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      Antibacterial activity of plasma from crocodile ( Crocodylus siamensis) against pathogenic bacteria

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          Abstract

          Background

          The Siamese crocodile ( Crocodylus siamensis) is a critically endangered species of freshwater crocodiles. Crocodilians live with opportunistic bacterial infection but normally suffer no adverse effects. They are not totally immune to microbial infection, but their resistance thereto is remarkably effective. In this study, crude and purified plasma extracted from the Siamese crocodile were examined for antibacterial activity against clinically isolated, human pathogenic bacterial strains and the related reference strains.

          Methods

          Crude plasma was prepared from whole blood of the Siamese crocodile by differential sedimentation. The crude plasma was examined for antibacterial activity by the liquid growth inhibition assay. The scanning electron microscopy was performed to confirm the effect of crude crocodile plasma on the cells of Salmonella typhi ATCC 11778. Effect of crude crocodile plasma on cell viability was tested by MTT assay. In addition, the plasma was purified by anion exchange column chromatography with DEAE-Toyopearl 650 M and the purified plasma was tested for antibacterial activity.

          Results

          Crude plasma was prepared from whole blood of the Siamese crocodile and exhibited substantial antibacterial activities of more than 40% growth inhibition against the six reference strains of Staphylococcus aureus, Salmonella typhi, Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, and Staphylococcus epidermidis, and the four clinical isolates of Staphylococcus epidermidis, Pseudomonas aeruginosa, Salmonella typhi, and Vibrio cholerae. Especially, more than 80% growth inhibition was found in the reference strains of Salmonella typhi, Vibrio cholerae, and Staphylococcus epidermidis and in the clinical isolates of Salmonella typhi and Vibrio cholerae. The effect of the crude plasma on bacterial cells of Salmonella typhi, a certain antibacterial material probably penetrates progressively into the cytoplasmic space, perturbing and damaging bacterial membranes. The effect of the crude plasma was not toxic by the yellow tetrazolium bromide (MTT) assay using a macrophage-like cell, RAW 264.7. The pooled four fractions, designated as fractions D1-D4, were obtained by column chromatography, and only fraction D1 showed growth inhibition in the reference strains and the clinical, human pathogenic isolates.

          Conclusions

          The crude and purified plasma from the Siamese crocodile significantly showed antibacterial activity against pathogenic bacteria and reference strains by damage cell membrane of target bacterial cells. From the MTT assay, the Siamese crocodile plasma was not cytotoxic to the cells.

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          Most cited references20

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          Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity.

          The antimicrobial peptide LL-37 belongs to the cathelicidin family and is the first amphipathic alpha-helical peptide isolated from human. LL-37 is considered to play an important role in the first line of defence against local infection and systemic invasion of pathogens at sites of inflammation and wounds. Understanding its mode of action may assist in the development of antimicrobial agents mimicking those of the human immune system. In vitro studies revealed that LL-37 is cytotoxic to both bacterial and normal eukaryotic cells. To gain insight into the mechanism of its non-cell-selective cytotoxicity, we synthesized and structurally and functionally characterized LL-37, its N-terminal truncated form FF-33, and their fluorescent derivatives (which retained structure and activity). The results showed several differences, between LL-37 and other native antimicrobial peptides, that may shed light on its in vivo activities. Most interestingly, LL-37 exists in equilibrium between monomers and oligomers in solution at very low concentrations. Also, it is significantly resistant to proteolytic degradation in solution, and when bound to both zwitterionic (mimicking mammalian membranes) and negatively charged membranes (mimicking bacterial membranes). The results also showed a role for the N-terminus in proteolytic resistance and haemolytic activity, but not in antimicrobial activity. The LL-37 mode of action with negatively charged membranes suggests a detergent-like effect via a 'carpet-like' mechanism. However, the ability of LL-37 to oligomerize in zwitterionic membranes might suggest the formation of a transmembrane pore in normal eukaryotic cells. To examine this possibility we used polarized attenuated total reflectance Fourier-transform infrared spectroscopy and found that the peptide is predominantly alpha-helical and oriented nearly parallel with the surface of zwitterionic-lipid membranes. This result does not support the channel-forming hypothesis, but rather it supports the detergent-like effect.
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            Eggerthella hongkongensis sp. nov. and eggerthella sinensis sp. nov., two novel Eggerthella species, account for half of the cases of Eggerthella bacteremia.

            Eggerthella, one of the human gut flora, was rarely reported to cause bacteremia in the literature. We describe the application of 16S ribosomal RNA gene sequencing in defining the epidemiology and clinical significance of Eggerthella bacteremia during a 4-year period. Among 55 clinically significant blood culture isolates of anaerobic Gram-positive bacilli, 5 were identified as E. lenta and 5 belonged to 2 novel Eggerthella species, proposed as E. hongkongensis and E. sinensis, respectively. The 10 patients with Eggerthella bacteremia were adults, and 9 had underlying diseases. In all cases, the source of the bacteremia was likely from endogenous flora. Septic shock was a complication in 4 patients, and 3 patients died. The present study suggests that Eggerthella bacteremia is much more common than expected and is associated with significant morbidity and mortality. Moreover, the 2 novel species account for half of the cases of Eggerthella bacteremia.
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              Antibacterial properties of serum from the American alligator (Alligator mississippiensis).

              Treatment of alligator (Alligator mississippiensis) and human serum samples with Escherichia coli resulted in a time- and concentration-dependent inhibition of bacterial proliferation. When inoculated with E. coli, alligator serum exhibited 10-fold lower bacterial survival rates after 1 h than human serum. In addition, the antibacterial spectrum of alligator serum was shown to be much broader than that of human serum, with growth inhibition occurring in 100% of bacterial strains tested (compared to only 35% for human serum). Additional results showed that the antimicrobial activities of alligator serum could be completely inhibited by preincubation with proteases, indicating the proteinaceous nature of the antimicrobial activities. Furthermore, incubation of alligator serum at 56 degrees C for 30 min (classical human serum complement inactivation conditions) obliterated all antimicrobial properties of the alligator serum. The antibacterial activities occurred relatively quickly in vitro, with significant activity occurring within 5 min of inoculation with E. coli and maximal activity at 20 min. Also, the antimicrobial activity exhibited temperature dependence, with a substantial decrease in activity below 15 degrees C. These data suggest that the antimicrobial properties of alligator serum may be due to an active serum complement system.
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                Author and article information

                Journal
                Ann Clin Microbiol Antimicrob
                Ann. Clin. Microbiol. Antimicrob
                Annals of Clinical Microbiology and Antimicrobials
                BioMed Central
                1476-0711
                2012
                30 July 2012
                : 11
                : 22
                Affiliations
                [1 ]Department of Biochemistry, Faculty of Science, Protein Proteomic Research Group, Khon Kaen University, Khon Kaen, 40002, Thailand
                [2 ]Department of Microbiology, Faculty of liberal arts and science, Kasetsart University Kamphaeng saen Campus, Nakhon Pathom, 73140, Thailand
                [3 ]Sriracha Moda Farm, Chon Buri, 20110, Thailand
                [4 ]Faculty of Humanities and Social Sciences, Khon Kaen University, Khon Kaen, Thailand, 40002
                [5 ]BIOTEC Culture Collection (BCC), National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani, 12120, Thailand
                [6 ]JICA Senior Overseas Volunteer, Japan International Cooperation Agency (JICA), Shibuya-ku, Tokyo, 151-8558, Japan
                [7 ]Shizuoka University, Suruga-ku, Shizuoka, 422-8529, Japan
                Article
                1476-0711-11-22
                10.1186/1476-0711-11-22
                3490821
                22846342
                1d5624f8-b48d-479c-b8f7-e512a253615c
                Copyright ©2012 Kommanee et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 1 May 2012
                : 1 July 2012
                Categories
                Research

                Infectious disease & Microbiology
                cytotoxicity,crocodile (crocodylus siamensis),antibacterial activity,pathogenic bacteria

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