I.Y. Mahmoud

Bacterial flora and antibiotic resistance from eggs of green turtles Chelonia mydas: An indication of polluted effluents

Sea turtles migrate to various habitats where they can be exposed to different pollutants. Bacteria were collected from turtle eggs and their resistance to antibiotics was used as pollutant bio-indicators of contaminated effluents. Eggs were collected randomly from turtles when they were laying their eggs. A total of 90 eggs were collected and placed into sterile plastic bags (3 eggs/turtle) during June-December of 2003. The bacteria located in the eggshell, albumen and yolk were examined, and 42% of the eggs were contaminated with 10 genera of bacteria. Pseudomonas spp. were the most frequent isolates. The albumen was found to be the part of the egg to be the least contaminated by bacterial infection. Bacterial isolates tested with 14 antibiotics showed variations in resistance. Resistance to ampicillin was the highest. The presence of antibiotic resistant bacteria in eggs indicates that the green turtle populations were subjected to polluted effluents during some of their migratory routes and feeding habitats. Scanning electron microscopy revealed that Salmonella typhimurium penetrated all eggshell layers.

Coastal sewage discharge and its impact on fish with reference to antibiotic resistant enteric bacteria and enteric pathogens as bio-indicators of pollution.

Presence of antibiotic resistant bacteria as bio-indicators of pollution was monitored in fish (colons and gills) and in sewage treated effluent (STE) in two marine sites in the Gulf of Oman, Muscat. One site was polluted as the sewage effluent STE was directly discharged into the sea while the unpolluted site was 10 km away. A total of 42 water samples (250 mL each) were collected from both sites. In the polluted site, 14 samples were taken directly from the STE discharge point and 14 from the surrounding area near the STE discharging point (7-10 m). Water and fish samples were collected from polluted and unpolluted sites. Chlorine and nitrate levels were significantly higher in STE samples. A significant difference (P<0.005) was found in total bacterial count of fish colon and gills in the two sites. Most probable number (MPN) was significantly higher in STE and polluted seawater over unpolluted. Coliforms were detected in the polluted site only. A total of 320 target enteric normal flora of animals and pathogenic species were isolated from 10 genera taken from water and fish samples. The isolates were exposed to 14 antibiotics. Most of the isolates were resistant to at least one antibiotic with ampicillin was the dominant antibiotic. This is a clear indication that sewage effluent causes contamination of marine wildlife along coastal lines.

Biomonitoring marine habitats in reference to antibiotic resistant bacteria and ampicillin resistance determinants from oviductal fluid of the nesting green sea turtle, Chelonia mydas

During the egg-laying process, oviductal fluid was collected using a non-invasive procedure from the cloacal vent of the green turtles. Forty-two independent isolates of antibiotic-resistant bacteria from 11 genera were obtained from 20 turtles during nesting. The dominant isolate was Citrobacter (52.4%), followed by Pseudomonas, Proteus, Enterobacter, Salmonella, Escherichia coli, Shigella, Edwardsiella, Morganella, Providencia and Arcomobacter. Most of the isolates were resistant to ampicillin. Ampicillin-resistant isolates showed variations in their resistance for the following classes of β-lactamases: extended-spectrum β-lactamases (EBSLs), AmpC type β-lactamases C (AmpC), and screen-positive β-lactamase. None of the isolates produced metallo β-lactamase. Some ampicillin-resistant genes were detected by multiplex polymerase chain reaction (PCR) only. Inhibitor based test (IBT) categorized some isolates as AmpC β-lactamase producers. β-Lactamase genes were detected from a few strains. The sequencing of those genes revealed the presence of cephamycinase (CMY) and AmpC β-lactamases. The oviductal fluid was used in this study as a source of bacterial antibiotic-resistant determinants for biomonitoring marine turtles exposed to contaminated effluents. This data can be of value in understanding the decline of this endangered species as a result of exposure to marine pollution which is threatening their survival.

Viability of multiple antibiotic resistant bacteria in distribution lines of treated sewage effluent used for irrigation.

Viability of multiple antibiotic resistant bacteria (MARB) in tertiary treated sewage effluent (TTSE) used for irrigation, was investigated at the Sultan Qaboos University sewage treatment plant (STP). This water recycle system is used here as a model for the systems commonly used throughout Oman and the Gulf region. Samples of TTSE were collected weekly from four sites, 1.5 km from each other. Chlorine levels declined gradually at the three sites with increasing distance from the STP. Viable bacteria, coliforms and nitrate concentrations increased significantly while biological oxygen demand (BOD) declined after STP chlorination. Mean values of turbidity changed slightly. Trace elements values were insignificant. A total of 336 bacteria from 8 genera revealed that the dominant isolates were Enterobacter spp., Pseudomonas spp., and Aeromonas spp. Among the isolates 59.8% were multiply resistant to several antibiotics. Resistance was higher to ampicillin followed by sulphamethoxazole, carbenicillin, streptomycine and minocycline. Frequency of resistance to the 14 antibiotics varied among the isolates. The present system related to the viability of MARB in TTSE used for irrigation may have serious implications for public health and wildlife. Results of this investigation will be of value in modifying current STPs systems and thus avoiding serious health issues.

Antibiotic resistant bacteria as bio-indicator of polluted effluent in the green turtles, Chelonia mydas in Oman.

Antibiotic resistant bacteria were studied as bio-indicators of marine polluted effluents during egg-laying in green turtles. A non-invasive procedure for sampling oviductal fluid was used to test for exposure of turtles to pollution in Ras Al-Hadd, Oman, which is one of the most important nesting beaches in the world. Each sample was obtained by inserting a 15 cm sterile swab gently into the cloacal vent as the sphincter muscle is relaxed and the cloacal lining is unfolded to the outside. Forty turtles were sampled. A hundred and thirty-two species of bacteria from 7 genera were isolated. The dominant isolate was Citrobacter. Among the isolates 60.6% were multiple resistant to 15 tested antibiotics. The dominant resistance to antibiotics was ampicillin followed by streptomycin and sulphamethoxazole. Sampling oviductal fluid for resistant bacteria to antibiotics is valuable way to assess exposure to polluted effluents during feeding and migratory in turtles. Polluted effluents using bacteria as bio-indicator may influence reproductive potential in this endangered species.

Diversity and trapping efficiency of nematophagous fungi from Oman.

Summary. A survey of the nematophagous mycobiota biodiversity of 82 soil and leaf-litter samples in the Sultanate of Oman yielded ten species of nematode trapping fungi belonging to three genera. The species are: Arthrobotrys eudermata, A. thaumasia, A. musiformis, A. oligospora, A. oligospora var. oligospora, A. oudemansii, A. multiformis, A. javanica, Drechslerella brochopaga and Gamsylella geophyropaga. This is the first record of these species in Oman. Arthobotrys multiformis represents the second record of this species worldwide. A systematic study showed that A. oudemansii, A. multiformis and A. javanica were morphologically more variable than was so far known. In four days, A. oligospora, A. thaumasia, D. brochopaga and A. eudermata trapped all nematodes added to the Petri dishes (Panagrellus redivivus, 100 specimens per dish). No significant differences were found in the trapping efficiency among the species tested.

Ultrastructural features and elemental distribution in eggshell during pre and post hatching periods in the green turtle, Chelonia mydas at Ras Al-Hadd, Oman

Eggshells were randomly collected from turtle nests immediately after oviposition and at the end of incubation to examine the ultrastructural features using scanning JSM-5600LV microscopy. Three layers were recognized; an outer calcareous, a middle multistrata and an inner membrane. The calcareous layer had loose nodular units varying in shape and size without interlocking attachments. In freshly laid eggs, each nodular unit had spicules arranged in folded stacks. The spicules became unfolded during incubation, to form radiating configurations. Elemental composition and mapping of the layers were analyzed using energy dispersive spectroscopy (EDS). The elements were unevenly distributed throughout the eggshell and Ca(2+) decreased significantly after hatching. X-ray diffraction was used to identify the crystals of the eggshells. It revealed that nodular units of the calcareous were made up of CaCO(3), as aragonite (91%), calcite (6%) and vaterite (3%). The middle layer consisted of organic amorphous material with aragonite (89%) and calcite (11%). The shell membrane consisted of reticular fibers with crystals predominantly of NaCl halite. Thermogravimetry analysis of the calcareous layer indicated a complete evaporation of bonded H(2)O at 480 degrees C and CO(2) at 830 degrees C. Using the differential thermal analysis (DTA), aragonite was transformed to stable calcite at 425 degrees C.

Detection of low plasma estradiol concentrations in nesting green turtles (Chelonia mydas) by HPLC/Ms-Ms

In previous studies on nesting green turtles under natural conditions from different geographical regions, 17-β-estradiol (E(2) ) was either undetectable or detected at very low levels. RIA and other related techniques were not sensitive enough to measure low E(2) values in the green turtles. In this study, a sensitive method was used in detecting low hormone concentrations: high performance liquid chromatography with tandem quadruple mass spectrometry (HPLC-MS/MS). Using this technique, estradiol for the first time was detected in nesting green turtles during the peak season (June-October) at Ras Al-Hadd Reserve, Oman. The E(2) values recorded from this study were the highest ever recorded from nesting green turtles in any geographical region, but the levels did not vary significantly throughout different phases of nesting. The presence of E(2) during nesting presumably plays a role in the physiology and behavior of this species. Ras Al-Hadd hosts one of the largest nesting populations of green turtles in the world, and an understanding of their nesting patterns may be of value in conservation and management programs for this endangered species.

Seasonal changes in plasma steroid levels in relation to ovarian steroidogenic ultrastructural features and progesterone receptors in the house gecko, Hemidactylus flaviviridis, in Oman

A comprehensive study of the annual ovarian cycle in the house gecko, Hemidactylus flaviviridis, was conducted in Oman. Circulating estradiol (E(2)), progesterone (P), and testosterone (T) were measured during the active and inactive phases of the cycle. The steroid levels were related to the steroidogenic ultrastructural features such as the abundance of smooth endoplasmic reticulum (SER), the presence of cisternal whorls, and close association of lipid droplets with swollen vesiculated mitochondria and SER. The steroids were measured using a sensitive detection technique HPLC-MS/MS. E(2) levels began to rise in January at the onset of vitellogenesis and continued to rise between February and May relative to ovulation, postovulation, gravidity and oviposition. Afterwards, E(2) remained low during the inactive phase (June-December). P levels increased significantly in March and peaked in April, which coincided with luteinization. P levels began to decline relative to luteolysis (May-June). Afterwards, it remained low throughout the inactive phase. T levels rose significantly in March-April coinciding with vitellogenesis, but decreased rapidly and significantly in May and remained low during the inactive phase. Progesterone receptors (PR), identified using immunohistochemistry, were strongly expressed during the breeding period, but were absent during the non-breeding period. The appearance of the steroidogenic ultrastructural features in the preovulatory and lutein granulosa cells was correlated with the significant rise in the three steroid levels and the PR. As the steroid levels declined, the granulosa cells underwent a general degeneration and disruption of the associated steroidogenic features.

Analysis of elemental composition of the eggshell before and after incubation in the loggerhead turtle ( Caretta caretta) in Oman.

To date, there are limited studies on loggerhead turtle (Caretta caretta) eggshell ultrastructure and its elemental composition. Eggs were collected from turtle nests immediately after oviposition and post hatching. Three eggshell layers were recognized. The outer calcareous layer consists of loose nodular units of different shapes and sizes with loose attachment between the units, resulting in numerous spaces and openings. Each unit consists of CaCO3 crystals in aragonite (99%) and calcite (1%). The middle layer has several strata with numerous openings connecting the calcareous and the inner shell membrane. Crystallites of the middle layer are a mix of amorphous material with aragonite (62%) and calcite (38%). The inner shell membrane has numerous reticular fibers mixed predominantly with halite (NaCl) and small amounts of sylvite. Thermogravimetry analysis of the calcareous showed a low exothermic peak at 425°C, which corresponds to a transitional phase from aragonite to calcite. A high endothermic peak at 814°C corresponds to decomposition of calcite CaCO3 to CaO and CO2. Electron diffraction confirmed the presence of NaCl halite crystal. A significant difference was found in the percentage of elements and crystal configurations in the three layers. This study has value in assessing the emergence success in this endangered species.