In this report, we describe the detection of AmpC and CMY-2 beta-lactamases with the loss of OmpK35 porin among seven sporadic strains of ceftazidime-resistant Klebsiella pneumoniae and ceftazidime-resistant Escherichia coli. Cefoxitin, which was used as a marker of resistance toward 7-alpha-methoxy-cephalosporins, exhibited high minimum inhibitory concentration (MIC) values ranging between 128 microg/ml and >256 microg/ml in all the strains. The presence of hyperproducing AmpC enzymes was indicated by the positive three-dimensional test. Isoelectric focusing (IEF) study confirmed the presence of AmpC enzymes in all the strains. The ampC gene was detected by PCR in all the strains and confirmed by DNA sequencing. Large plasmids in all the strains, ranging from 60 kb to 150 kb in size, most likely encode the ampC gene. Two E. coli strains out of the seven strains showed positive amplification of the bla(CMY-2) gene, an AmpC variant, and was confirmed by DNA sequence analyses. DNA hybridization confirmed the bla(CMY-2) gene to be plasmid-mediated in both of these strains. However, one of these two strains also mediated a chromosomal CMY gene. All the strains showed an absence of OmpK35 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and was confirmed by western blot analyses using raised polyclonal anti-OmpK35 antiserum. This suggests that, apart from CMY production, absence of OmpK35 porin also contributed to cefoxitin resistance resulting in extended-spectrum beta-lactam resistance among these isolates.
In continuation of investigation for environmentally benign protocol for new solvents termed deep eutectic solvents (DESs), it is herein reported results concerning the toxicity and cytotoxicity of choline chloride (ChCl) based DESs with four hydrogen bond donors including glycerine, ethylene glycol, triethylene glycol and urea. The toxicity was investigated using two Gram positive bacteria Bacillus subtilis and Staphylococcus aureus, and two Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa. The cytotoxicity effect was tested using the Artemia salina leach. It was found that there was no toxic effect for the tested DESs on all of the studied bacteria confirming their benign effects on these bacteria. Nevertheless, it was found that the cytotoxicity of DESs was much higher than their individual components (e.g. glycerine, ChCl) indicating that their toxicological behavior is different. For our best knowledge this is the first time that toxicity and cytotoxicity of DESs were studied. The toxicity and cytotoxicity of DESs varied depending on the structure of components. Careful usage of the terms non-toxicity and biodegradability must be considered. More investigation on this matter is required.
Quorum sensing is a mechanism for regulating proteobacterial gene expression in response to changes in cell population. In proteobacteria, N-acyl homoserine lactone (AHL) appears to be the most widely used signalling molecules in mediating, among others, the production of extracellular virulence factors for survival. In this work, the genome of B. cepacia strain GG4, a plasmid-free strain capable of AHL synthesis was explored. In silico analysis of the 6.6 Mb complete genome revealed the presence of a LuxI homologue which correspond to Type I quorum sensing. Here, we report the molecular cloning and characterization of this LuxI homologue, designated as BurI. This 609 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was approximately 25 kDa and is highly similar to several autoinducer proteins of the LuxI family among Burkholderia species. To verify the AHL synthesis activity of this protein, high resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-hexanoylhomoserine lactone, N-octanoylhomoserine lactone and 3-hydroxy-octanoylhomoserine lactone from induced E. coli BL21 harboring the recombinant BurI. Our data show, for the first time, the cloning and characterization of the LuxI homologue from B. cepacia strain GG4 and confirmation of its AHL synthesis activity.
L-asparaginase (LA) catalyzes the degradation of asparagine, an essential amino acid for leukemic cells, into ammonia and aspartate. Owing to its ability to inhibit protein biosynthesis in lymphoblasts, LA is used to treat acute lymphoblastic leukemia (ALL). Different isozymes of this enzyme have been isolated from a wide range of organisms, including plants and terrestrial and marine microorganisms. Pieces of information about the three-dimensional structure of L-asparaginase from Escherichia coli and Erwinia sp. have identified residues that are essential for catalytic activity. This review catalogues the major sources of L-asparaginase, the methods of its production through the solid state (SSF) and submerged (SmF) fermentation, purification, and characterization as well as its biological roles. In the same breath, this article explores both the past and present applications of this important enzyme and discusses its future prospects.
The enzymes involved in synthesizing the bacterial cell wall are attractive targets for the design of antibacterial compounds, since this pathway is essential for bacteria and is absent in animals, particularly humans. A survey of the genome of a bacterium that belongs to the phylum Verrucomicrobia, the closest free-living relative to bacteria from the Chlamydiales phylum, shows genetic evidence that Verrucomicrobium spinosum possesses a novel fusion open reading frame (ORF) annotated by the locus tag (VspiD_010100018130). The ORF, which is predicted to encode the enzymes UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and UDP-N-acetylmuramate:l-alanine ligase (MurC) that are involved in the cytoplasmic steps of peptidoglycan biosynthesis, was cloned. In vivo analyses using functional complementation showed that the fusion gene was able to complement Escherichia coli murB and murC temperature sensitive mutants. The purified recombinant fusion enzyme (MurB/C Vs ) was shown to be endowed with UDP-N-acetylmuramate:l-alanine ligase activity. In vitro analyses demonstrated that the latter enzyme had a pH optimum of 9.0, a magnesium optimum of 10 mM and a temperature optimum of 44-46°C. Its apparent K m values for ATP, UDP-MurNAc, and l-alanine were 470, 90, and 25 μM, respectively. However, all attempts to demonstrate an in vitro UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) activity were unsuccessful. Lastly, Hidden Markov Model-based similarity search and phylogenetic analysis revealed that this fusion enzyme could only be identified in specific lineages within the Verrucomicrobia phylum.
Periplasmic expression of soluble proteins in Escherichia coli not only offers a much-simplified downstream purification process, but also enhances the probability of obtaining correctly folded and biologically active proteins. Different combinations of signal peptides and target proteins lead to different soluble protein expression levels, ranging from negligible to several grams per litre. Accurate algorithms for rational selection of promising candidates can serve as a powerful tool to complement with current trial-and-error approaches. Accordingly, proteomics studies can be conducted with greater efficiency and cost-effectiveness. Here, we developed a predictor with a two-stage architecture, to predict the real-valued expression level of target protein in the periplasm. The output of the first-stage support vector machine (SVM) classifier determines which second-stage support vector regression (SVR) classifier to be used. When tested on an independent test dataset, the predictor achieved an overall prediction accuracy of 78% and a Pearson's correlation coefficient (PCC) of 0.77. We further illustrate the relative importance of various features with respect to different models. The results indicate that the occurrence of dipeptide glutamine and aspartic acid is the most important feature for the classification model. Finally, we provide access to the implemented predictor through the Periscope webserver, freely accessible at http://lightning.med.monash.edu/periscope/.
The completion of Escherichia coli K1 genome has identified several genomic islands that are present in meningitis-causing E. coli RS218 but absent in the non-pathogenic E. coli MG1655. In this study, the role of various genomic islands in E. coli K1 interactions with intestinal epithelial cells (Caco-2) and kidney epithelial cells (MA104) was determined. Using association assays, invasion assays, and intracellular survival assays, the findings revealed that the genomic island deletion mutants of RS218 related to P fimbriae, S fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, protein secretion system (T1SS for hemolysin; T2SS; T5SS for antigen 43), Iro system and hmu system), invasins (CNF1, IbeA), toxins (α-hemolysin), K1 capsule biosynthesis, metabolism (d-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism), prophage genes, showed reduced interactions with both cell types. Next, we determined the role of various genomic islands in E. coli K1 resistance to serum. When exposed to the normal human serum, the viability of the genomic island deletion mutants related to adhesins such as S fimbriae, P fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, antigen 43 and T5SS for antigen 43, T2SS, and T1SS for hemolysin, Iro system and hmu system, prophage genes, metabolism (sugar metabolism and d-serine catabolism), K1 capsule biosynthesis, and invasins such as CNF1 was affected, suggesting their role in bacteremia. The characterization of these genomic islands should reveal mechanisms of E. coli K1 pathogenicity that could be of value as therapeutic targets.
Acinetobacter baumannii is a Gram-negative nosocomial pathogen of importance due to its uncanny ability to acquire resistance to most antimicrobials. These include carbapenems, which are the drugs of choice for treating A. baumannii infections, and polymyxins, the drugs of last resort. Whole genome sequencing was performed on two clinical carbapenem-resistant A. baumannii AC29 and AC30 strains which had an indistinguishable ApaI pulsotype but different susceptibilities to polymyxin. Both genomes consisted of an approximately 3.8 Mbp circular chromosome each and several plasmids. AC29 (susceptible to polymyxin) and AC30 (resistant to polymyxin) belonged to the ST195 lineage and are phylogenetically clustered under the International Clone II (IC-II) group. An AbaR4-type resistance island (RI) interrupted the comM gene in the chromosomes of both strains and contained the bla OXA-23 carbapenemase gene and determinants for tetracycline and streptomycin resistance. AC29 harbored another copy of bla OXA-23 in a large (~74 kb) conjugative plasmid, pAC29b, but this gene was absent in a similar plasmid (pAC30c) found in AC30. A 7 kb Tn1548::armA RI which encodes determinants for aminoglycoside and macrolide resistance, is chromosomally-located in AC29 but found in a 16 kb plasmid in AC30, pAC30b. Analysis of known determinants for polymyxin resistance in AC30 showed mutations in the pmrA gene encoding the response regulator of the two-component pmrAB signal transduction system as well as in the lpxD, lpxC, and lpsB genes that encode enzymes involved in the biosynthesis of lipopolysaccharide (LPS). Experimental evidence indicated that impairment of LPS along with overexpression of pmrAB may have contributed to the development of polymyxin resistance in AC30. Cloning of a novel variant of the bla AmpC gene from AC29 and AC30, and its subsequent expression in E. coli also indicated its likely function as an extended-spectrum cephalosporinase.
Cell-cell communication is also known as quorum sensing (QS) that happens in the bacterial cells with the aim to regulate their genes expression in response to increased cell density. In this study, a bacterium (L8A) isolated from dental plaque biofilm was identified as Citrobacter amalonaticus by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Its N-acylhomoserine-lactone (AHL) production was screened by using two types of AHL biosensors namely Chromobacterium violaceum CV026 and Escherichia coli [pSB401]. Citrobacter amalonaticus strain L8A was identified and confirmed producing numerous types of AHL namely N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-hexadecanoyl-L-homoserine lactone (C16-HSL). We performed the whole genome sequence analysis of this oral isolate where its genome sequence reveals the presence of QS signal synthase gene and our work will pave the ways to study the function of the related QS genes in this bacterium.
The study explored on the commonly available weed plant Commelina nudiflora which has potential in-vitro antioxidant and antimicrobial activity. The different polar solvents such as ethanol, chloroform, dichloromethane, hexane and aqueous were used for the soxhlet extraction. The extracts were identified pharmacologically as important bioactive compounds and their potential free radical scavenging activities, and antimicrobial properties were studied. C. nudiflora extracts were monitored on their in-vitro antioxidant ability by DPPH and ABTS radical scavenging assay. Aqueous extract shows significant free radical scavenging activity of 63.4 mg/GAE and 49.10 mg/g in DPPH and ABTS respectively. Furthermore, the aqueous crude extract was used in antibacterial studies, which shows the highest inhibitory activity against Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi. Among all the extracts, aqueous extract of C. nudiflora has significant control over free radical scavenging activity and inhibition of the growth of food pathogenic bacteria. Also, the aqueous extract contains abundance of phenolics and flavonoids higher than other extracts. This study explored weed plant C. nudiflora as a potential source of antioxidant and antibacterial efficacy and identified various therapeutic value bioactive compounds from GC-MS analysis.
Myriad proteobacteria use N-acyl homoserine lactone (AHL) molecules as quorum sensing (QS) signals to regulate different physiological functions, including virulence, antibiotic production, and biofilm formation. Many of these proteobacteria possess LuxI/LuxR system as the QS mechanism. Recently, we reported the 3.89 Mb genome of Acinetobacter sp. strain GG2. In this work, the genome of this long chain AHL-producing bacterium was unravelled which led to the molecular characterization of luxI homologue, designated as aciI. This 552 bp gene was cloned and overexpressed in Escherichia coli BL21(DE3). The purified protein was ∼20.5 kDa and is highly similar to several autoinducer proteins of LuxI family among Acinetobacter species. To verify the AHL synthesis activity of this protein, high-resolution liquid chromatography-mass spectrometry analysis revealed the production of 3-oxo-dodecanoyl-homoserine lactone and 3-hydroxy-dodecanoyl-homoserine lactone from induced E. coli harboring the recombinant AciI. Our data show for the first time, the cloning and characterization of the luxI homologue from Acinetobacter sp. strain GG2, and confirmation of its AHLs production. These data are of great significance as the annotated genome of strain GG2 has provided a valuable insight in the study of autoinducer molecules and its roles in QS mechanism of the bacterium.
Systems metabolic engineering and in silico analyses are necessary to study gene knockout candidate for enhanced succinic acid production by Escherichia coli. Metabolically engineered E. coli has been reported to produce succinate from glucose and glycerol. However, investigation on in silico deletion of ptsG/b1101 gene in E. coli from glycerol using minimization of metabolic adjustment algorithm with the OptFlux software platform has not yet been elucidated. Herein we report what is to our knowledge the first direct predicted increase in succinate production following in silico deletion of the ptsG gene in E. coli GEM from glycerol with the OptFlux software platform. The result indicates that the deletion of this gene in E. coli GEM predicts increased succinate production that is 20% higher than the wild-type control model. Hence, the mutant model maintained a growth rate that is 77% of the wild-type parent model. It was established that knocking out of the ptsG/b1101 gene in E. coli using glucose as substrate enhanced succinate production, but the exact mechanism of this effect is still obscure. This study informs other studies that the deletion of ptsG/b1101 gene in E. coli GEM predicted increased succinate production, enabling a model-driven experimental inquiry and/or novel biological discovery on the underground metabolic role of this gene in E. coli central metabolism in relation to increasing succinate production when glycerol is the substrate.
Extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae are often susceptible in vitro to β-lactam/β-lactamase inhibitor (BLBLI) combination antibiotics, but their use has been limited by concerns of clinical inefficacy. We aimed to compare outcomes between patients treated with BLBLIs and carbapenems for bloodstream infection (BSI) caused by cefotaxime non-susceptible (likely ESBL- or AmpC β-lactamase-producing) Escherichia coli and Klebsiella pneumoniae.
Thirty-five veterinary isolates of Salmonella enteritidis were characterized by their susceptibility to 10 antimicrobial agents and by their plasmid profiles on agarose gel electrophoresis. All were susceptible to carbenicillin, chloramphenicol and nalidixic acid but 89% were resistant to tetracycline. When examined, 91% of the isolates harboured plasmids, with sizes ranging from 9.8 to 60 MDa. However, it was only possible to associate the presence of plasmids with tetracycline resistance; plasmids occurring in 90% of the tetracycline-resistant isolates. In conjugation experiments, with Escherichia coli K12 Nal(r) as recipient, the tetracycline resistance in three selected S. enteritidis isolates was observed to transfer at frequencies of 3.0×10(-3) to 1.0×10(-2)/donor cell. The concomitant transfer of a 56-MDa or 60-MDa plasmid in these three S. enteritidis isolates was also detected.
In a case study involving preliminary investigations for the feasibility of a beach resort complex in the west coast of Sarawak, Malaysia, since the acceptable quality of recreational water quality had not been specified, existing international standards and practices were reviewed to arrive at acceptable microbiological and physicochemical parametric levels. Water samples were collected on a weekly basis in the proposed beach complex at Tanjong Batu Coastal Reserve and also along the nearby Sungei Batang Kemena and its estuary. It was ascertained that the swimming water quality was acceptable in terms of faecal Coliforms, temperature, pH and dissolved oxygen. However E. Coli counts did indicate a potential risk of 1.68% for gastrointestinal illness and the ubiquitous presence of faecal Streptococci pointed to recent pollution of human or animal origin. Besides, grease and oil contents exceeded the WHO Guideline values. It was hence recommended that an appropriate sanitation or pollution survey should be carried out in the adjacent coastal catchment area and the beach. Also routine water sampling should be undertaken. Such action will help to pinpoint sources of pollution and lead to antipollution measures, thus helping to upgrade swimming water quality and establish swimming water quality standards.
The pattern of antibiotic resistance amongst gram-negative bacteria (GNB) in paediatric units, which have heavy empirical usage of broad-spectrum antibiotics, was studied prospectively over a 6-month period. A total of 200 consecutive, non-duplicate gram-negative isolates were obtained from 109 patients admitted to intensive care and oncology units in two hospitals. The commonest isolates were Klebsiella spp (36.5 per cent) and Pseudomonas (20.0 per cent). The isolates showed lower susceptibility rates to the third-generation cephalosporins (47-62 per cent) compared with cefepime (91 per cent), imipenem (90 per cent) and ciprofloxacin (99 per cent). Fifty-four (52.8 per cent) Klebsiella and Escherichia coli isolates were determined to be extended-spectrum beta-lactamase (ESBL) producing strains. Antibiotics found to be effective against ESBL-producers were imipenem and ciprofloxacin. The high resistance rate amongst GNB to third-generation cephalosporins is a likely consequence of heavy empirical usage of this group of antibiotics. The carbapenems and quinolones remain useful agents in the management of patients admitted to these units.
Bacteriological isolation and identification were performed on 60 cloacal swabs and 15 aquarium water samples of pet red-eared sliders (Trachemys scripta elegans) obtained from aquarium shops in the Klang Valley, Central Peninsula Malaysia. The most common bacteria isolated was Aeromonas spp., which was present in both cloacal swabs (70%) and aquarium water (86.7%). Klebsiella spp. (50%), Escherichia coli (33.3%), Yersinia spp. (16.7%) and Salmonella spp. (15%) obtained form cloacal swabs were identified as pathogenic to both humans and animals. Salmonella spp. were isolated from both cloacal swabs and aquarium water. The Salmonella serotypes identified were S. tennessee, S. typhimurium, S. brezany, S. pomona, S. corvallis and S. schwarzengrund. Bacterial infections in humans associated with handling exotic pets directly or indirectly in contact with aquarium water have been described regularly, hence the zoonotic significance of owning a turtle infected with Salmonella spp. or any pathogenic bacteria therefore cannot be ignored.
Introduction:Staphylococcus aureus is a Gram-positive staphylococci that form biofilms. Bacteria that dwell in bio-films tend to be highly resistant towards the action of antibiotics. S. aureus is a main cause of infections in the oral cavity such as angular cheilitis, endodontic infections, osteomyelitis of the jaw, parotitis and oral mucositis. Previous studies reported that S. aureus also spread to the other parts of the body through the circulatory system, which may lead to chronic infections. Hence the search for new antibacterial agents remains high and needs urgent attention to treat this problem. Plants offer a rich source of antimicrobial agents and bioactive compounds. In this study, aque-ous oil palm leaf extracts (OPLE) has been used as an alternative antibacterial agent against oral infections mainly caused by Staphylococcus aureus. Many studies report the potential use of oil palm leaf extracts in treating bacterial infections such as Escherichia coli, Salmonella sp., Staphylococcus aureus (isolated from other part of the body), Pseudomonas aeruginosa and Bacillus sp. Although previous studies have documented the antimicrobial properties of oil palm leaf extracts, to date no study has been reported on the effect of oil palm leaf extract on oral microbes. Methods: The agar diffusion method, minimum inhibitory concentration (MIC) and minimal bactericidal concen-tration (MBC) assay were conducted in order to observe the antibacterial activity of aqueous oil palm leaf extract. The crystal violet assay was used to determine the anti-biofilm activity of the extracts. Chlorhexidine and deionised distilled water were used as the positive and negative control respectively. For agar diffusion method, the diameter of inhibition zone was measured. Results: The inhibition zone of the tested bacteria was observed between 0-20mm. The MIC and MBC assay were used to know the lowest concentrations of the extract that inhibit the growth and killed the tested bacteria respectively. The MIC and MBC values for the tested bacteria were observed between 0-7.813mg/mL. While for anti-biofilm assays, OPLE aqueous extract acts as a potent anti-biofilm agent with dual actions, pre-venting and eradicating the biofilm of the tested bacteria. Conclusion: In conclusion, the tested plant extracts could serve as alternative natural antibacterial and anti-biofilm agent against oral infections.
Biofouling on the membrane surface leads to performance deficiencies in membrane filtration. In this study, the application of ginger extract as a bio-based additive to enhance membrane antibiofouling properties was investigated. The extract was dispersed in a dimethyl acetamide (DMAc) solvent together with polyvinylidene fluoride (PVDF) to enhance biofouling resistance of the resulting membrane due to its antibiotic property. The concentrations of the ginger extract in the dope solution were varied in the range of 0-0.1 wt %. The antibacterial property of the resulting membranes was assessed using the Kirby Bauer disc diffusion method. The results show an inhibition zone formed around the PVDF/ginger membrane against Escherichia coli and Staphylococcus aureus demonstrating the efficacy of the residual ginger extract in the membrane matrix to impose the antibiofouling property. The addition of the ginger extract also enhanced the hydrophilicity in the membrane surface by lowering the contact angle from 93° to 85°, which was in good agreement with the increase in the pure water flux of up to 62%.
The evolution of cosmetic products results in the growing demands for cosmetics that are preservatives free. Plant essential oils were found to be a promising antimicrobial and also antioxidant agent. In this study, Cymbopogon citratus (lemongrass), Laurus nobilis (bay leaf) and Backhousia citriodora (lemon myrtle) essential oils were selected and evaluated for their antimicrobial properties. It was found that Laurus nobilis exhibited strong antimicrobial activity against the selected bacteria Streptococcus saprophyticus (ATCC 49619), Streptococcus aureus (ATCC 22923), Streptococcus pyogenes (ATCC 29436), Pseudomonas aeruginosa (ATCC 13048), Klebsiella pneumoniae (ATCC 700603), Escherichia coli (ATCC 22922) with MIC ranging between 7.8 ug/mL to 250 μg/mL. The antioxidant activity of selected essential oils was determined by antioxidant assays which were 1,1-Diphenyl-2-picrylhydrazyl assay (DPPH), determination of ferric reducing antioxidant power assay (FRAP) and β-Carotene/linoleic acid bleaching assay. Backhousia citriodora and Laurus nobilis showed the highest antioxidant activity.
n-Octanal and β-Selinene were identified to be the major components with peak area of 26.37 % and 13.92 % respectively in secondary metabolites analysis by Gas Chromatography-Mass Spectrometry (GCMS).