Monday, 17 February 2014

Antibiotic resistant bacteria as a bio-indicator of marine pollution

 Anthropogenic pollution, in particular sewage effluents, imposes huge pressures on the marine environment.  This pollution is often assessed against water quality standards using microbial indicators of human pathogens.

The current study however uses the trait of resistance within bacteria as a bio-indicator of polluted effluents; antibiotic resistant bacteria remain viable even after disinfection, and are transmitted to the marine environment. Resistance within bacteria is attributed to horizontal gene transfer and/or mutational events. The widespread release of the ever increasing spectrum of antibiotics used in human and vetinary medicine has been shown to lead to the development of multiple resistant bacteria.

Such bacteria have previously been reported in a range of marine animals, including in the egg shells of green turtles, Chelonia mydas.  The oviduct is the reproductive tract in turtles. A gland within the oviduct secretes fluid, known as oviductal fluid, over the eggs periodically both during and following egg formation to retain moisture. It has previously been shown that the bacteria present in the oviductal fluids penetrate all the egg components, and such bacteria can be deposited within the eggs prior to them being laid.
The study investigated the marine pollution by contaminated effluents using antibiotic resistant bacteria within the oviductal fluid of green turtles.

To collect samples of the oviductal fluid a sterile cotton swap was taken from within the oviduct. Samples were taken in turtles before and after egg-laying. Sand proximal to the nests was also sampled.

Using nutrient agar and differential selective media, bacteria were isolated and identified. Oviductal fluid was found to be heavily contaminated with bacteria. From the samples, 132 different species of bacteria were identified from 7 different genera. In pre egg-laying turtles, the microbial composition was as follows: Citrobacter spp. (51.4%), Pasteurella spp. (16.3%), Pseudomonas spp. (11.6%), Salmonella spp. (11.6%), Proteus spp. (4.7%), Aeromonas spp. (2.1%), and Shigella spp. (2.3%). Citrobacter spp dominated the other isolates.

Post egg-laying turtles contained a completely different bacterial composition. However, this bacterial profile was very similar to the profile found in sand samples. It was noted that sand had contaminated these samples thus the results of post egg-laying turtles did not represent the true endogenous bacteria present.

The susceptibility of these bacterial isolates to 15 commonly used antibiotics was assessed using disc diffusion method, with inhibition zones measured after 24hours incubation.
60.6% of bacterial isolates were resistant to the tested antibiotics. Any bacteria found to be resistant to more than 1 of the 15 tested antibiotics was considered ‘multiple resistant bacteria’. The bacteria showed varying degrees of multiple resistance (Figure 1.) Of all bacteria tested, P. aeruginosa had the highest resistance, demonstrating resistance to 12 antibiotics.

Figure 1. Percentage number of resistant bacteria found in oviductal fluid to a number of antibiotics

Of all the anitibiotics tested, ampicillin, streptomycin and sulphamethoxazole were the antibiotics to which the majority of isolates showed resistance to (Figure 2).

Figure 2. Percentage frequency of resistant bacteria to different tested antibiotics after 24h incubation. Amp- Ampicillin, S – Streptomycin, Smx – Sulphamethoxazole, Te – Tetracycline, Cn – Carbenicillin, K – Kanamycin, Min – Minocyline, Ctx – Cephotaxime, Na – Nalidixic acid, C – Chloramphenicol, Tmp – Trimethoprim, Ak – Amikacin, Tob – Tobramycin, Gm – Gentamicin, N – Neomycin

The authors state that measuring the resistant bacteria within turtles’ oviductal fluid will enable them to detect the magnitude of pollution and will be valuable in tracing the source of polluted effluents along the migratory routes of the turtles. Personally, however, whilst the underlying use of antibiotic resistant bacteria has potential, I believe the current methodology to be somewhat limited. It’s capacity to quantify pollution is uncertain and dependant on a number of factors including the amount antibiotics released into the specific effluents, the exposure time of the bacteria, the quantity of bacteria that become resistant in comparison to pollution levels, the specific bacteria which best identify pollution etc. In addition to this, turtles cover a large geographical range on their migratory routes thus unless samples were taken at regular intervals, it would be difficult to determine the point source of the pollution. Further studies would be required to assess the use of resistant bacteria as bio-indicators of pollution.

Al-Bahry S., Mahmoud I., Al-Zadjali M., Elshafie A., Al-Harthy A. and Al-Alawi W. (2011) Antibiotic resistant bacteria as bio-indicator of polluted effluent in the green turtles, Chelonia mydas in Oman. Marine Environmental Research, 71, 139-144


  1. This is an interesting study and quite a different approach to look for human pollution in the ocean environments by somewhat investigating the spread of antibiotic resistance genes. How far away from human settlements were the samples taken from? It's possible that the turtles have picked up the bacteria with resistance close to the place where they were laying the eggs. I agree with you that the conclusions that can be drawn about the magnitude of pollution from the results are rather limited due to factors you mentioned.

    From this study, it should also be investigated to what extent the turtles are carriers of the resistance genes if the bacteria survive long enough in the animals.It's possible that the genes can be transported by the turtles via their migratory routes. Maybe future research could try to track turtles and assess the bacteria in their oviduct as regular as possible and not just once to give clue about the spread of resistance genes.

    1. The proximity of the turtle nests to human settlement was not stated in the paper, no. As you suggest, by attaining information on the survival time of bacteria within the turtle could help to paint a clearer picture of the pollution along the migratory route. For instance, if perhaps the bacteria only survived 3 days within the oviductal fluid of turtles and samples were taken periodically, the geographical scope for the source of the pollution would be greatly reduced. The use of oviductal fluid is limited in that as far as i am aware, is produced in egg laying turtles. Unless some form of tagging system was used, it would be difficult to assess which tutles in the ocean are actually egg bearing.

  2. I also agree that this is an interesting study which looks at a different perspective of antibiotic resistance, but there only seems to be an inference to the link between human pollution and the presence of these bacteria. This study could have provided more evidence for this by looking for a causal link between the two. Expanding on Malin's point on where the samples were taken from, I think it would also be worth doing another study on another population of green turtles at a site which is known to be less affected by pollution to see whether the microbial composition changes, or if the amount of resistance that the bacteria have differs significantly, as this could give more support for their claim about resistance being a bio-indicator for polluted effluents.
    Perhaps it would also be worth conducting the same tests on different species of turtles to see whether some species are more susceptible to resistance in bacteria?

    1. I very much agree Ellie. A correlation between pollution levels and antibiotic resistant genes is definately neccessary, without this, it is difficult for the data to be used as a bioindicator of pollution. The microbial consortium in pre-egg laying and post-egg laying (though the samples were sand contaminated) were tested, though information on the microbial consortium based on different levels of pollution would be more valuable information. It could be that the population of anitiobic resistant bacteria differs with pollution levels? It is clear that a lot more research needs to be conducted within this area before the validity of antibitotic resistant bacteria as a bio-indicator for pollution can be said with any confidence. Using another species, and perhaps another physiological substance e.g. fish mucous secretions may be more beneficial: fish are more ubiquitous and measurements wouldn't be dependant upon reproductive stage, such as it is in the present study.