Siamese crocodile plasma synergizes with ceftazidime against ceftazidime-resistant Enterobacter cloacae

Purpose: To evaluate whether Siamese crocodile plasma exhibits antibacterial properties and if it synergizes with ceftazidime against ceftazidime-resistant Enterobacter cloacae (CREnC).Methods: Protein fractions were from crocodile plasma and tested them on CREnC strains. Multiplex polymerase chain reaction (PCR) screening test was performed for extended-spectrum β-lactamase (ESBL) phenotype and AmpC gene. The effects of the antibacterial agents were analyzed using a bacterial suspension standard curve, minimum inhibitory concentration (MIC), Checkerboard assays, viability curves, membrane permeability assays, enzyme assays, and transmission electron microscopy.Results: CREnC strains expressed ESBL-AmpC gene combinations. The MICs of resuspended protein 1 (P1), protein 5 (P5), ceftazidime, cefotaxime, and benzylpenicillin against all tested CREnC and E. coli strains were in the range of > 1024 μg/mL, indicating resistance. However, P1 and P5 exhibited a synergistic effect against test CREnC and E. coli strains when used in combination with ceftazidime and cefotaxime, with fraction inhibitory concentration indices of < 0.062 and 0.28, respectively. A kill curve demonstrated that the combination treatments had synergistic activity and inhibited β-lactamase.Conclusion: The synergistic activity of P1 and P5 in combination with ceftazidime is achieved in multiple ways, including increased cytoplasmic and outer membrane permeability, β-lactamase inhibition, and peptidoglycan damage. Therefore, the combination therapy of Siamese crocodile plasma and ceftazidime may be a novel therapeutic approach for treating recalcitrant E. cloacae infection.Keywords: Crocodylus siamensis, ceftazidime-resistant Enterobacter cloacae, synergistic activity, β-lactamase activit

Survival and Growth of American Alligator (Alligator mississippiensis) hatchlings after artificial incubation and repatriation

Hatchling American Alligators (Alligator mississippiensis) produced from artificially incubated wild eggs were returned to their natal areas (repatriated). We compared artificially incubated and repatriated hatchlings released within and outside the maternal alligator’s home range with naturally incubated hatchlings captured and released within the maternal alligator’s home range on Lake Apopka, Lake Griffin, and Orange Lake in Florida. We used probability of recapture and total length at approximately nine months after hatching as indices of survival and growth rates. Artificially incubated hatchlings released outside of the maternal alligator’s home range had lower recapture probabilities than either naturally incubated hatchlings or artificially incubated hatchlings released near the original nest site. Recapture probabilities of other treatments did not differ significantly. Artificially incubated hatchlings were approximately 6% shorter than naturally incubated hatchlings at approximately nine months after hatching. We concluded that repatriation of hatchlings probably would not have long-term effects on populations because of the resiliency of alligator populations to alterations of early age-class survival and growth rates of the magnitude that we observed. Repatriation of hatchlings may be an economical alternative to repatriation of older juveniles for population restoration. However, the location of release may affect subsequent survival and growth

Antibacterial activity of plasma from crocodile (<it>Crocodylus siamensis</it>) against pathogenic bacteria

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.</p

Population structure of Siamese and Saltwater crocodiles from 69 crocodile individuals.

<p>(a) Evanno's Δ<i>K</i> graph. (b) STRUCTURE bar plots depicting the model-based clustering results for inferred <i>K</i> = 2. Inferred genetic clusters are displayed as different colors. Each vertical bar on the <i>x</i>-axis represents an individual, and the <i>y</i>-axis presents the proportion of membership (posterior probability) in each genetic cluster. Recovered crocodile species, including clusters α and β, are superimposed on the plot, with black vertical lines indicating the boundaries. Detailed information for all crocodile individuals is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184526#pone.0184526.s002" target="_blank">S1 Table</a>.</p

Microsatellite-based phylogenetic relationships for 52 Siamese crocodiles and 17 Saltwater crocodiles generated by the unweighted pair group with arithmetic mean (UPGMA) clustering method.

<p>Support values at each node are bootstrap values. “*” indicates a bootstrap value < 50%. The genetic similarity matrices are shown by Jaccard's coefficient. Detailed information for all crocodile individuals is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184526#pone.0184526.s002" target="_blank">S1 Table</a>.</p

Population structure of Siamese and Saltwater crocodiles from 69 crocodile individuals.

<p>(a) Mean Ln P(<i>K</i>) graph. (b) STRUCTURE bar plots depict the model-based clustering results for inferred <i>K</i> = 13. Inferred genetic clusters are displayed as different colors. Each vertical bar on the <i>x</i>-axis represents an individual, and the <i>y</i>-axis presents the proportion of membership (posterior probability) in each genetic cluster. Recovered crocodile species, including clusters A–M, are superimposed on the plot, with black vertical lines indicating the boundaries. Detailed information for all crocodile individuals is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184526#pone.0184526.s002" target="_blank">S1 Table</a>.</p

Observed distribution of pairwise relatedness for 52 Siamese crocodiles and 17 Saltwater crocodiles plotted against expected distributions.

<p>Observed distribution of pairwise relatedness for 52 Siamese crocodiles and 17 Saltwater crocodiles plotted against expected distributions.</p

High genetic diversity and demographic history of captive Siamese and Saltwater crocodiles suggest the first step toward the establishment of a breeding and reintroduction program in Thailand

<div><p>The Siamese crocodile (<i>Crocodylus siamensis</i>) and Saltwater crocodile (<i>C</i>. <i>porosus</i>) are two of the most endangered animals in Thailand. Their numbers have been reduced severely by hunting and habitat fragmentation. A reintroduction plan involving captive-bred populations that are used commercially is important and necessary as a conservation strategy to aid in the recovery of wild populations. Here, the genetic diversity and population structure of 69 individual crocodiles, mostly members of captive populations, were analyzed using both mitochondrial D-loop DNA and microsatellite markers. The overall haplotype diversity was 0.924–0.971 and the mean expected heterozygosity across 22 microsatellite loci was 0.578–0.701 for the two species. This agreed with the star-like shaped topology of the haplotype network, which suggests a high level of genetic diversity. The mean ratio of the number of alleles to the allelic range (<i>M</i> ratio) for the populations of both species was considerably lower than the threshold of 0.68, which was interpreted as indicative of a historical genetic bottleneck. Microsatellite markers provided evidence of introgression for three individual crocodiles, which suggest that hybridization might have occurred between <i>C</i>. <i>siamensis</i> and <i>C</i>. <i>porosus</i>. D-loop sequence analysis detected bi-directional hybridization between male and female individuals of the parent species. Therefore, identification of genetically non-hybrid and hybrid individuals is important for long-term conservation management. Relatedness values were low within the captive populations, which supported their genetic integrity and the viability of a breeding and reintroduction management plan. This work constitutes the first step in establishing an appropriate source population from a scientifically managed perspective for an <i>in situ</i>/<i>ex situ</i> conservation program and reintroduction of crocodile individuals to the wild in Thailand.</p></div