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Marine Turtle Newsletter 115:9-11, © 2007

Marine Turtle Newsletter-Online

Identification of Bacterial Isolates from Unhatched Loggerhead (Caretta caretta) Sea Turtle Eggs in Georgia, USA

Kathryn S. Craven, Judy Awong-Taylor, Laura Griffiths, Crystal Bass & Mario Muscarella
Armstrong Atlantic State University, Dept. Biology, 11935 Abercorn St., Savannah, GA 31419 USA (E-mail: cravenka@mail.armstrong.edu)

Loggerhead sea turtles (Caretta caretta) are the most common species nesting in Georgia and represent part of the temperate nesting population in the United States (Bowen & Karl 1997). Worldwide, more than 80% of sea turtle eggs hatch successfully (Miller 1997), while mean loggerhead hatch success during the 2002-2005 nesting seasons in Georgia was 66.5%. On Jekyll Island, one of Georgia’s southernmost barrier islands, mean hatch success during the same seasons was only 62.2% (Dodd & Mackinnon 2002; 2003; 2004; 2005). Mathematical models have predicted that egg loss and juvenile mortality can reduce recruitment into the adult breeding population (Heppell et al. 2003). Causes of egg failure during development are not always clear. While many anecdotal reports place the blame on bacterial contamination, very little data exist to support these claims. Three previous studies list bacteria found associated with unhatched sea turtle eggs (leatherback, Girondot et al. 1990; olive ridley, Mo et al. 1990; loggerhead, Wyneken et al. 1988). This study reports on the presence of potentially pathogenic microbes cultured from unhatched loggerhead sea turtle eggs.

Sixty-seven unhatched intact loggerhead turtle eggs from 15 nests were collected during August and September in 2004 and 2005 in an effort to characterize and identify their bacterial populations. All nests were laid on Jekyll Island, Georgia, USA and allowed to go to full term. Samples were collected 3-5 days after emergence of the hatchlings was confirmed, or after 70 days of incubation. Eggs were kept in sand from the nest, placed in sterile paper bags and transported to Armstrong Atlantic State University in Savannah, GA, USA. Bacterial sampling and assessment of embryonic development was completed within 2-3 hours of egg collection. Data from controlled healthy nests were not available due to the designation of loggerheads as a threatened species.

Sterile cotton swabs were used to remove bacterial samples from the outside surface of the eggs. Eggs were surface sterilized with hydrogen peroxide (five minutes) and 95% ethanol (three minutes). Betadine was applied to a portion of shell that was then aseptically removed with sterile scissors. A sterile cotton swab was used to obtain a fluid sample from the interior of the egg. Bacteria were grown and isolated on tryptic soy agar (TSA) and marine agar (MA) using a quadrant isolation technique. Bacterial isolates were maintained on TSA and MA slants.

After fluid samples were taken, eggs were dissected using scissors and tweezers. All contents were examined for signs of fertility (yolk metabolism, blood vessel development or visible embryonic tissue). All visible embryos representing >10 days of development were removed and preserved in 10% formalin. Straight line carapace length (SCL) was measured using stainless steel calipers. Egg contents were classified as infertile (no metabolism of albumin or yolk), fertile (yolk metabolized, no visible embryo), embryonic disc (mass of tissue, no eye spots), early embryonic death (EED, SCL less than 1.0 cm), mid-embryonic death (MED, SCL 1.01-2.0 cm), and late embryonic death (LED, SCL > 2.01).

Bacterial characteristics such as growth patterns, texture, pigmentation, and oxygen utilization were determined using tryptic soy broth (TSB), TSA, marine broth (MB) and MA mediums. Gram stains were used to determine shape and arrangement of cells. Isolates were classified as gram-positive or gram-negative. Isolates of were identified using the API 20 Enteric and Nonenteric Identification System for Enterobacteriaceae and other non-fastidious and non-enteric gram-negative rods (bioMerieux). Positive (Escherichia coli) and negative (deionized water) controls were run with these kits. Gram-positive isolates were grown on Manganese agar, subjected to endospore staining and identified as endospore positive or negative. Gram-positive isolates are currently being identified using either the API 50 CHB/E for gram-positive, endospore forming Bacillus or API 50 CHL for gram-positive, nonendospore forming Lactobacillus.


Table 1. Bacteria isolated and identified from unhatched loggerhead eggs collected in 2004 (n= 16 eggs from 5 nests) and 2005 (n=13 nests from 3 nests). A large amount of overlap of species existed between seasons. Bacteria found in the internal fluid of the eggs are marked as I and those found on the outside shell are marked O. Pathogenic bacteria are marked with an asterisk. Motility of bacteria has been indicated (Kreig & Holt 1984).

Multiple isolates were obtained from eight of the nests sampled (2004 nests: 1-5; 2005 nests: 6-8). Cultures from the other seven nests sampled died during processing. Bacteria were isolated from sand surrounding the eggs, the outside shell and fluid inside the eggs (Table 1). Preliminary identification of the bacteria isolated from samples of nest sand indicated that they were identical to species on the outside surface of the eggs, so sampling of sand was discontinued. Eighteen gram-negative and two gram-positive species of bacteria were identified. Of the 12 species isolated in 2004, eight were found again in 2005 (67%), with eight new additions. Bacteria cultured were diverse (2-11 species per nest). All bacteria were known to be common soil/environmental species and 15 of the 20 isolates (75%) were potential pathogens (Kreig & Holt 1984). Five of the 15 potential pathogens were found only inside the eggs.

Four of the genera of gram-negative bacteria identified have been previously reported in unhatched loggerhead sea turtle eggs (Table 1, Wyneken et al. 1988). Wyneken et al. (1988) speciated only one microbe and sampled eggs from only one nesting season. The current study sampled during two nesting seasons in order to collect data from different nesting cohorts. Forty percent overlap of the species was found between seasons (8/20 species). Four specimens were isolated in 2004 only, and eight species were solely found in 2005, suggesting that the bacterial diversity does vary by nesting season.

Bacteria could enter eggs in the oviduct, may contaminate the shell as eggs pass through the cloaca or gain access to the eggs from the surrounding sand during incubation. It is not known which species of bacteria routinely inhabit the oviduct or the intestinal tract of adult loggerhead sea turtles. Many of the bacteria identified were small (0.4-5.0 μm in length), motile gram-negative rods (Kreig & Holt 1984) with flagella that could facilitate their entrance into the eggs. Penetration of eggs by bacteria is called horizontal contamination and has been documented in birds. Humidity and shell quality are two variables that can affect bacterial invasion of eggs (Cox et al. 2000). The species identified in the current study have been associated with medical problems in mammals and non-mammals including respiratory, wound and gastrointestinal infections, inflamed fetal membranes and sepsis (Kreig & Holt 1984). These data suggest that the environmental bacteria that may be resident in the adult females or present on nesting beaches have the potential to be opportunistic pathogens and may contribute to embryonic death in sea turtles.

Known pathogens identified in fluid samples from the egg interior included Pseudomonas aeruginosa and Serratia marcescens. Growth characteristics were particularly useful for identifying these microbes. S. marcescens grew with a distinct red/pink pigment and P. aeruginosa had a green pigment. Color made these two species recognizable as common inhabitants of nests examined on barrier islands in Georgia. In 2005, Jekyll nest 46 had a 2 % hatch success. Fifty five of the 81 unhatched eggs from this nest had a distinct green hue throughout their contents. Bacteria were not cultured from nest 46, however P. aeruginosa may have been present.

All study nests with the exception of Jekyll nest 5 from 2004, had eggs which were contaminated internally. Embryonic development in contaminated eggs ranged from fertile to late embryonic death. In all of the nests examined, this one stood apart from the rest. The majority of the eggs from Jekyll nest 5 had near full term embryos (>45 days of development, LED) that had ceased development. Samples of fluid from 4 eggs collected from the nest revealed no internal bacterial presence. By correlating the excavation date with the development of the embryos, it was concluded that this nest drowned during the storm surge associated with Hurricane Frances but otherwise could have hatched successfully. This nest could indirectly be used as a control since collection of a control nest was not possible.

A variety of bacteria were found in fertile eggs from 8 nests sampled on Jekyll Island, GA. (Table 1). Because the sources of the bacteria could have been the reproductive and digestive tract of the nesting female or the nesting beach, the next step in the investigation will be to swab the cloaca of nesting loggerheads to determine their resident flora. Sampling from the oviduct and collection of control nests is limited by state and federal permits. Seventy-five percent of the bacteria found in eggs were potential pathogens, some of which have been reported in non-mammalian vertebrates including fish, amphibians, birds and reptiles. This opens the door to the possibility that bacteria could act as opportunistic pathogens in sea turtle eggs. By the nature of opportunism, when present in the wrong place at the wrong time, normal environmental bacteria can cause harm. Further investigation is needed to clarify the nature and extent of the interaction.

BOWEN, B & S. KARL. 1997. Population genetics, phylogeography, and molecular evolution. In: P. Lutz & J. Musick (Eds.). The Biology of Sea Turtles. CRC Press, New York. pp. 29-50.

COX, N. A., BERRANG, M. E. & J. A. CASON. 2000. Salmonella penetration of egg shells and proliferation in broiler hatching eggs- a review. Poultry Science 79: 1571-1574.

DODD, M. & A. MACKINNON. 2005. Loggerhead turtle (Caretta caretta) nesting in Georgia. Annual report to U.S. Fish & Wildlife Serv. 47 pp.

DODD, M. & A. MACKINNON. 2004. Loggerhead turtle (Caretta caretta) nesting in Georgia. Annual report to U.S. Fish & Wildlife Serv. 44 pp.

DODD, M. & A. MACKINNON. 2003. Loggerhead turtle (Caretta caretta) nesting in Georgia. Annual report to U.S. Fish & Wildlife Serv. 46 pp.

DODD, M .& A. MACKINNON. 2002. Loggerhead turtle (Caretta caretta) nesting in Georgia. Annual report to U.S. Fish & Wildlife Serv. 46 pp.

GIRONDOT, M, J. FRETEY, I. PROUTEAU & J. LESCURE. 1990. Hatching success for Dermochelys coriacea in a French Guiana hatchery. In: T. H. Richardson, J. I. Richardson & M. Donnelly (Comps) Proceedings of the Tenth Annual Workshop on Sea Turtle Biology and Conservation. NOAA Technical Memorandum NMFS-SEFSC-278. pp. 229-232.

HEPPELL, S. S., SNOVER, M. L. & L. B. CROWDER. 2003. In: P. Lutz, J. Musick & J. Wyneken (Eds.). The Biology of Sea Turtles Volume II. CRC Press, New York pp. 275-306.

KREIG, N. R. & J. G. HOLT, Eds. 1984. Bergey’s manual of systematic bacteriology 1st edition. Vol. 1. Williams & Wilkins, Baltimore.

MILLER, J. 1997. Reproduction in sea turtles. In: P. Lutz and J. Musick (Eds.). The Biology of Sea Turtles. CRC Press, New York pp. 51-82.

MO, C. L., I. SALAS, & M. CABALLERO. 1990. Are fungi and bacteria responsible for olive ridley’s egg loss? In: T. H. Richardson, J. I. Richardson & M. Donnelly (Comps) Proceedings of the Tenth Annual Workshop on Sea Turtle Biology and Conservation. NOAA Tech. Mem. NMFS-SEFSC-278 pp. 249-252.

WYNEKEN, J., T. J. BURKE, M. SALMON & D. K. PEDERSON. 1988. Egg failure in natural and relocated sea turtle nests. Journal of Herpetology 22: 88-96.