Ingestion of metal-nanoparticle contaminated food disrupts endogenous microbiota in zebrafish

Nanoparticles and the gastrointestinal tract of zebra fish

The use of nano-particle technology is growing in favour while their effects on the environment remain relatively under studied. Materials such as Ag-NP (Silver nano-particles) are utilised by clothing companies and in food packaging because of their antimicrobial effects. This is obviously beneficial for us but what happens when these nano-particles (NP) enter the environment. As they are designed to be antimicrobial (in some cases) we can assume that they will have an effect on the microbial community. Microbes rarely exist on their own and complex relationships with other microbes as well as higher organisms exist. It’s a safe assumption that all vertebrates will have some level of microbial symbiosis for example in the gastrointestinal tract (GIT), where microbes help with the assimilation of key nutrients into the body.

A commonly studied vertebrate is the zebra fish, Danio rerio, which is used as a model organism in many studies. Using this model species Merrifield et al., (2013) investigated the effect that two of these NP had on the GIT microbiota. This was done by through feeding with either CuSO₄, AgNO₃, Ag-NP, Cu-NP or untreated food. The use of both elemental and NP forms was to differentiate between effects caused by NPs and those caused by the presence of the element. After one fortnights feeding the gut microbiota was analysed and a portion of the 16S region was amplified. From this the species richness and diversity could be determined. This was found to be highest in Cu-NP exposed fish. However this does not signify a healthy gut. In fact some key species were missing such as Cetobacterium somerae.  This microbe produces large quantities of vitamin B12 and is predicted to be a useful probiotic. The increase in diversity could then be linked to the removal of a dominant competitor. Further to this the CuSO₄ treatment had no effect on C. somerae indicating an effect of the NP not the element. Further investigation as to how this NP inhibition occurs is required.

Silver is a powerful antimicrobial agent in some environments, however neither Ag treatments had any effect. This is likely because of Ag speciation occurring in the gut lumen. High intestinal chloride concentration could be causing Ag to precipitate as silver chloride before any effect on microbiota happens. Also –SH ligands in food will bind to any ionic silver leading to low dissolved concentrations and preventing further interactions with micro organisms.

This study is the first of its kind, investigating the effect nanoparticles have on microbiota. As a preliminary experiment it opens the door for much further research into the subject. For example a metagenomic approach could be used to further identify organisms and processes affected with in the gut micrbiome. This kind of information will be useful when determining the future use of nanoparticles, i.e. how much can be released into the environment, what does this mean for wild fisheries, how can we better manage aquaculture based on this knowledge. For example this kind of work may help us to formulate feeds that incorporate nanoparticles that inhibit the growth of pathogenic organisms while promoting that of beneficial ones. To do this a much better understanding of the GIT microbiome is necessary.

Merrifield, D. L., Shaw, B. J., Harper, G. M., Saoud, I. P., Davies, S. J., Handy, R. D., & Henry, T. B. (2013). Ingestion of metal-nanoparticle contaminated food disrupts endogenous microbiota in zebrafish (Danio rerio). Environmental Pollution, 174, 157–163.

ALTERNATIVE INDICATOR SPECIES OF HUMAN FAECAL POLLUTION

Faecal pollution has always been a major problem and detection of traditional indicator species such as Escherichia coli or species of the genus Enterococci results in closure of coastal beaches to public. As these easily culturable bacteria are present in both animals and humans, even if they are found in water samples, their presence cannot tell us anything about source of faecal pollution. This is a limitation of using E. coli like traditional indicators because we need to efficiently use our finite resources for reducing human health risks; that requires data about the specific sources of faecal pollution. Finding human-specific indicators is important also because human faeces is a major reservoir for human pathogens.

As previous studies have found specific gut microbiota of specific host, which is also dependant on factors like diet; characterization of these host-specific microbes may identify bacteria that can be used as host-specific indicators of faecal pollution. Molecular techniques like terminal restriction fragment length polymorphism, family-specific cloning and sequencing of nearly full-length 16S rRNA gene have been used in many previous studies to identify host-specific phylotypes within the order Bacteroidales for humans, cows, pigs, dogs etc. Similarly, subtractive hybridization and genomic enrichment of the metagenome have been previously used for identifying alternative host-specific indicator microbes including a human-specific species from Bacteroides spp. Apart from Bacteroidales, other taxonomic groups from which alternative host-specific indicator bacterial species have been described include, Bifidobacteriaceae and Lachnospiraceae. Human microbiom project revealed staggering variety and complexity of microbes from the human gut. Though, Bacteroidales and Clostridiales have been reported as among the most abundant faecal anaerobes; there is lot of interrelated variation among these. Thus, identification of the most common and abundant human-specific bacterial indicator species has turned out to be difficult.

The authors of this study examined 38 sewage samples collected over a 4 year period from the city of Milwaukee, Wisconsin USA and 11 other samples collected from different geographic locations of USA to characterize microbial community structure specific to humans. Mainly, a taxonomic group from Clostridiales which is very specific to humans was on focus. Sewage sampling ideally represents a random, composite sampling of several individuals and hence there is no issue of individual variability in indentifying most common human-specific microbes. To understand distribution of Clostridiales species, previously published data sets for human, cattle and chicken, were used.   

Majority of Clostridiales from sewage samples and human faecal samples were identified as Lachnospiraceae which were rarely found in the samples of both cattle and chicken. Network analysis revealed that Lachnospiraceae family shared most pyrotags among human faecal and sewage samples which means, members of Lachnospiraceae were mostly originating from humans. Among Lachnospiraceae, the two most abundant species were Roseburia sp and Blautia wexlerae.

Thus, members of Lachnospiraceae can be seen as potential alternative indicators, which could give information about human-specific source of faecal pollution. The criteria for host-specific indicators include their abundance in the host of interest so that sensitivity for their detection is maximised, their almost nonexistence in other hosts making them specific to host of interest and their robustness over a large geographic region. Lachnospiraceae is estimated to make 19% to 50% human faecal microbiota and previous investigation on Lachnospiraceae support the notion of this study that they can be used as alternative indicators. The authors conducted even further molecular analysis and provided evidence of chronic human faecal pollution of surface waters by targeting a gene (Lachno2) from one of the most abundant members of Lachnospiraceae.

The authors have suggested that sewage sampling could be used as a measure of microbial community patterns in the human population linked with age, health and dietary habits. Being high in diversity and abundance, functional roles of members of Lachnospiraceae in human gut have been discussed. In conclusion, they are the most promising candidates of human-specific indicators among Clostridiales. It is unlikely that single host-specific single species would be the most ideal indicator with the necessary sensitivity for quick detection methods. This study proposes effective alternative indicators using highly advanced molecular technologies. The question remains is that how cost-effective these molecular methods would be for practical routine use? This question becomes even more important for developing countries like Bangladesh where problem of faecal pollution would be a big challenge, I guess.

McLellan, S. L., Newton, R. J., Vandewalle, J. L., Shanks, O. C., Huse, S. M., Eren, A. M., & Sogin, M. L. (2013). Sewage reflects the distribution of human faecal Lachnospiraceae. Environmental microbiology, 15(8), 2213-2227.

The incidence of antibiotic resistant bacterial strains in Italian fish farms

The increase in intensive large-scale fish aquaculture has been linked to the development of antibiotic resistance within some marine microbial communities. In Italy, a number of environmentally different areas in the coastal North Adriatic region have been established as semi-intensive and extensive fish farms. These farms normally operate either with productive management of the coastal environment (mostly lagoons) in marine and brackish water using extensive and semi-intensive methods, or by intensively farming commercially valuable fish and shellfish species in tanks and cages.

Several environmental hazards can arise from these techniques, as the intensive conditions can result in the spread of infectious diseases in the farmed fish and shellfish species, which are caused by several types of Vibrio, Aeromonas and Photobacterium. The subsequent use of the antibiotics tetracycline (TET) and oxytetracycline (OXTET), flumequine (FLU), trimethoprim (TIM) and the association of trimethoprim with sulfadiazide (TIM-SUL), which have been linked to the induction of antimicrobial resistance (AR) in marine bacteria by horizontal gene transfer and mobile genetic elements. Pollution in coastal areas causes significant problems in the marine environment, and can result in contaminated water that used for drinking, irrigation and recreation, and hence reduce health quality.

Fig. 1. Different incidences of antibiotic resistance strains in water, sediment and biofilm samples obtained from the aquaculture centres.

This study measured the global incidence of antimicrobial resistance and the frequency of AR bacteria to either individual or multiple antibiotics commonly used in Italian fish farms at a number of different sites within each centre that are distributed along the Adriatic Sea between the Venetian Lagoon and the Gargano area in Puglia (Italy), as well as in a coastal site in the Veneto region. Samples were taken from sediment, biofilms and water from inlet water (W­_in) and outlet (W_out) entering and leaving the fish tank/ farm at each aquaculture centre.

Under the selective pressure of the antibiotics used in fish farms, AR marine bacterial strains can persist and form an environmental reservoir directly involved in the seafood chain. AR bacteria exist in natural environments where antibiotics affect bacterial metabolism, so comparisons were done between bacterial strains from coastal aquaculture centres and those from an area 2 km away from the coastline. This meant the data from the more intensively- and recently-treated aquaculture centres could be compared to a baseline AR incidence. There was a higher level of susceptibility to FLU, as the resistance frequency was <1% (0.3%), compared to TET and OXTET, which averaged out at 20% resistance (but ranged between 10 to 50%).

Veneto I is a fish farm located in a small “valle di pesca”, (valley/ brackish lake) in the north of the Adriatic region, where only experimental techniques to produce new fish species are used. This experimental aquaculture approach seems to be effective, as it is the centre with the lowest global incidence of AR strains (24%). Veneto II is also a brackish lake where the fish are farmed in a semi-intensive culture. In comparison with the first site within the same area, there was a global incidence of 45% of AR strains to at least one antibiotic, but the most significant result that has been found for Veneto II is the 18% of multi-resistant strains in the centre. The authors suggest that this large difference is because there is a more frequent use of antibiotics in the second farm, whereas the first one had no records of usage in at least a year.

Gargano I is an open-sea site located 2km away from the Puglia coastline that sporadically used antibiotics for intensively-farmed fish in 35 cages up to 2009-2010, and the AR resistance incidence was similar to other centres, but the level of multi-resistance was similar to that in the coastal sites. Finally, Gargano II is located in a lagoon in the Gargano peninsula area. Horses and sheep were being extensively bred in the surrounding area, and were also being given TET; therefore this external source of TET-resistant bacteria could be why 50% of strains were resistant to the antibiotic. This site was found to have the highest incidence of AR, with 79% of strains showing resistance to at least one antibiotic and a third with multiple resistances, but individually were still all susceptible to TEM, TEM-SUL and FLU, which are only used in aquaculture.

Further research could look into more detail at how the community composition is altered by the different aquaculture methods and the antibiotics used (i.e. which microbial species are dominant when a certain antibiotic is introduced). In addition, they could’ve also investigated whether some strains of the same bacteria are resistant to at least one of the antibiotics at one site, but still susceptible to the same ones at other sites, because some antibiotics have been used for longer periods of time/ higher frequencies (i.e. so that random mutations and/ or horizontal gene transfer can occur).

Labella, A., Gennari, M, Ghidini, Trento, I., Manfrin, A., Berrego, J.J., and Lleo, M.M. (2013) High incidence of antibiotic multi-resistant bacteria in coastal areas dedicated to fish farming. Marine Pollution Bulletin, 70: 197-203

Relevance of temporal and spatial variability for monitoring the microbiological water quality in an urban bathing area

This paper was all about variability in indicator bacterium being dependent on temporal and spatial sampling. Four beaches (Pastoras, Gondarem, Castelo and Matosinhos) on a 4.5km near-shore stretch were hourly sampled over an 11 hour time period. Sampling occurring during peak bathing season (June-August) and for each beach, the same sampling site with a number of repeated samples was used throughout.

Intestinal enterococci and E. coli were the two indicator organisms used in this study.  Whilst the intestinal enterococci concentrations did not exhibit any relevant temporal patterns with regards to the months E.coli concentrations varies monthly – for example Castelo was most polluted in June, Gondarem showed its highest faecal contamination in July/August, and the two remaining beaches were most polluted in August. 

This variation was statistically significant, however the general trend looks to be that in August months there was the highest faecal contamination (with regards to E. coli,) coincidentally this occurred alongside the highest mean water temperatures recorded. It is important to note that water temperature within the 10km surveying zone could vary considerably within a single survey (e.g. in August, measured temperatures varied from 12.7°C to 21.5°C)

There was a trend of contamination being higher (but not statically so) in morning samples than after noon samples which was explained due to the fact that indicator bacteria is known to exhibit a diel cycle. Also noted was an association between lower salinity and higher bacterial concentration.

Short term (referred to as ‘hourly’) temporal variation was observed at Matosinhos beach, for example in June an 08:00 survey showed a maximum E. coli concentration of 5 100 cfu 100 ml^-1 , but by 15:00 the minimum E. coli concentration was 21 cfu 100 ml^-1. If using classification techniques (the European Directive 2006/7/EC) these two samples would be known as “excellent” and “poor” respectively.

General conclusions were that temporal variation explained most (76%) of the total variance seen in water quality – with monthly variance (44.3% and 46.3%) explaining slightly more of bacterium variability (E. coli and intentional enterococci respectively)  than inconsistencies in sampling hours (32.5%, 30.1%). Lastly, and in this experiment least importantly, the spatial variance explained 23.3% and 23.6% of bacterium concentration differences. Percentages were obtained from a nested ANOVA.

Spatial and monthly variance causing bacterium variations are common sense ideas, but it is nice to have this paper as some hard evidence. However, I feel the most important idea to come from this paper is the effect hourly variation may have on the results of a faecal water quality test – meaning that the time a sample is taken can push a beach from a “poor” to a “good” rating or vice versa.  Due to this reason I am feeling very pro the new EU bathing water directive 2006 idea of continuously testing a water body throughout four years to get a more accurate sample.

Amorim E., Ramon S., & Bordalo A. A. (2014) Relevance of temporal and spatial variability for monitoring the microbiological water quality in an urban bathing area. Ocean & Coastal Management, 91(1), 41-49

Toxicity and distribution of tetrodotoxin-producing bacteria in puffer fish Fugu rubripes collected from the Bohai Sea of China.

Overview:

Tetrodotoxin (TTX) is an extremely potent low-molecular weight non-protein neurotoxin. TTX has been isolated from many different organisms, including fish, chaetognaths, gastropod molluscs and the list goes on. The fascinating property of TTX is its appearance in many genetically unrelated animals, leading to questions of the origin of TTX. An interesting theory is that bacteria may produce the substance, rather than an innate production mechanism within the organism.

Previous studies had established a direct link between TTX (including TTX derivatives) and the presence of TTX-producing bacteria. In their 2005 paper, Zhenlong Wu et al take this further to prove that distribution and toxicity of TTX-producing bacteria in each tissue of the puffer fish F. rubripes are closely related to the toxicity of the puffer fish. They also show that toxicity is higher/more potent in the liver and ovaries than in other organs, and that this corresponds with a higher number and % of total bacteria that produce sodium channel blocking toxins in these organs.

Results:

The Bacteria strain with the highest level of toxicity, 1.6MU/g compared to 0.1 (lowest) was found in the ovaries of the puffer fish, which is known to be one of the most potent organs, along with the liver. The cultured Bacteria included Bacillus (32 strains), followed by Actiomyces spp. (3 strains), and vibrio spp. (1 strain). Among 35 bacterial strains, 20 strains (13 strains from the ovaries, 6 from livers and 1 from intestines) were found to produce sodium channel blocking toxins. The percentages of toxin-producing strains from each organ were found to be 68% for the ovaries, 55% for the livers and 25% for intestines.

These results, although somewhat simple, were the first that proved that distribution and toxicity of TTX-producing bacteria were related to toxicity in the organism.

Moving Forward:

Zhenlong Wu et al put forward several projects that would help improve understanding in the future, including elucidating the exact mechanism of the synthesis of TTX by bacteria and the role of TTX in the bacteria themselves as well as discovering whether the TTX-producing bacteria accumulate through the food chain or come from the marine environment needs more research.

In the other blog posts, several papers more recent than this have been summarised and show how the TTX field has moved forward, but is still somewhat still mysterious. I feel like this is a very simple paper but one that may have been instrumental to the beginnings of this research topic. They also suggest some important future tests.

Zhenlong Wu, Ying Yang, Liping Xie, Guoliang Xia, Jiangchun Hu, Shujin Wang, Rongqing Zhang, Toxicity and distribution of tetrodotoxin-producing bacteria in puffer fish Fugu rubripes collected from the Bohai Sea of China, Toxicon, Volume 46, Issue 4, 15 September 2005, Pages 471-476.

A Review: Bacteriophages infecting Bacteroides as a marker for microbial source tracking.

Identifying the sources of faecal pollution is necessary in order to control the spread of human pathogens in the environment, and especially ground and surface water resources are affected by it. Human faecal pollution is a major factor contributing to disease outbreaks and tracking the sources of pollution can also help to estimate potential health risks. Microbial source tracking (MST) is a developing tool for this and this paper focused on the tracking bacteriophages infecting strains of Bacteroides spp. which are very common in human faeces and therefore waters contaminated with human waste are expected to have high abundance of bacteriophages.

As discussed in the last lecture, faecal pollution indicators have to fulfil certain criteria so that they can be used as most accurate markers. For instance, they have to be countable and associated with the source of contamination (e.g. human gut). However, none of the currently used indicators are perfect and hence several markers are used to track the sources of pollution and give good estimates of the abundance of contaminants. Phages that infect gut bacteria in warm blooded animal are not expected to be able to replicate outside the gut environments and therefore the abundance detected can be considered as somewhat accurate indicators. In addition, studies have shown that Bacteroides spp. infecting phages were steady in abundance throughout the year.

Most phages that infect Bacteroides species have a narrow range of strains they infect which can allow tracking specific sources of contamination when infecting strains are host-specific as well (e.g. strains that are only present in humans). For example, phages B40-8 and ΦB124.14 infecting strains B. fragilis HSP40 and Bacteroides sp. GB-124 (respectively) have been found to be human gut specific phages which was revealed by metagenomic analyses. Until now, it is not fully understood why some phages infecting Bacteroides are only found in certain host animals. It was hypothesised that host animal associated bacteria and their phages may have co-evolved more separately than other bacterial groups.

Studies have investigated bacteriophages infecting B. fragilis and were able to detect and enumerate them with commonly utilised plaque assays and enrichment methods after the bacteria were cultivated under anaerobic conditions. Identifying specific bacteriophages associated to hosts and therefore determining their source can also be done with molecular techniques sequencing their DNA which is more useful to examine non-culturable bacteria and their phages. 

In summary, bacteriophages infecting Bacteroides spp. have shown to make reasonably good indicators of faecal pollution in the environment, however there need to be more research in this field to give more representative data. Host-specificity of these phages was found in the example mentioned above and in other studies described in the paper, which is very useful for determining the sources of contamination. This paper summarises very nicely what kind of studies have been conducted with these bacteriophages and why and how they are used. Unfortunately, it is also a fairly long review paper so I couldn’t highlight all aspects of it, but I would highly recommend reading it to get a better understanding of this current field of research. 

Jofre, J. et al. (2014) Bacteriophages infecting Bacteroides as a marker for microbial source tracking. Water Research, doi: 10.1016/j.watres.2014.02.006.

The article came out recently and hasn't been published in a journal yet but is accessible here:
http://www.sciencedirect.com/science/article/pii/S004313541400116X

Next Generation Sequencing Technologies for the Marine Enviroment

Next generation sequencing (NGS) platforms are commercially introduced high throughput devices that allow DNA sequences to be recovered directly from marine environmental samples (amongst others!). NGS are also without need of vector based cloning procedure normally used for amplifying DNA templates. This eliminates cloning bias to sample evenness, but incurs other limitations for each NGS platform.

Despite diverse chemistry and base incorporation/detection techniques, all NGS platforms follow 2 steps; library fragmentation/amplicon library preparation and detection of the incorporated nucleotides

Shokralla et al. (2012) describes and examines NGS techniques for their pros and cons in regard to environmental DNA research.

All PCR-based NGS systems have bias’ introduced during amplification. Bias is initially introduced during amplicon library preparation. PCR bias is also strongly affected by the number of replicating cycles and by annealing temperature. These however can be reduced by using high template concentrations, wise primer selection, low cycle number, low annealing temperature and mixed replicate reaction preparations. Bias’ from amplicon preparation can by exaggerated in the library amplification step. Any bias from library amplification alone is likely eradicated by use of universal probes.

454 pyrosequencing is the most common platform for NGS. Some big advantages are its long read length, short run time, and that it doesn’t necessitate an extra chemical debunking step for DNA extension by DNA polymerase, which is unique among NGS platforms. This reduces the chance of dephasing by lowering the likelihood of premature chain termination, and non-simultaneous extension. Long sequences generated in 454 pyrosequencing will mean higher accuracy when identifying nonmodel organisms in ecological applications. Drawbacks are its high cost per megabase sequencing output, and its lack of terminating moiety to stop the extension run, making it a challenge to read homopolymer regions. The most common error type however is insertion-deletion instead of substitution, causing errors in analysis of environmental DNA by mimicking haplotypes of rare biota. This howeveris significantly reduced with computational tools to highlight and extract such sequences.

Illumia an d SOLiD systems perform individual necleuotide detection, meaning homopolymer regions are accurately sequenced. These systems also have a high output per run compared to 454 pyrosequencing However optical signal decay and dephasing in these platforms leads to short reading lengths. This infers situations were no reference sequence is available to align assaign and annotate generated short sequences.  As well as this most NGS workflows are time consuming, tedious and require highly skilled personnel.

Another PCR-based NGS platform not assessed by Shokralla et al.  is Life Technologies Ion Torrent, a post light sequencing technology. This relies on real-time detection of hydrogen ion concentration, released as a by-product when a nucleotide is incorporated into a strand of DNA by polymerase action.

Other NGS platforms mentioned by Shokralla et al. include Single-molecule DNA-sequencing technologies, which don’t require a PCR-amplification step and so remove such formerly stated bias’. These are Helicos biosciences HeliScope, which works by sequencing-by-synthesis on a single DNA molecule, and Pacific Biosciences SMRT DNA sequencer, which uses less steps and so a faster process than Heliscope, using the natural capacity of DNA polymerase to incorporate ten or more nucleotides per second in several thousand parallel nano-structures.

An enhancement for NGS technologies is target selection (Sequence Capture), which eliminates initial amplification steps and allows selective analysis of large numbers of target sequences. Sequence capture involves hybridization-based methods using oligonucleotide probes. These are either immobilized to a solid array ‘Capture arrays’ or in solution ‘Baits’ to capture the sequencing targets. The latter is preferable due to higher specificity and uniformity of sequences, and cheaper hardware costs. Target enrichment sequence capture eliminates the initial PCR Step, however it is necessary to start library preparation with a relatively large amount of DNA.

Examples of sequence capture systems are: Roche’s NimbleGen, Agilent’s SureSelect, Rain-Dance Technologies’ RainStorm and Illumina’s TruSeq Exome Enrichment system. Though currently used in studies of human and other model organisms, they show great promise for environmental DNA research.

Modification of sample preparation protocals, like library construction, could also take shape as further enhancements to NGS platforms.

Multiplexing of different target gene markers of a single bulk sample, or multiplexing of a single marker from multiple samples, by tagging or bar coding mixtures of DNA templates could have great ecological application. This process can also be carried out at a reasonable price, however potential biases caused by the addition of multiplexing identifier tags to primer oligos should be considered.

The authors highlight many instances of marine application of such NGS technologies in ecological research of bacterial and viral natures, from surface waters to coral reefs. Following Colin’s last lecture, such applications could also help identify indicator species of disease in the ocean; helping beach goers (locals and tourists alike) make informed decisions before taking a dip!

- Shokralla, S., Spall, J. L., Gibson, J. F., & Hajibabaei, M. (2012). Next‐generation sequencing technologies for environmental DNA research. Molecular Ecology, 21(8), 1794-1805.

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

Empirical evidence to support pole-ward bound pathogenic Vibrios?

An increase in Vibrio related infections pushing pole-wards has been linked to increasing temperatures, however Baker-Austin et al. (2012) have now provided some empirical evidence linking long-term temperature increases around the Baltic sea with increased incidences of Vibrio related infections, particularly V. vulnificus.  The study looked at multi-decadal sea surface temperature (SST) data sets of the Baltic sea, which suggested that this area is warming at unprecedented rates, more so than virtually any other body of water on Earth.  Authors suggest that this study provides evidence that anthropogenic change is driving the increased incidents of Vibrio disease in temperate regions and in turn, affecting the distribution of human pathogenic bacteria globally.

V. vulnificus abundance peaks at temperatures > 19 °C and recent studies suggest that the pathogenic properties of some Vibrios is mediated by temperature and certainly emergence of increased Vibrio outbreaks in temperate regions such as Chile, Peru and NW Spain have been linked with warming patterns.  Many human pathogens, such as Vibrios, grow and replicate in warm (>15 °C), low salinity (< 25 ppt) marine environments, with the Baltic being semi-enclosed and predicted rising and warming seas, the authors predict increased Vibrio related incidents.  The study analysed the potential risk and presence of V. vulnificus in the Baltic area using a model based on low salinity (<25 ppt as is common in the region) and observed SST of >19 °C.  This was compared to models with temperature projections for 2050 and results suggest that temperature can be used as a predictor of Vibrio outbreaks.  Moreover, results predict significant expansion of waters capable of hosting large populations of pathogenic Vibrios. 

Whilst I am in agreement that changing temperatures are undoubtedly linked to increases in Vibrio clinical cases (Fig. 1), I am not convinced that this study provides the conclusive evidence that it proclaims to, in relation to humans driving the change.  Don’t get me wrong, I think humans are driving the temperature increases at unprecedented rates, but this paper doesn’t offer any evidence in this regard.  Other limitations of this study included the sourcing of epidemiological data, which was collated from several different sources, including ‘grey’ literature, and it was not clear whether all cases included in the study were actually from the Baltic region (eg, may have been infected abroad), potentially confounding results.   Furthermore, the models used offer some possible projections of pathogenic Vibrio distribution around the Baltic region however they are only applicable if SSTs continue along the same trend.   What may be beneficial however, as suggested by the authors, is the use of near-real-time remote sensing data reporting SSTs, which may serve as an indicator of the risk of pathogen outbreaks, allowing local authorities to inform bathers accordingly.  

 

Figure 1. Vibrio cases and SST. a, The relationship between Vibrio infections reported around the Baltic Sea area and maximum annual SST. Stars show observed data, dashed line shows GLM model predictions (based on the influence of SST alone). b, Time series of Baltic Sea Vibrio cases. Solid line shows observed cases and dotted line shows GLM model predictions based on the influence of maximum SST and time.

 

Baker-Austin, C., Trinanes, J. A., Taylor, N. G., Hartnell, R., Siitonen, A., & Martinez-Urtaza, J. (2012). Emerging Vibrio risk at high latitudes in response to ocean warming. Nature Climate Change, 3(1), 73-77.