Partially purified actinomycetes compounds enhance the intracellular damages in multi-drug resistant P. aeruginosa and K. pneumoniae

Based on the excellent nutrient level, the current study was focused on isolation and anti-bacterial activity of the actinomycetes from marine mangrove soil samples. As result, 10 different strains of actinomycetes strains were identified on actinomycetes isolation agar plates. The identified strains were shown with white, clear, uncontaminated well matured spore producing ability. Based on the initial observation, the isolated colonies were actinomycetes. The partially extracted crude compound shown excellent anti-bacterial activity against P. aeruginosa and K. pneumoniae with 15 mm and 13 mm zone of inhibitions were observed at 500 μL concentrations. The minimum inhibition concentration result was also confirmed the 500 μL concentration against both the tested concentration with high inhibition rate. Then, the intracellular damages, decreased cell growth of the crude actinomycetes extract treated bacterial strains were clearly observed by confocal laser scanning electron microscope. The extracellular damages of bacterial cell wall and shape of the both the pathogens were clearly shown by scanning electron microscope. Therefore, all the results were clearly supported to the partially extracted crude compound and it has excellent anti-bacterial activity against tested multi drug resistant bacteria.     

Synergistic effect of Myrtus communis L. essential oils and conventional antibiotics against multi-drug resistant Acinetobacter baumannii wound isolates

Acinetobacter baumannii is a rapidly emerging, highly resistant clinical pathogen with increasing prevalence. In recent years, the limited number of antimicrobial agents available for treatment of infections with multi-drug resistant (MDR) strains reinforced tendency for discovery of novel antimicrobial agents or treatment strategies. The aim of the study was to determine antimicrobial effectiveness of three Myrtus communis L. essential oils, both alone and in combination with conventional antibiotics, against MDR A. baumannii wound isolates. The results obtained highlighted the occurrence of good antibacterial effect of myrtle oils when administered alone. Using checkerboard method, the combinations of subinhibitory concentrations of myrtle essential oils and conventional antibiotics, i.e. polymixin B and ciprofloxacine were examined. The results proved synergism among M. communis L. essential oils and both antibiotics against MDR A. baumannii wound isolates, with a FIC index under or equal 0.50. Combination of subinhibitory concentrations of essential oils and ciprofloxacin most frequently reduced bacterial growth in synergistic manner. The similar has been shown for combination with polymyxin B; furthermore, the myrtle essential oil resulted in re-sensitization of the MDR wound isolates, i.e. MICs used in combination were below the cut off for the sensitivity to the antibiotic. Time-kill curve method confirmed efficacy of myrtle essential oil and polymyxin B combination, with complete reduction of bacterial count after 6 h. The detected synergy offers an opportunity for future development of treatment strategies for potentially lethal wound infections caused by MDR A. baumannii.     

Preparative HPLC fraction of Hibiscus rosa-sinensis essential oil against biofilm forming Klebsiella pneumoniae

Recent years Klebsiella pneumoniae (K. pneumoniae) biofilm formation (BF) is emerging thread worldwide. For tackling this problem, we have chosen Hibiscus rosa-. pneumoniae. The HPLC purified essential oils (EOs sinensis (H. rosa-sinensis) (HRS) to inhibit the BF K) of H. rosa-sinensis was performed against BF K. pneumoniae and showed concentration dependent biofilm inhibition. At the MBIC of EOs (90 µg/ml), the biofilm inhibition was showed at 92% against selected BF K. Pneumoniae. The biofilm metabolic assay, exopolysaccharide quantification and hydrophobicity index variation results exhibited with 88%, 92% and 89% reduction at 90 μg/mL was observed respectively. In addition, the morphological modification of MBIC treated K. pneumoniae was clearly viewed by scanning electron microscope (SEM). Overall, all the invitro experiments result were confirmed that the MBIC of H. rosa-sinensis EOs was very effective against BF K. pneumonia.     

Anti-ESBL activity of silver nanoparticles biosynthesized using soil Streptomyces species

Emergence of antibiotic resistance by bacteria has become a serious threat for public health worldwide. In this study, Streptomyces isolated from fertile soil sample was tested for biosynthesis of silver nanoparticles (AgNps) using cell-free supernatant and synthesized AgNps were screened for anti-ESBL (extended spectrum β-lactamase) activity against multi-drug resistant (MDR) ESBL-producing strain Klebsiella pneumoniae (ATCC 700603) and other medically important pathogens. Synthesis of AgNps was confirmed by change in pale yellow color to dark brown color and characteristic absorption spectra at 420 nm. The XRD spectrum displayed typical peaks of crystalline silver and EDAX analysis showed a major signal for silver. FTIR spectra revealed prominent peaks at 3,294 cm^?1 (NH stretching due to amide group), 2,952 cm^?1 (aldehydic C–H stretching) 1,658 cm^?1 indicating the presence of carbonyl group. AgNps were spherical in shape with size ranging from 20 to 70 nm. The synthesized AgNps showed significant antimicrobial activity against standard ESBL pathogen K. pneumoniae (22 mm), 21 mm against clinical ESBL isolate E. coli and 16 mm against clinical ESBL isolates K. pneumoniae and Citrobacter species, respectively. The results of this study suggest that AgNps synthesized by Streptomyces sp. VITSJK10 can be used as a potential alternative to control MDR ESBL pathogens. The present study aimed for green synthesis of AgNps using Streptomyces species and to explore its anti-ESBL activity.     

Novel strategies of essential oils,chitosan, and nano- chitosan for inhibition of multi-drug resistant: E. coli O157:H7 and Listeria monocytogenes

Despite the wide range of available antibiotics, food borne bacteria demonstrate a huge spectrum of resistance. The current study aims to use natural components such as essential oils (EOs), chitosan, and nano-chitosan that have very influential antibacterial properties with novel technologies like chitosan solution/film loaded with EOs against multi-drug resistant bacteria. Two strains of Escherichia coli O157:H7 and three strains of Listeria monocytogenes were used to estimate antibiotics resistance. Ten EOs and their mixture, chitosan, nano-chitosan, chitosan plus EO solutions, and biodegradable chitosan film enriched with EOs were tested as antibacterial agents against pathogenic bacterial strains. Results showed that E. coli O157:H7 51,659 and L. monocytogenes 19,116 relatively exhibited considerable resistance to more than one single antibiotic. Turmeric, cumin, pepper black, and marjoram did not show any inhibition zone against L. monocytogenes; Whereas, clove, thyme, cinnamon, and garlic EOs exhibited high antibacterial activity against L. monocytogenes with minimum inhibitory concentration (MIC) of 250–400 μl 100^?1 ml and against E. coli O157:H7 with an MIC of 350–500 μl 100^?1 ml, respectively. Among combinations, clove, and thyme EOs showed the highest antibacterial activity against E. coli O157:H7 with MIC of 170 μl 100^?1 ml, and the combination of cinnamon and clove EOs showed the strongest antibacterial activity against L. monocytogenes with an MIC of 120 μl 100^?1 ml. Both chitosan and nano-chitosan showed a promising potential as an antibacterial agent against pathogenic bacteria as their MICs were relatively lower against L. monocytogenes than for E. coli O157:H7. Chitosan combined with each of cinnamon, clove, and thyme oil have a more effective antibacterial activity against L. monocytogenes and E. coli O157:H7 than the mixture of oils alone. Furthermore, the use of either chitosan solution or biodegradable chitosan film loaded with a combination of clove and thyme EOs had the strongest antibacterial activity against L. monocytogenes and E. coli O157:H7. However, chitosan film without EOs did not exhibit an inhibition zone against the tested bacterial strains.     

First Emergence of acrAB and oqxAB Mediated Tigecycline Resistance in Clinical Isolates of Klebsiella pneumoniae Pre-Dating the Use of Tigecycline in a Chinese Hospital

Tigecycline is one of the few therapeutic options for treating infections caused by some multi-drug resistant pathogens, such as Klebsiella pneumoniae. However, tigecycline-resistant K. pneumoniae has been discovered recently in China. From 2009 to 2013, nine tigecycline-resistant K. pneumoniae isolates were identified in our hospital. Six of nine strains were identified before using tigecycline. To investigate the efflux-mediated resistance mechanisms of K. pneumoniae, the expression of efflux pump genes (acrA, acrB, tolC, oqxA and oqxB) and pump regulators (acrR, marA, soxS, rarA, rob and ramA) were examined by real-time RT-PCR. Molecular typing of the tigecycline resistant strains was performed. ST11 was the predominant clone of K. pneumoniae strains, while ST1414 and ST1415 were novel STs. Efflux pump inhibitor (EPI)-carbonyl cyanide chlorophenylhydrazone (CCCP) was able to reverse the resistance patterns of 5 resistant K. pneumoniae strains. In comparison with strain A111, a tigecycline-susceptible strain (negative control), we found that the expression levels of efflux pump genes and pump regulators were higher in a majority of resistant strains. Higher expression levels of regulators rarA (2.41-fold, 9.55-fold, 28.44-fold and 18.31-fold, respectively) and pump gene oqxB (3.87-fold, 31.96-fold, 50.61-fold and 29.45-fold, respectively) were observed in four tigecycline resistant strains (A363, A361, A368, A373, respectively). Increased expression of acrB was associated with ramA and marA expression. To our knowledge, studies on tigecycline resistance mechanism in K. pneumoniae are limited especially in China. In our study, we found that both efflux pump AcrAB-TolC and OqxAB contributed to tigecycline resistance in K. pneumoniae isolates.     

Antioxidant and antibacterial activities of eugenol and carvacrol‐grafted chitosan nanoparticles

Essential oils are known to possess antimicrobial and antioxidant activity while chitosan is a biocompatible polymer with antibacterial activity against a broad spectrum of bacteria. In this work, nanoparticles with both antioxidant and antibacterial properties were prepared by grafting eugenol and carvacrol (two components of essential oils) on chitosan nanoparticles. Aldehyde groups were first introduced in eugenol and carvacrol, and the grafting of these oils to chitosan nanoparticles was carried out via the Schiff base reaction. The surface concentration of the grafted essential oil components was determined by X‐ray photoelectron spectroscopy (XPS). The antioxidant activities of the carvacrol‐grafted chitosan nanoparticles (CHCA NPs) and the eugenol‐grafted chitosan nanoparticles (CHEU NPs) were assayed with diphenylpicrylhydrazyl (DPPH). Antibacterial assays were carried out with a representative gram‐negative bacterium, Escherichia coli (E. coli) and a gram‐positive bacterium, Staphylococcus aureus (S. aureus). The grafted eugenol and carvacrol conferred antioxidant activity to the chitosan nanoparticles, and the essential oil component‐grafted chitosan nanoparticles achieved an antibacterial activity equivalent to or better than that of the unmodified chitosan nanoparticles. Cytotoxicity assays using 3T3 mouse fibroblast showed that the cytotoxicity of CHEU NPs and CHCA NPs were significant lower than those of the pure essential oils. Biotechnol. Bioeng. 2009; 104: 30–39 © 2009 Wiley Periodicals, Inc.     

Novel fabrication of morphology tailored nanostructures with Gelatin/Chitosan Co-polymeric bio-composited hydrogel system to accelerate bone fracture healing and hard tissue nursing care management

In this study, we are successfully fabricated on a hydrogel consisting of TiO₂ nanoparticles loaded onto a gelatin/chitosan matrix to control the acceleration of bone fracture healing and improved the nursing care applications. Each specimen (chitosan, gelatin and titanium dioxide) were characterized and confirmed by using different techniques, Fourier transforms infrared spectroscopy, X-ray diffraction analysis, Scanning Electron Microscopy with Elemental dispersive X-ray analysis, Thermo-gravimetric and Differential thermal analysis. In addition, the cell cytotoxicity results verified that the TiO₂/gelatin-chitosan hydrogel are nontoxic to osteoblasts. And cell fixation outcome after 5 days of incubation condition revels that the enhanced in vitro cell survival and cell spreading on the prepared TiO₂ incorporated hydrogel with respect to gelatin/chitosan hydrogel. Furthermore, TiO₂/gelatin-chitosan hydrogel nanostructures can modulate the bone fracture healing, indicating a potential application on nursing care.     

Anti-biofilm activity of LC-MS based Solanum nigrum essential oils against multi drug resistant biofilm forming P. mirabilis

Urinary tract infections are second most important diseases worldwide due to the increased amount of antibiotic resistant microbes. Among the Gram negative bacteria, P. mirabilis is the dominant biofilm producer in urinary tract infections next to E. coli. Biofilm is a process that produced self-matrix of more virulence pathogens on colloidal surfaces. Based on the above fact, this study was concentrated to inhibit the P. mirabilis biofilm formation by various in-vitro experiments. In the current study, the anti-biofilm effect of essential oils was recovered from the medicinal plant of Solanum nigrum, and confirmed the available essential oils by liquid chromatography-mass spectroscopy analysis. The excellent anti-microbial activity and minimum biofilm inhibition concentration of the essential oils against P. mirabilis was indicated at 200 µg/mL. The absence of viability and altered exopolysaccharide structure of treated cells were showed by biofilm metabolic assay and phenol–sulphuric acid method. The fluorescence differentiation of P. mirabilis treated cells was showed with more damages by confocal laser scanning electron microscope. Further, more morphological changes of essential oils treated cells were differentiated from normal cells by scanning electron microscope. Altogether, the results were reported that the S. nigrum essential oils have anti-biofilm ability.     

Antibacterial activity of chitosan tripolyphosphate nanoparticles loaded with various metal ions

The aim of this study was to prepare and select chitosan nanoparticles loaded metal ions with high antibacterial activities. Chitosan nanoparticles were prepared based on ionic gelation between chitosan and sodium tripolyphosphate. Then, Ag⁺, Cu²⁺, Zn²⁺, Mn²⁺, or Fe²⁺ was individually loaded onto chitosan nanoparticles. Their particle sizes and zeta potentials were measured. Their antibacterial activities were evaluated by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli 25922, Salmonella choleraesuis ATCC 50020 and Staphylococcus aureus 25923 in vitro. Results showed that antibacterial activity was significantly enhanced by the metal ions loaded, except for Fe²⁺. Especially for chitosan nanoparticles loaded Cu²⁺, the MIC and MBC against E. coli 25922, S.choleraesuis ATCC 50020 and S. aureus 25923 were 21–42 times lower than that of Cu²⁺, respectively. Moreover, it was found that antibacterial activity was directly proportional to zeta potential.     

Gefitinib loaded PLGA and chitosan coated PLGA nanoparticles with magnified cytotoxicity against A549 lung cancer cell lines

In the current study, gefitinib loaded PLGA nanoparticles (GFT-PLGA-NPs) and chitosan coated PLGA nanoparticles (GFT-CS-PLGA-NPs) were synthesized to investigate the role of surface charge of NPs for developing drug delivery system for non-small-cell lung cancer (NSCLC). The developed NPs were evaluated for their size, PDI, zeta potential (ZP), drug entrapment, drug loading, DSC, FTIR, XRD, in vitro release profile, and morphology. The anti-cancer activity of GFT loaded PLGA NPs and GFT loaded CS-PLGA-NPs were examined in human A549 lung cancer cell lines. In vitro release studies of GFT-CS-PLGA-NPs showed more sustained release in comparison to GFT-PLGA-NPs due surface charge attraction of chitosan. In addition, viability of A549 cells decreases significantly with the increasing concentration of GFT-PLGA NPs and GFT-CS-PLGA-NPs when compared to that of pure GFT and blank PLGA NPs. In addition, the microscopic analysis and counting of viable cells also validate the cytotoxicity of the developed NPs. This investigation proved that the developed NPs would be efficient carriers to deliver GFT with improved efficacy against NSCLC.     

Sensitivity of nonfermentative gram-negative bacteria to essential oils of different origin

The sensitivity of MDR (multi drug resistant) strains of Stenotrophomonas maltophilia, Acinetobacter baumannii, and Pseudomonas aeruginosa to essential oils and their individual components was studied; bacteriostatic and bactericidal concentrations of 16 substances were determined. Crimean rose oil exhibited the highest activity, with the minimum inhibitory concentration of 1.95 μL/ mL. Growth of bacterial batch cultures in the presence of subinhibitory concentrations of essential oils or their individual components was studied. Kinetic models analysis revealed positive correlations of growth characteristics of the studied bacteria the effects of essential oils (p-level < 0.05). Correlations between lag phase duration and the death of bacterial cultures and correlations between the concentration of linalool (a component of essential oils) and the degree of growth suppression for S. maltophilia and A. baumannii were revealed.     

Antimicrobial activity of Fe‐loaded chitosan nanoparticles

To increase the antimicrobial activities of chitosan, chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ were prepared by surfactant‐assisted chitosan chelating Fe²⁺, Fe³⁺ and ionic gelation chitosan. Their chelating rates were determined by spectrophotometry. The particle sizes and zeta potentials of chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ were measured by size and zeta potential analysis. The nanoparticles antimicrobial activities were evaluated by different concentration against Escherichia coli, Staphylococcus aureus, Candida albicans in vitro. Results showed that the mean diameter of chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ were 206.4 and 195.2 nm, respectively. Their zeta potentials were +28.82 and +28.26 mV, respectively. The chelating rate of chitosan nanoparticles loaded with Fe²⁺ was greatly higher than with Fe³⁺. Their antimicrobial activity was showed greatly higher at lower concentrations compared to chitosan, and the antibacterial effect of chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ was preliminary observed.     

Biological activity of Cymbopogon schoenanthus essential oil

IntroductionA number of plant species, including Cymbopogon schoenanthus, are traditionally used for the treatment of various diseases. C. schoenanthus is currently, traded in the Saudi markets, and thought to have medicinal value. This study aimed at investigating the biological activities of C. schoenanthus against both Gram-positive and Gram-negative bacteria and to identify its chemical ingredients.Materials and methodsThe inhibitory effects of water extracts of C. schoenanthus essential oils were evaluated against ten isolates of both Gram-positive and Gram-negative bacteria using the agar well diffusion and dilution methods. The minimum inhibitory concentration (MIC) was assayed using the Broth microdilution test on five of the ten isolates. The death rates were determined by the time kill assay, done according to the Clinical Laboratory Standards Institute (CLSI) guidelines. The chemical composition of the essential oils of the plant was performed using GC/MS.ResultsThe C. schoenanthus essential oil was effective against Escherichia coli, Staphylococcus aureus, methicillin-sensitive (MSSA) S. aureus (MRSA) and Klebsiella pneumoniae. The essential oil was not effective against Staphylococcus saprophyticus at the highest concentration applied of >150 μg/ml. The MIC values were as follows: 9.37 μg/ml for E. coli 4.69 μg/ml for S. aureus (MRSA), 2.34 mg/ml for MSSA and 2.34 μg/ml for K. pneumoniae. The time-kill assay indicated that there was a sharp time dependent decline in K. pneumoniae counts in the presence of the oil. This is in contrast to a gradual decline in the case of S. aureus under the same conditions. The eight major components of the essential oil were: piperitone (14.6%), cyclohexanemethanol (11.6%), β-elemene (11.6%), α-eudesmol (11.5%), elemol (10.8%), β-eudesmol (8.5%), 2-naphthalenemethanol (7.1%) and γ-eudesmol (4.2%).ConclusionThe results of the present study provide a scientific validation for the traditional use of C. schoenanthus as an antibacterial agent. Future work is needed to investigate and explore its application in the environmental and medical fields. In addition, to evaluating the efficacy of the individual ingredients separately to better understand the underlying mechanism.     

Synthesis of silver nanoparticles using plant derived 4-N-methyl benzoic acid and evaluation of antimicrobial,antioxidant and antitumor activity

The present study is to investigate the antitumor, antioxidant and antibacterial potential of silver nanoparticles (Ag NPs) synthesized from a phenolic derivative 4-N-methyl benzoic acid, isolated from a medicinal plant (Memecylon umbellatum Burm F). The Bio-inspired nanoparticles (NPs) were analyzed by using UV–vis spectroscopy, FTIR, HRTEM, Zeta potential and XRD techniques. The UV–vis spectroscopy study at the band of 430 nm confirmed the nanoparticles formation. HRTEM report showed that the AgNPs synthesized were in the size range 7–23 nm. The harvested nanoparticles were subjected to anti-bacterial assay and a dose dependent inhibitory action was observed against the tested human pathogens. Among the tested bacteria, Acinetobacter baumannii was found to be highly sensitive to AgNPs (diameter of zone of inhibition was 31 mm). Further, the silver nanoparticles exhibited a good anti-tumor activity against the breast cancer cell line (MCF 7) with an IC₅₀ value of 42.19 µg/mL. As the present study confirmed a good antibacterial, antioxidant and antitumor activity in the nanoparticles synthesized using 4-N-methyl benzoic acid derived from a medicinal plant, the product can be further tested to formulate a good lead compound for biomedical applications.     

Complement mediated Klebsiella pneumoniae capsule changes

The Gram-negative bacterium Klebsiella pneumoniae is an opportunistic pathogen, which can cause life-threatening infections such as sepsis. Worldwide, emerging multidrug resistant K. pneumoniae infections are challenging to treat, hence leading to increased mortality. Therefore, understanding the interactions between K. pneumoniae and the immune system is important to develop new treatment options.We characterized ten clinical K. pneumoniae isolates obtained from blood of bacteremia patients. The interaction of the isolates with human serum was investigated to elucidate how K. pneumoniae escapes the host immune system, and how complement activation by K. pneumoniae changed the capsule structure. All K. pneumoniae isolates activated the alternative complement pathway despite serum resistance of seven isolates. One serum sensitive isolate activated two or all three pathways, and this isolate was lysed and had numerous membrane attack complexes in the outer membrane. However, we also found deposition of complement components in the capsule of serum resistant isolates resulting in morphological capsule changes and capsule shedding. These bacteria did not lyse, and no membrane attack complex was observed despite deposition of C5b-9 within the capsule, indicating that the capsule of serum resistant K. pneumoniae isolates is a defense mechanism against complement-mediated lysis.     

Biocompatible, biodegradable and thermo-sensitive chitosan-g-poly (N-isopropylacrylamide) nanocarrier for curcumin drug delivery

A nano formulation of curcumin loaded biodegradable thermoresponsive chitosan-g-poly (N-isopropylacrylamide) co-polymeric nanoparticles (TRC-NPs) (150 nm) were prepared by ionic cross-linking method and characterized. The in vitro drug release was prominent at above LCST. Cytocompatibility of TRC-NPs (100-1000 μg/ml) on an array of cell line is proved by MTT assay. The drug loaded TRC-NPs showed specific toxicity on cancer cells. The cell uptake studies were confirmed by fluorescent microscopy. Flowcytometric analysis of curcumin loaded TRC-NPs showed increased apoptosis on PC3 cells. These results indicated that TRC-NPs could be a potential nanovehicle for curcumin drug delivery.     

Essential Oils,Silver Nanoparticles and Propolis as Alternative Agents Against Fluconazole Resistant Candida albicans,Candida glabrata and Candida krusei Clinical Isolates

Development of effective and safe therapeutic treatment of fungal infections remains one of the major challenge for modern medicine. The aim of presented investigation was to analyze the in vitro antifungal activity of selected essential oils, ethanolic extracts of propolis and silver nanoparticles dropped on TiO₂ against azole-resistant C. albicans (n = 20), C. glabrata (n = 14) and C. krusei (n = 10) clinical isolates. Among tested essential oils, the highest activity has definitely been found in the case of the oil isolated from the bark of Cinnamomum cassia, with MIC and MFC values for all tested strains in the range of 0.0006–0.0097 % (v/v) and 0.0012–0.019 % (v/v), respectively. High activity was also observed for the Lemon, Basil, Thyme, Geranium and Clove (from buds) essential oils. Significant differences in fungicidal activity have been observed in the case of four tested propolis samples. Only one of them revealed high activity, with MFC values in the range from 0.156 to 1.25 % (v/v). Satisfactory fungicidal activity, against C. albicans and C. glabrata isolates, was also observed in the case of silver nanoparticles, however C. krusei isolates were mostly resistant. We also revealed that constituents of most of essential oils and propolis as well as silver nanoparticles are not substrates for drug transporters, which belong to the most important factors affecting resistance of Candida spp. clinical isolates to many of conventional antimycotics. To conclude, the results of our investigation revealed that essential oils, propolis and silver nanoparticles represent high potential for controlling and prevention candidiasis.     

Characterization of Streptococcus pneumoniae N-acetylglucosamine-6-phosphate deacetylase as a novel diagnostic marker

The identification of novel diagnostic markers of pathogenic bacteria is essential for improving the accuracy of diagnoses and for developing targeted vaccines. Streptococcus pneumoniae is a significant human pathogenic bacterium that causes pneumonia. N-acetylglucosamine-6-phosphate deacetylase (NagA) was identified in a protein mixture secreted by S. pneumoniae and its strong immunogenicity was confirmed in an immuno-proteomic assay against the anti-serum of the secreted protein mixture. In this study, recombinant S. pneumoniae NagA protein was expressed and purified to analyze its protein characteristics, immunospecificity, and immunogenicity, thereby facilitating its evaluation as a novel diagnostic marker for S. pneumoniae. Mass spectrometry analysis showed that S. pneumoniae NagA contains four internal disulfide bonds and that it does not undergo post-translational modification. S. pneumoniae NagA antibodies successfully detected NagA from different S. pneumoniae strains, whereas NagA from other pathogenic bacteria species was not detected. In addition, mice infected with S. pneumoniae generated NagA antibodies in an effective manner. These results suggest that NagA has potential as a novel diagnostic marker for S. pneumoniae because of its high immunogenicity and immunospecificity.     

Suppression of stop codon UGA in acrB can contribute to antibiotic resistance in Klebsiella pneumoniae ATCC10031

We previously reported that Klebsiella pneumoniae MGH78578 exhibited higher resistance against various antimicrobials than K. pneumoniae ATCC10031. In this study, we showed that the plasmid, pKPN5, in K. pneumoniae MGH78578 played an important role in resistance against aminoglycosides, ampicillin, tetracycline, and chloramphenicol, while genome-derived β-lactamases and drug efflux pumps appeared to be more important in resistance to cloxacillin. acrAB, encoding a potent multidrug efflux pump, was cloned from K. pneumoniae MGH78578 and ATCC10031, to investigate reasons for the high drug resistance of K. pneumoniae MGH78578, and the results revealed that AcrAB from K. pneumoniae ATCC10031 conferred weaker drug resistance than AcrAB from K. pneumoniae MGH78578. DNA sequencing revealed that acrB from K. pneumoniae ATCC10031 carried the nonsense mutation, UGA, which was not found in acrB from K. pneumoniae MGH78578. However, acrB from K. pneumoniae ATCC10031 conferred slightly elevated resistant levels to several antimicrobials. The intact length of AcrB was detected in K. pneumoniae ATCC10031 by Western blot analysis, even though its quantity was small. Therefore, the stop codon UGA in acrB was thought to be overcome to some extent in this strain. We artificially introduced the nonsense mutation, UGA to the cat gene on pACYC184, and the plasmid also elevated the MIC of chloramphenicol in K. pneumoniae ATCC10031. These results suggest that a mechanism to overcome the nonsense mutation in acrB sustained resistance against a few β-lactams, dyes, and cholic acid in K. pneumoniae ATCC10031.