Gram-negative Bacterial Species Research Articles

Presence of pathogenicity island related and plasmid encoded virulence genes in cytolethal distending toxin producing Escherichia coli isolates from diarrheal cases.

Context:Mobile genetic elements such as plasmids, bacteriophages, insertion elements, and genomic islands play a critical role in virulence of bacterial pathogens. These elements transfer horizontally and could play an important role in the evolution and virulence of many pathogens. A broad spectrum of gram-negative bacterial species has been shown to produce a cytolethal distending toxin (CDT). On the other hand, Shiga toxin producing Escherichia coli are the one carry virulence genes such as stx 1 and stx 2 (Shiga toxin) and these genes can be acquired by horizontal gene transfer.Aim:The aim of this study was to investigate the presence of other virulence associated genes among CDT producing E. coli strains.Materials and Methods:Thirty CDT positive strains isolated from patients with diarrhea were characterized. Thereafter, the association with virulent genetic elements in known pathogenicity islands (PAIs) was assessed by polymerase chain reaction.Results:In this study, it was shown that the most CDT producing E. coli isolates express Shiga toxin. Moreover, the presence of prophages framing cdt genes (like P2 phage) was also identified in each cdt-type genomic group. Flanked regions of cdt-I, cdt-IV, and cdt-V-type was similar to plasmid sequences while cdt-II and cdt-III-type regions similarity with hypothetical protein (orf3) was observed.Conclusion:The occurrence of each cdt-type groups with specific virulence genes and PAI genetic elements is indicative of horizontal gene transfer by these mobile genetic elements, which could lead to diversity among the isolates.

Volatiles from the Rare Australian Desert Plant Prostanthera centralis B.J.Conn (Lamiaceae): Chemical Composition and Antimicrobial Activity

Hydrodistilled essential oils and dichloromethane (DCM) extracted volatiles were taken from cultivated specimens of Prostanthera centralis, endemic to central Australia. All volatiles were chemically characterised by Gas Chromatography-Mass Spectroscopy (GC-MS) with the use of authentic standards, followed by Nuclear Magnetic Resonance (NMR) Spectroscopy. The antimicrobial activity of the essential oils was measured against a range of Gram-negative and Gram-positive bacterial species using a micro-titre plate broth dilution assay. Twenty-two compounds were identified as components of the sweet smelling aromatic essential oil and DCM extracts, both showing a relatively high abundance of prostantherol. The volatiles extracted using DCM, differed only in the relative abundance of the major components and the lack of ledol and squamulosone. This study constitutes the first time ledol and squamulosone have been identified in a Prostanthera species. Antimicrobial assays showed moderate to high inhibitory activity against some Gram-positive bacteria and the yeast Candida albicans.

Rapid Molecular Detection of Pathogens in 516 Consecutive Haematological Patients with Febrile Neutropenia

▪ BACKGROUNDFebrile neutropenia and sepsis are frequent and life-threatening complications in patients with haematological malignancies. Although the proportion of infectious deaths in haematological patients has decreased over the last two decades, much remains to be done to further reduce these events. More effective preventive and prophylactic strategies for high-risk patients are necessary. Blood cultures (BC) identify a pathogen in only 20 to 30% of febrile episodes, the culturing and pathogen identification process is lengthy, postponing the start of a pathogen-targeted treatment. Thereby, a sensitive tool to promptly recognize pathogens causing sepsis is of high clinical relevance. METHODSWe assessed the diagnostic usefulness of the LightCycler SeptiFast test (SF; Roche Molecular Systems), a PCR-based multiplex assay performed on peripheral blood and capable of detecting 25 among the most common species isolated in sepsis. The assay uses dual fluorescent resonance energy transfer (FRET) probes against the species-specific internal transcribed spacer (ITS) regions, a non-coding sequence interspaced among highly conserved bacterial and fungal RNA. Time from processing to result is remarkably short (less than 6 hours). In this study, blood samples from febrile haematological patients were concomitantly tested by traditional blood culture (BacT/Alert 3D; bioMerieux). RESULTSA total of 1941 blood samples were collected from 516 consecutive haematological patients treated for febrile neutropenia at the San Raffaele Haematology and Bone Marrow Transplantation Unit, from 2009 to 2013. Out of the total 1941 episodes examined, positive results were detected in 537 samples by SF (28%), and in 263 by BC (14%). Together, the two methods identified a total of 586 microorganisms in 509 (26%) episodes: Gram-positive bacterial species (80%), Gram-negative bacterial species (17%), and fungal species (3%). Concordance between the two methods was 77%, with most of the discordant samples that tested negative by culture but positive using the molecular approach (50% of the total positive samples). The cases positive by SF alone were mostly samples from patients already receiving antimicrobial therapy, or, importantly, sample positive for fungal pathogens such as Aspergillus fumigatus, which is hard to detect by the traditional approaches. CONCLUSIONThis analysis demonstrates a significant correlation between the molecular test and the standard BC in haematological patients with febrile neutropenia. A molecular test such as SF, in combination with traditional assays, can play an important role in the diagnosis of sepsis, particularly in persistent fever despite antibacterial therapy, when a non-responding bacterial infection or an invasive fungal infection is suspected, therefore leading to a rapid diagnosis and an earlier targeted antimicrobial therapy. DisclosuresNo relevant conflicts of interest to declare.

Pyrene Schiff base: photophysics, aggregation induced emission, and antimicrobial properties.

Pyrene containing Schiff base molecule, namely 4-[(pyren-1-ylmethylene)amino]phenol (KB-1), was successfully synthesized and well characterized by using (1)H, (13)C NMR, FT-IR, and EI-MS spectrometry. UV-visible absorption, steady-state fluorescence, time-resolved fluorescence, and transient absorption spectroscopic techniques have been employed to elucidate the photophysical processes of KB-1. It has been demonstrated that the absorption characteristics of KB-1 have been bathochromatically tuned to the visible region by extending the π-conjugation. The extended π-conjugation is evidently confirmed by DFT calculations and reveals that π→π* transition is the major factor responsible for electronic absorption of KB-1. The photophysical property of KB-1 was carefully examined in different organic solvents at different concentrations and the results show that the fluorescence of this molecule is completely quenched due to photoinduced electron transfer. Intriguingly, the fluorescence intensity of KB-1 increases enormously by the gradual addition of water up to 90% with concomitant increase in fluorescence lifetime. This clearly signifies that this molecule has aggregation-induced emission (AIE) property. The mechanism of AIE of this molecule is suppression of photoinduced electron transfer (PET) due to hydrogen bonding interaction of imine donor with water. A direct evidence of PET process has been presented by using nanosecond transient absorption measurements. Further, KB-1 was successfully used for antimicrobial and bioimaging studies. The antimicrobial studies were carried out through disc diffusion method. KB-1 is used against both Gram-positive (Rhodococcus rhodochrous and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial species and also fungal species (Candida albicans). The result shows KB-1 can act as an excellent antimicrobial agent and as a photolabeling agent. S. aureus, P. aeruginosa, and C. albicans were found to be the most susceptible microorganisms at 1 mM concentration among the bacteria used in the present investigation.

Dissemination of NDM metallo-β-lactamase genes among clinical isolates of Enterobacteriaceae collected during the SMART global surveillance study from 2008 to 2012.

The prevalence of carbapenemase enzymes continues to increase. Among the Ambler class B enzymes is the New Delhi metallo-β-lactamase (NDM). This particular enzyme is capable of hydrolyzing nearly all β-lactam antimicrobial agents and has spread rapidly, becoming a global problem. Therapeutic treatment options for patients infected with isolates which produce this enzyme are difficult to manage, as cross-resistance to other antimicrobial classes is common. The Study for Monitoring Antimicrobial Resistance Trends (SMART) is a global surveillance study evaluating the antimicrobial susceptibilities of numerous Gram-negative bacterial species recovered from people with intra-abdominal and urinary tract infections. The Clinical and Laboratory Standards Institute methods and a molecular analysis identified 134 isolates of Enterobacteriaceae (nine species) and one Acinetobacter sp. with blaNDM genes. These isolates were collected in nine countries, and >95% of the isolates possessed the NDM-1 variant. The MIC90 values were >4 mg/liter and >8 mg/liter for ertapenem and imipenem, respectively. No tested β-lactam or β-lactamase inhibitor combination had activity against these isolates. Resistance to amikacin (79.9%) and levofloxacin (82.8%) was common. Nearly all the isolates encoded additional enzymes, including AmpC cephalosporinases and extended-spectrum β-lactamases. There is an urgent need for infection control and continued global monitoring of isolates which harbor the NDM enzyme, as evidenced by recent outbreaks.

In vitro antibacterial activity of AZD0914, a new spiropyrimidinetrione DNA gyrase/topoisomerase inhibitor with potent activity against Gram-positive, fastidious Gram-Negative, and atypical bacteria.

AZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potent in vitro antibacterial activity against key Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae and Neisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance in S. aureus, and if mutants were obtained, the mutations mapped to gyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, β-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration and in vitro time-kill studies. In in vitro checkerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potent in vitro antibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development.

Zebrafish fed on recombinant Artemia expressing epinecidin-1 exhibit increased survival and altered expression of immunomodulatory genes upon Vibrio vulnificus infection

Artemia has been used extensively in aquaculture as fodder for larval fish, shrimp, and shellfish. Epinecidin-1, an antimicrobial peptide, was isolated from grouper (Epinephelus coioides) in 2005. Epinecidin-1 has been previously reported to possess antimicrobial activity against several Gram-positive and Gram-negative bacterial species, including Staphylococcus coagulase, Pseudomonas aeruginosa, Streptococcus pyogenes, and Vibrio vulnificus. In this study, we used electroporation to introduce plasmid DNA encoding a green fluorescent protein (EGFP)-epinecidin-1 fusion protein under the control of the cytomegalovirus (CMV) promoter into decapsulated Artemia cysts. Optimization of various properties (including cyst weight (0.2 g), plasmid concentration (50 μg/100 μl), and pulse voltage (150 V), length (10 ms), and number (2)) resulted in a hatching rate of 41.15%, a transfection efficiency of 49.81%, and a fluorescence intensity (A.U.) of 47.46. The expression of EGFP-epinecidin-1 was first detected by quantitative RT-PCR at 120 h post-electroporation, and protein was identified by Western blot at the same time. Furthermore, the EGFP-epinecidin-1 protein inhibited V. vulnificus (204) growth, as demonstrated by zone of inhibition studies. Zebrafish fed on transgenic Artemia expressing CMV-gfp-epi combined with commercial fodder were more resistant to infection by V. vulnificus (204): survival rate was enhanced by over 70% at 7, 14, and 21 days post-infection, and bacterial numbers in the liver and intestine were reduced. In addition, feeding of transgenic Artemia to zebrafish affected the immunomodulatory response to V. vulnificus (204) infection; expression of immune-responsive genes, including hepcidin and defbl2, was altered, as shown by qPCR. These findings suggest that feeding transgenic Artemia expressing CMV-gfp-epi to larval fish has antimicrobial effects, without the drawbacks of introducing drug residues or inducing bacterial drug resistance.

Structural Features of a Highly Conserved Omp16 Protein of Pasteurella multocida Strains and Comparison with Related Peptidoglycan-associated Lipoproteins (PAL)

Bacterial lipoproteins of varying size and functional role in physiology/pathogenesis are widely prevalent among Gram-negative bacterial species. Among virulent bacteria, Pasteurella multocida is known to be associated with diverse infectious diseases of livestock worldwide and possess several outer membrane proteins (OMPs) as virulence factors. In the present study, we cloned and sequenced omp16 gene of different P. multocida strains (n = 9) of Indian origin and compared with representative related bacterial species from Pasteurellaceae (n = 28) as well as others (n = 11). Phylogenetic and multiple sequence alignment revealed high degree conservation of Omp16 among P. multocida strains. However, it significantly differed from similar peptidoglycans-associated lipoproteins of other Gram-negative bacterial species. Notably, a conversed lipobox at N-terminus of proteins was noticed among all bacterial species. Further, three dimensional homology model of Omp16 was predicted and its structural features were analyzed. The study indicated the potential possibilities to use either native or recombinant Omp16 protein in developing species-specific diagnostic assay or broadly active subunit vaccine along with suitable adjuvant for pasteurellosis in livestock.

Synthesis, characterization and magnetic properties of hematite (α-Fe2O3) nanoparticles on polysaccharide templates and their antibacterial activity

The present study is to synthesize iron oxide nanoparticles on different polysaccharide templates calcined at controlled temperature, characterizing them for spectroscopic and magnetic studies leading to evaluate their antibacterial property. The synthesized iron oxide nanoparticles were characterized by X-ray diffractometer (XRD), Fourier transform infrared spectroscopy, high resolution scanning electron microscopy (HRSEM), high resolution transmission electron microscopy (HRTEM) and vibrating sample magnetometer. The iron oxide nanoparticles were tested for antibacterial activity against gram-positive and gram-negative bacterial species. The XRD confirms the crystalline nature of iron oxide nanoparticles with the mean crystallite size of 10 nm. The functional groups of the synthesized iron oxide nanoparticles were 547, 543 and 544 cm–1 characterizing the Fe–O and the broad bands at 3,398, 3,439 and 3,427 cm–1 were attributed to the stretching vibrations of hydroxyl group absorbed by iron oxide nanoparticles. HRTEM analyses revealed that the average particle size of the hematite nanoparticles are about 85, 92 and 77 nm for AF, DF and GF, respectively, which was a coincident with the results obtained from the HRSEM analysis. Magnetic measurement exhibited ferromagnetic behavior of the α-Fe2O3 at the room temperature with higher coercivity of HC = 2,303, 2,333 and 1,019 Oe for AF, DF and GF, respectively. Antibacterial test showed the inhibition against Aeromonas hydrophila and Escherichia coli with significant antagonistic activity.

Role of Cholera Toxin in <i>Vibrio cholerae</i> Infection in Humans - A Review

Vibrio cholerae, the causative agent of Asiatic cholera, is a gram-negative motile bacterial species acquired via oral ingestion of contaminated food or water sources. Cholera has spread from the Indian subcontinent where it is endemic to involve nearly the whole world seven times during the past 185 years. V. cholerae serogroup O1, biotype El Tor, has moved from Asia to cause pandemic disease in Africa and South America during the past 35 years. A new serogroup, O139, appeared in south Asia in 1992, has become endemic there, and threatens to start the next pandemic. The facultative human pathogen V. cholerae represents a paradigm that evolved from environmental non-pathogenic strains by acquisition of virulence genes. The major virulence factors of V. cholerae, cholera toxin (CTX) encoded by the ctxAB genes residing in the genome of filamentous lysogenic bacteriophage (CTXɸ) and toxin coregulated pilus (TCP) encoded by vibrio pathogenicity island (VPI). CTX, a potent stimulator of adenylate cyclase, causes the intestine to secrete watery fluid rich in sodium, bicarbonate, and potassium, in volumes far exceeding the intestinal absorptive capacity, by ADP-ribosylation of the alpha subunit of the GTP-binding protein. Thus intestinal infection with V. cholerae results in the loss of large volumes of watery stool, leading to severe and rapidly progressing dehydration and shock. Without adequate and appropriate rehydration therapy, severe cholera kills about half of affected individuals. Today, cholera still remains a burden mainly for underdeveloped countries, which cannot afford to establish or to maintain necessary hygienic and medical facilities. During the last three decades, intensive research has been undertaken to unravel the virulence properties and to study the epidemiology of this significant human pathogen. This review provides an overview of the role of CTX in the occurrence of this disease in humans.

Role of Cholera Toxin in <i>Vibrio cholerae</i> Infection in Humans - A Review

Vibrio cholerae, the causative agent of Asiatic cholera, is a gram-negative motile bacterial species acquired via oral ingestion of contaminated food or water sources. Cholera has spread from the Indian subcontinent where it is endemic to involve nearly the whole world seven times during the past 185 years. V. cholerae serogroup O1, biotype El Tor, has moved from Asia to cause pandemic disease in Africa and South America during the past 35 years. A new serogroup, O139, appeared in south Asia in 1992, has become endemic there, and threatens to start the next pandemic. The facultative human pathogen V. cholerae represents a paradigm that evolved from environmental non-pathogenic strains by acquisition of virulence genes. The major virulence factors of V. cholerae, cholera toxin (CTX) encoded by the ctxAB genes residing in the genome of filamentous lysogenic bacteriophage (CTXɸ) and toxin coregulated pilus (TCP) encoded by vibrio pathogenicity island (VPI). CTX, a potent stimulator of adenylate cyclase, causes the intestine to secrete watery fluid rich in sodium, bicarbonate, and potassium, in volumes far exceeding the intestinal absorptive capacity, by ADP-ribosylation of the alpha subunit of the GTP-binding protein. Thus intestinal infection with V. cholerae results in the loss of large volumes of watery stool, leading to severe and rapidly progressing dehydration and shock. Without adequate and appropriate rehydration therapy, severe cholera kills about half of affected individuals. Today, cholera still remains a burden mainly for underdeveloped countries, which cannot afford to establish or to maintain necessary hygienic and medical facilities. During the last three decades, intensive research has been undertaken to unravel the virulence properties and to study the epidemiology of this significant human pathogen. This review provides an overview of the role of CTX in the occurrence of this disease in humans.

Genomics-guided discovery of potent anticancer natural products from exotic bacterial species

for 2014 UNTHSC RAD (March 21, 2014; Fort Worth, TX), No. 1144 GENOMICS-GUIDED DISCOVERY OF POTENT ANTICANCER NATURAL PRODUCTS FROM EXOTIC BACTERIAL SPECIES Xiangyang Liu 1,2 , Cheng Wang 2 , Sreya Biswas 2 , Christopher M. Antapli 2 , Hui Zhu 1 , Santosh Thapa 3 and Yi-Qiang Cheng 1,2,4* 1 UNT System College of Pharmacy, 3 Graduate School of Biomedical Sciences, 4 Institute for Cancer Research, University of North Texas Health Science Center, 3500 Camp Bowie Blvd, Fort Worth, TX 76107. 2 Department of Biological Sciences and Department of Chemistry and Biochemistry, University of Wisconsin–Milwaukee, PO Box 413, Milwaukee, WI 53201 Tel: (817) 735-0165; E-mail: yiqiang.cheng@unthsc.edu Abstract. Natural products are small chemical molecules produced by bacteria, fungi or plants. Natural Natural products are small chemical molecules produced by bacteria, fungi or plants. Natural products have made great contributions to medicine, particularly as anticancer drugs or anti-infective drugs. Currently there is a renaissance of natural product discovery due to the development of new discovery technologies and unmet medical needs. We hypothesized that exotic Gram-negative bacterial species can be a good source of diverse natural products. We analyzed the genomes of two bacterial species originally isolated from central Thailand or north Australia, and found that each genome contains multiple natural product biosynthetic gene clusters. Those information facilitated our discovery of two groups of new natural products, named thailandepsins and thailanstatins, that were found to possess potent antiproliferative activities against an array of human cancer cell lines. Additional studies are being conducted in animal models through collaborations. We thus concluded that Gram-negative bacterial species is a good source of diverse natural products, and genomics-guided discovery approach is effective and particularly suitable for small research laboratories with limited resources. Purpose. We hypothesize that exotic Gram-negative bacterial species can be a good source of diverse natural products. The purpose of the research is thus to discover new bioactive natural products from exotic bacterial species. Materials and Methods. Burkholderia thailandensis E264, a Gram-negative beta-proteobacterium strain originally isolated from a rice paddy in central Thailand, was purchased from the American Type Culture Collection (ATCC); Burkholderia thailandensis MSMB43, another Gram-negative beta-proteobacterium strain originally isolated from a water source in north Australia, was obtained from the US Centers for Disease Control (CDC). Bacterial genome analysis and natural product discovery and identification were performed according to standard procedures. Results. Mining the genome of B. thailandensis E264 revealed a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS–PKS) biosynthetic gene cluster that resembles that of FK228 (romidepsin, drug name Istodax) in Chromobacterium violaceum No. 968, which led us to discover thailandepsins A–F, natural analogues of FK228, and potent histone deacetylase inhibitors and antiproliferative agents with GI50 values in the sub-nM range. Mining the genome of B. thailandensis MSMB43 revealed at least 13 biosynthetic gene clusters. Among them one hybrid NRPS–PKS gene cluster is highly homologous to that of FR901464 (a prototype spliceosome inhibitor) in Pseudomonas sp. No. 2663, which led us to discover thailanstatins A–D, natural and more stable analogues of FR901464, and potent pre-mRNA splicing inhibitors and antiproliferative agents with GI50 values in the low nM range. Selected members of those natural products are under intensive collaborative investigations as anticancer drug candidates, and preliminary results are encouraging. Metabolic engineering approach is being undertaken to increase the yield of those potent compounds that are often produced in minute amounts by the wild-type bacteria. Conclusions. Potent new anticancer natural products have been discovered from exotic bacterial species via a genomics-guided discovery approach, which is effective and particularly suitable for small research laboratories with limited resources. We seek additional collaborations for identifying the best possible use of our small collection of potent natural products; we also seek to establish a “Texas Network for Collaborative Natural Product Discovery and Development” for sharing the resources, risks and rewards.

Identification of pBC218/pBC210 Genes of Bacillus cereus G9241 in Five Florida Soils Using qPCR.

The distribution of the virulent plasmid pBC210 of B. cereus that carries several B. anthracis genes and has been implicated in lethal anthrax-like pulmonary disease is unknown. We screened our collection of 103 B. cereus isolates and 256 soil samples using a quantitative PCR (qPCR) assay that targeted three open reading frames putatively unique to pBC210. When tested with DNA from 2 B. cereus strains carrying pBC210, and 64 Gram-positive and 55 Gram-negative bacterial species, the assay had 100% sensitivity and specificity. None of the DNA from the B. cereus isolates yielded positive amplicons but DNA extracted from five soils collected in Florida gave positive results for all three target sequences of pBC210. While screening confirms that pBC210 is uncommon in B. cereus, this study is the first to report that pBC210 is present in Florida soils. This study improves our knowledge of the distribution of pBC210 in soils and, of public health importance, the potential threat of B. cereus isolates carrying the toxin-carrying plasmid. We demonstrated that sequences of pBC210 can be found in a larger geographical area than previously thought and that finding more B. cereus carrying the virulent plasmid is a possibility in the future.

Assembly of the bacterial type III secretion machinery

Many bacteria that live in contact with eukaryotic hosts, whether as symbionts or as pathogens, have evolved mechanisms that manipulate host cell behaviour to their benefit. One such mechanism, the type III secretion system, is employed by Gram-negative bacterial species to inject effector proteins into host cells. This function is reflected by the overall shape of the machinery, which resembles a molecular syringe. Despite the simplicity of the concept, the type III secretion system is one of the most complex known bacterial nanomachines, incorporating one to more than hundred copies of up to twenty different proteins into a multi-MDa transmembrane complex. The structural core of the system is the so-called needle complex that spans the bacterial cell envelope as a tripartite ring system and culminates in a needle protruding from the bacterial cell surface. Substrate targeting and translocation are accomplished by an export machinery consisting of various inner membrane embedded and cytoplasmic components. The formation of such a multimembrane-spanning machinery is an intricate task that requires precise orchestration. This review gives an overview of recent findings on the assembly of type III secretion machines, discusses quality control and recycling of the system and proposes an integrated assembly model.

Antimicrobial consumption and resistance in five Gram-negative bacterial species in a hospital from 2003 to 2011.

The misuse of antimicrobial agents increases drug resistance in bacteria. The correlation between antimicrobial agent consumption and related resistance in the Gram-negative bacteria Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus mirabilis was analyzed during the period 2003-2011. Among these five bacteria, overall E. coli and K. pneumoniae were more commonly isolated from bloodstream than the other species. Regarding Enterobacteriaceae, E. coli and K. pneumoniae showed annual increases of resistance to the tested antimicrobial agents; conversely, P. mirabilis exhibited reduced resistance to cefuroxime, ceftriaxone and cefepime. In contrast to the relatively low antimicrobial resistance in P. aeruginosa, A. baumannii revealed high resistance, which was over 85% resistant rate to the tested antimicrobial agents and over 80% carbapenem resistance in 2011. E. coli, K. pneumoniae, and P. mirabilis differed in development of antimicrobial resistance after consumption of the antimicrobial agents. K.pneumoniae developed resistance to all antimicrobial groups, whereas resistance in P. mirabilis was not related to any antimicrobial consumption. P. aeruginosa developed resistance to β-lactam antimicrobials and aminoglycosides, whereas A. baumanii developed resistance to carbapenems after their use. The development of antimicrobial resistance was related to antimicrobial agents and bacterial species.

Rapid diagnosis and discrimination of bacterial meningitis in children using gram probe real-time polymerase chain reaction.

In this study, we developed a method of simultaneous detection and discrimination of bacteria in cerebrospinal fluid (CSF) with gram probe real-time polymerase chain reaction (PCR). Our results showed 25 clinical strains representing 13 gram-positive and 12 gram-negative bacterial species. They were identified correctly with the corresponding gram probe. The standard curve showed that the amplification efficiency of templates with different concentrations of bacteria was almost the same with a potential detection limit of 10 colony-forming units/mL. A total of 482 children who were clinically suspected of bacterial meningitis were included in this study. A total of 1.0 mL of CSF was collected from every child and was subjected to gram probe-based PCR (GP-PCR), CSF culture, and CSF routine analysis. The positive rate of the GP-PCR array was (32/482, 6.64%) significantly higher than that of CSF culture (23/482, 4.77%). GP-PCR was proved to be an excellent technique for rapid and accurate diagnosis and discrimination of bacterial meningitis, and hence its use as a diagnostic tool in future seems very promising.

Clinical investigation of bacterial species and endotoxin in endodontic infection and evaluation of root canal content activity against macrophages by cytokine production

This study investigated the presence of different Gram-negative bacterial species and the levels of endotoxins found in primary endodontic infection (PEI), determining their stimulation ability against macrophages through the levels of interleukin (IL)-1, IL-6, IL-10, and tumor necrosis factor alpha (TNF-α), and evaluated their relationship with clinical and radiographic findings. Samples were taken from 21 root canals with primary endodontic infection with apical periodontitis (PEIAP). Molecular techniques were used for bacterial detection. Limulus amebocyte lysate assay was used to measure endotoxins. Pro-inflammatory cytokines were measured by ELISA assay. All samples were positive for bacterial DNA (21/21). Prevotella nigrescens (57.2%) was the most frequent species. Higher levels of endotoxins were found in teeth with pain on palpation and exudation (all p < 0.05). Positive correlations were found between endotoxins and the levels of TNF-α and IL-1β, whereas a negative correlation was found between endotoxin and the amount of IL-10 (p < 0.05). Endotoxin levels were found to be a risk factor for exudation and increased the number of Gram-negative bacterial species for the presence of a larger area of bone destruction (all p < 0.05). A wide variety of Gram-negative bacterial species are involved in primary endodontic infection, with participation of different Treponema species. Thus, the levels of endotoxins and the number of Gram-negative bacteria species present in root canals were considered risk factors for the severity of endodontic infection. The present study revealed that Gram-negative bacterial species and endotoxins play an important role in the development of signs/symptoms and the severity of bone destruction, this knowledge is essential for the establishment of an effective therapy.

Modified clay minerals efficiency against chemical and biological warfare agents for civil human protection

Sorption efficiencies of modified montmorillonite and vermiculite of their mono ionic Na and organic HDTMA and HDP forms were studied against chemical and biological warfare agents such as yperite and selected bacterial strains. Yperite interactions with modified clay minerals were observed through its capture in low-density polyethylene foil-modified clay composites by measuring yperite gas permeation with using chemical indication and gas chromatography methods. The antibacterial activities of synthetized organoclays were tested against selected Gram-positive and Gram-negative bacterial species in minimum inhibitory concentration tests. The obtained results showed a positive influence of modified clay minerals on the significant yperite breakthrough-time increase. The most effective material was the polyethylene-Na form montmorillonite, while the polyethylene-Na form vermiculite showed the lowest efficiency. With increasing organic cations loading in the interlayer space the montmorillonite efficiency decreased, and in the case of vermiculite an opposite effect was observed. Generally the modified montmorillonites were more effective than modified vermiculites. The HDP cations seem to be more effective compare to the HDTMA. The antibacterial activity tests confirmed efficiency of all organically modified clay minerals against Gram-positive bacteria. The confirmation of antibacterial activity against Y. pestis, plague bacteria, is the most interesting result of this part of the study.

Trichoplaxin — A new membrane-active antimicrobial peptide from placozoan cDNA

A method based on the use of signal peptide sequences from antimicrobial peptide (AMP) precursors was used to mine a placozoa expressed sequence tag database and identified a potential antimicrobial peptide from Trichoplax adhaerens. This peptide, with predicted sequence FFGRLKSVWSAVKHGWKAAKSR is the first AMP from a placozoan species, and was named trichoplaxin. It was chemically synthesized and its structural properties, biological activities and membrane selectivity were investigated. It adopts an α-helical structure in contact with membrane-like environments and is active against both Gram-negative and Gram-positive bacterial species (including MRSA), as well as yeasts from the Candida genus. The cytotoxic activity, as assessed by the haemolytic activity against rat erythrocytes, U937 cell permeabilization to propidium iodide and MCF7 cell mitochondrial activity, is significantly lower than the antimicrobial activity. In tests with membrane models, trichoplaxin shows high affinity for anionic prokaryote-like membranes with good fit in kinetic studies. Conversely, there is a low affinity for neutral eukaryote-like membranes and absence of a dose dependent response. With high selectivity for bacterial cells and no homologous sequence in the UniProt, trichoplaxin is a new potential lead compound for development of broad-spectrum antibacterial drugs.

Type VI secretion system effectors: poisons with a purpose

The type VI secretion system (T6SS) mediates interactions between a broad range of Gram-negative bacterial species. Recent studies have led to a substantial increase in the number of characterized T6SS effector proteins and a more complete and nuanced view of the adaptive importance of the system. Although the T6SS is most often implicated in antagonism, in this Review, we consider the case for its involvement in both antagonistic and non-antagonistic behaviours. Clarifying the roles that type VI secretion has in microbial communities will contribute to broader efforts to understand the importance of microbial interactions in maintaining human and environmental health, and will inform efforts to manipulate these interactions for therapeutic or environmental benefit.