinsic electrical resistivity, over 1011 Ω cm. Large single crystals are grown using the vapor transport method, and each material shows excellent photo sensitivity under energetic photons. Detectors made from thin Hg3Q2I2 crystals show reasonable response under a series of radiation sources, including 241Am and 57Co radiation. The dimensionality of Hg-Q motifs (in terms of ordering patterns of Hg vacancies) has a strong influence on the conduction band structure, which gives the quasi one-dimensional Hg3Se2I2 a more prominently dispersive conduction band structure and leads to a low electron effective mass (0.20 m0). For Hg3Se2I2 detectors, spectroscopic resolution is achieved for both 241Am α particles (5.49 MeV) and 241Am γ-rays (59.5 keV), with full widths at half-maximum (FWHM, in percentage) of 19% and 50%, respectively. The carrier mobility-lifetime μτ product for Hg3Q2I2 detectors is achieved as 10-5-10-6 cm2/V. The electron mobility for Hg3Se2I2 is estimated as 104 ± 12 cm2/(V·s). On the basis of these results, Hg3Se2I2 is the most promising for room-temperature radiation detection.

Direct and Pollinator-Mediated Effects of Herbivory on Strawberry and the Potential for Improved Resistance.

The global decline in pollinators has partly been blamed on pesticides, leading some to propose pesticide-free farming as an option to improve pollination. However, herbivores are likely to be more prevalent in pesticide-free environments, requiring knowledge of their effects on pollinators, and alternative crop protection strategies to mitigate any potential pollination reduction. Strawberry leaf beetles (SLB) Galerucella spp. are important strawberry pests in Northern Europe and Russia. Given that SLB attack both leaf and flower tissue, we hypothesized pollinators would discriminate against SLB-damaged strawberry plants (Fragaria vesca, cultivar ‘Rügen’), leading to lower pollination success and yield. In addition we screened the most common commercial cultivar ‘Rügen’ and wild Swedish F. vesca genotypes for SLB resistance to assess the potential for inverse breeding to restore high SLB resistance in cultivated strawberry. Behavioral observations in a controlled experiment revealed that the local pollinator fauna avoided strawberry flowers with SLB-damaged petals. Low pollination, in turn, resulted in smaller more deformed fruits. Furthermore, SLB-damaged flowers produced smaller fruits even when they were hand pollinated, showing herbivore damage also had direct effects on yield, independent of indirect effects on pollination. We found variable resistance in wild woodland strawberry to SLB and more resistant plant genotypes than the cultivar ‘Rügen’ were identified. Efficient integrated pest management strategies should be employed to mitigate both direct and indirect effects of herbivory for cultivated strawberry, including high intrinsic plant resistance.

Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR.

Determination of N-acylhomoserine lactones of Pseudomonas aeruginosa in clinical samples from dogs with otitis externa.

BackgroundBacterial intercellular communication, called quorum sensing, takes place via the production and collective response to signal molecules. In Gram-negative bacteria, like Pseudomonas aeruginosa, these signaling molecules are N-acylhomoserine lactones (AHLs). P. aeruginosa is a common cause of inflammation of the ear canal (otitis externa) in dogs. It employs quorum sensing to coordinate the expression of host tissue-damaging factors, which are largely responsible for its virulence. The treatment of P. aeruginosa-associated otitis is challenging due to a high intrinsic resistance of P. aeruginosa to several antibiotics.Attenuation of quorum sensing signals to inhibit bacterial virulence is a novel strategy for the treatment of resistant bacterial pathogens, including P. aeruginosa. Therefore, it is important to recognize and define quorum sensing signal molecules in clinical samples. To date, there are no reports on determination of AHLs in the veterinary clinical samples. The purpose of this study was to validate an analytical procedure for determination of the concentration of AHLs in the ear rinses from dogs with P. aeruginosa-associated otitis externa.Samples were obtained with rinsing the ear canals with physiological saline solution. For validation, samples from healthy dogs were spiked with none or different known amounts of the selected AHLs. With the validated procedure, AHLs were analyzed in the samples taken in weekly intervals from two dogs, receiving a standard treatment for P. aeruginosa-associated otitis externa.ResultsValidation proved that the procedure enables quantification of AHLs in non-clinical and clinical samples. In addition, a time dependent reduction of AHL concentration was detected for the treated dogs.ConclusionsOur results indicate that liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is superior in detecting AHLs compared to other chromatographic techniques. This is the first report on determination of AHLs in the clinical samples of veterinary importance. The analytical procedure described in this paper is capable of supporting novel antimicrobial strategies, which target quorum sensing.

Load relaxation of olivine single crystals

AbstractSingle crystals of ferromagnesian olivine (San Carlos, AZ, peridot; ~Fo88‐90) have been deformed in both uniaxial creep and load relaxation under conditions of ambient pressure, T = 1500°C and = 10−10 atm; creep stresses were in the range 40 ≤ σ1 (MPa) ≤ 220. The crystals were oriented such that the applied stress was parallel to [011]c, which promotes single slip on the slowest slip system in olivine, (010)[001]. The creep rates at steady state match well the results of earlier investigators, as does the stress sensitivity (a power law exponent of n = 3.6). Dislocation microstructures, including spatial distribution of low‐angle (subgrain) boundaries, additionally confirm previous investigations. Inverted primary creep (an accelerating strain rate with an increase in stress) was observed. Load relaxation, however, produced a singular response—a single hardness curve—regardless of the magnitude of creep stress or total accumulated strain preceding relaxation. The log stress versus log strain rate data from load‐relaxation and creep experiments overlap to within experimental error. The load‐relaxation behavior is distinctly different than that described for other crystalline solids, where the flow stress is affected strongly by work hardening such that a family of distinct hardness curves is generated, which are related by a scaling function. The response of olivine for the conditions studied, we argue, indicates flow that is rate limited by dislocation glide, reflecting specifically a high intrinsic lattice resistance (Peierls stress).

Treatment of multi drug resistant gram negative bacilli with inhaled polymyxin-b

Introduction: A major challenge has arisen regarding the treatment of infections caused by Gram-negative bacilli, particularly those with high level intrinsic resistance to many antibiotic classes and extreme ability to acquire resistance. Aim: The present study was conducted to assess the safety and efficacy of aerosolized polymyxin-B in comparison to intravenous polymyxin-B therapy for treatment of MDR GNB. Materials and methods: Study was performed over a period of 18 months. A Comparative two group randomized clinical study with 50 patients with 25 patients in Group A and 25 patients in Group-B is undertaken to study the outcome of therapy, fever response to therapy and side effects. Results: Mean duration of ICU stay was 28.68±9.15 days in Group-A while it was 31.64±9.16 days in Group-B. This was not statistically significant with p-value 0.258786. Pseudomonas aeruginosa is the most common organism isolated .In this study improvement, cure and failure rates were 44%, 44% and 12% respectively in Group-A(inhaled polymyxin b). while improvement,cure and failure rates were 40%,20% and 40% in Group-B(iv Polymyxin B) which was statistically significant. fever response to study was better in Group-A (inhaled polymyxin B) which was statistically significant. Adverse events to polymyxin B Inhalation (bronchospasm) occurred in 16% of the patients in group-A , Nephotoxicity in 28% of patients in group –B.but did not lead to suspension of treatment. Conclusion: Inhaled polymyxin-B was useful in treatment of nosocomial pneumonia caused by MDR-GNB in mechanically ventilated patients. As the drug is given by inhalational route systemic side effects can be minimized.

Draft Genome Sequence of Bacillus farraginis R-6540T (DSM 16013), a Spore-Forming Bacterium Isolated at Dairy Farms.

Bacillus farraginis R-6540T is a Gram-positive, aerobic, and spore-forming bacterium with very high intrinsic heat resistance. Here, we report the 5.32-Mb draft genome sequence of B. farraginis R-6540T, which is the first genome sequence of this species and will promote its fundamental research.

A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important human pathogen whose infections are difficult to treat due to its high intrinsic resistance to many antibiotics. Here, we show that the disruption of PA4456, encoding the ATP binding component of a putative ATP-binding cassette (ABC) transporter, increased the bacterium's susceptible to tetracycline and other antibiotics or toxic chemicals. Fluorescence spectroscopy and antibiotic accumulation tests showed that the interruption of the ABC transporter caused increased intracellular accumulation of tetracycline, demonstrating a role of the ABC transporter in tetracycline expulsion. Site-directed mutagenesis proved that the conserved residues of E170 in the Walker B motif and H203 in the H-loop, which are important for ATP hydrolysis, were essential for the function of PA4456. Through a genome-wide search, the PhoPQ two-component system was identified as a regulator of the computationally predicted PA4456-4452 operon that encodes the ABC transporter system. A >5-fold increase of the expression of this operon was observed in the phoQ mutant. The results obtained also show that the expression of the phzA1B1C1D1E1 operon and the production of pyocyanin were significantly higher in the ABC transporter mutant, signifying a connection between the ABC transporter and pyocyanin production. These results indicated that the PhoPQ-regulated ABC transporter is associated with intrinsic resistance to antibiotics and other adverse compounds in P. aeruginosa, probably by extruding them out of the cell.

Draft Genome Sequence of Bacillus cecembensis PN5T (DSM 21993), a Psychrotolerant Bacterium Isolated from Soil Samples near the Pindari Glacier.

Bacillus cecembensis PN5T is a Gram-positive, aerobic, and spore-forming bacterium with very high intrinsic heat resistance. Here, we report the 4.72-Mb draft genome sequence of B. cecembensis PN5T, the first genome sequence of this species, which will promote its fundamental research.

Electrical Transport Mechanism in Au Modified Nano Porous Silicon

Electrical Transport Mechanism in Au Modified Nano Porous Silicon Nano porous silicon (PS) has drawn considerable attention as a potential material for different sensors and other device applications. A metal modified PS can be of certain scientific and practical interest in order to control its high intrinsic resistivity and current transport through it to make it an efficient electronic device material. We present here a study on the electrical transport in Au-modified nano PS layers obtained by anodic etching of p-Si with in-situ Au nano-particle incorporation from the dissolved Au salt in the etching solution. The current-voltage characteristics of the resulting Au-modified PS layers have been studied with reference to the unmodified PS layers. The results suggest that direct tunnelling is the dominating charge transport mechanism in Au-modified PS layers. With the increasing Au concentration, electrical transport mechanism deviates slowly from the hopping type in low applied field, Poole-Frenkel type in moderate applied field and Fowler-Nordheim type in high applied field range as observed in case of un-modified PS layers.