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 ß-lactamases: extended-spectrum ß-lactamases (EBSLs), AmpC type ß-lactamases C (AmpC), and screen-positive ß-lactamase. None of the isolates produced metallo ß-lactamase. Some ampicillin-resistant genes were detected by multiplex polymerase chain reaction (PCR) only. Inhibitor based test (IBT) categorized some isolates as AmpC ß-lactamase producers. ß-Lactamase genes were detected from a few strains. The sequencing of those genes revealed the presence of cephamycinase (CMY) and AmpC ß-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.

Opportunistic pathogens relative to physicochemical factors in water storage tanks.

Household water in Oman, as well as in other countries in the region, is stored in tanks placed on house roofs that can be subjected to physicochemical factors which can promote microbial growth, including pathogens and opportunistic pathogens which pose health risks. Water samples were collected from 30 houses in a heavily populated suburb of Muscat. The tanks used were either glass reinforced plastic (GRP), polyethylene or galvanised iron (GI). Heterotrophic bacteria, coliforms, faecal coliforms and iron sulphur bacteria varied significantly in the three tanks. Yeast and mould count showed significant variations. Isolation of Aeromonas spp., fluorogenic and pathogenic Pseudomonas, Pasteurella, Salmonella, Serratia and Tatumella, and Yersinia and Legionella in biofilms varied in the three tanks. The fungi isolates in the three tanks were Penicillium, Cladosporium and Aspergillus. Nephelometric turbidity unit, threshold odour number and free chlorine varied significantly in the three tanks. True colour unit values did not show a significant difference; however, GRP tanks had algae, autotrophic and pigmented microorganisms. In addition, GI tanks had sediments and corrosion. The results of this investigation are important to evaluate the status of the present household water tanks in countries with high annual temperatures, which may affect public health.

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.

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-ß-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.

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.

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.

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.

Antibiotic-resistant Salmonella spp. from human and non-human sources in Oman.

We investigated the antibiotic resistance of various strains of Salmonella isolated from different sources in Oman, namely sewage water, chicken and food handlers. Of 1242 isolates from food handlers, 15 (1.2%) were resistant to one or more antibiotic. Of these, 41% were resistant to ampicillin, 42% to co-trimoxazole and 22% to chloramphenicol. None of the isolates was resistant to ciprofloxacin. Of 515 isolates from chicken and 432 from sewage water, 23.7% and 14.1% respectively were resistant to one or more antibiotic. Susceptibility to the different antibiotics varied but none of the isolates from chicken was resistant to amikacin.

Antibiotic-resistant Salmonella spp. from human and non-human sources in Oman

We investigated the antibiotic resistance of various strains of Salmonella isolated from different sources in Oman, namely sewage water, chicken and food handlers. Of 1242 isolates from food handlers, 15 [1.2%] were resistant to one or more antibiotic. Of these, 41% were resistant to ampicillin, 42% to co-trimoxazole and 22% to chloramphenicol. None of the isolates was resistant to ciprofloxacin. Of 515 isolates from chicken and 432 from sewage water, 23.7% and 14.1% respectively were resistant to one or more antibiotic. Susceptibility to the different antibiotics varied but none of the isolates from chicken was resistant to amikacin

Adherence of Salmonella typhimurium to murine peritoneal macrophages is mediated by lipopolysaccharide and complement receptors.

Adherence of Salmonella typhimurium to mouse peritoneal macrophages (Mø) was monitored using a direct microscopic assay and flow cytometry. Competitive binding studies using wild-type lipopolysaccharide and derivatives confirmed a role for this moiety in bacterial adherence. Mø pretreated with 2-deoxy-D-glucose exhibited lower binding activity than did untreated controls, suggesting involvement of either Fc or complement receptors. Pre-exposing Mø to Fc fragments, however, failed to reduce bacterial binding, thus eliminating a role for Fc receptors in this process. Mø pretreated with neutrophil elastase exhibited a diminished ability to bind S. typhimurium, suggesting involvement of complement receptor 1. Monoclonal antibodies M1/70 and M18/2, specific for epitopes on the alpha and beta chains, respectively, of complement receptor 3, also blocked this adherence. In each case we were unable to eliminate completely bacterial adhesion to Mø. Monoclonal antibodies to two additional Mø receptors, Mac-2 and Mac-3, did not block bacterial attachment. These data indicate that multiple mechanisms are involved in the initial adhesion of S. typhimurium to mouse Mø.