Aquatic Reservoirs of Antibiotic Resistance

Since the 1940's many lives have been saved by the use of antibiotics, but due to increased use and poor disposal, antibiotic resistance has become an increasing global health concern. Every year approximately 25,000 European citizens die from infections caused by bacteria that have developed resistance to antimicrobials.

Bacteria become resistant to antibiotics through many mechanisms causing mutations by selecting or acquiring genes for resistance, through evolution, attributed mainly to horizontal gene transfer (HGT). Mis-use of these antibiotics and their release from wastewater treatment plants (WWTP) have contributed greatly to the emergence and spread of resistant bacteria, including bacteria that cause human and animal infections. Aquatic environments, especially, provide ideal conditions in the water column and sediment for the horizontal exchange of mobile genetic elements (MGEs).

Resistance in nature has an ancient origin in which organisms use as a defence mechanism to protect against harmful substances of others, encoded by a group of genes known as the resistome. Studies have collectively shown that the environment harbours a large majority of antibiotic-resistant genes (ARGs) but only in the last 10 years evidence has been demonstrating mobilisation of resistance into human pathogens.

Aquatic ecosystems are used as a way of disposal and therefore now play a significant role in ARG transfer, ecology and evolution. WWTPs provide an ideal environment for HGT as they contain high densities of bacteria, high oxygen levels, high nutrient concentrations and are in constant contact with antibiotics and resistant bacteria. A study conducted found that high concentrations of ARGs were found in biofilms downstream of a WWTP suggesting that they encourage the spread of resistance in the environment.

Biofilms are a natural dense community of microbes which could act as an ideal site for HGT of ARGs. Bacteria also aggregate in the sediments which facilitates the movement of genetic elements especially where antibiotics are used, in aquaculture and from other anthropogenic sources. Tetracycline resistant genes were found 100 times higher in sediments than in the water column, and these concentrations depended on the degree of anthropogenic stresses in that area.

Many other anthropogenic factors contribute to the spread of ARGs including sewage, agricultural runoff, heavy metal pollution and temperature. A natural phenomena that provides a surface for the movement of MGEs is the biofilms formed on chitin. Chitin is a very common element used in structural components on many crustaceans and molluscs and therefore these sites are abundant in which HGT can occur.

ARG's are very abundant in the environment but to prevent further mutations of human and animal pathogens, use and disposal of antibiotics into the environment needs to be monitored and limited as it could  pose a serious threat to global health.

Marti, E., Variatza, E. and Balcazar, J.L. (2013) The role of aquatic ecosystems as reservoirs of antibiotic resistance. Trends in Microbiology. XX:1-6

The squid –Vibrio story continues... squid derived chitin is important in colonisation

The squid –Vibrio story continues... squid derived chitin is important in colonisation

Euprymna scolopes, the Hawaiian bobtail squid, has a well known symbiotic relationship with bioluminescent bacteria Vibrio fischeri (recently reclassified as Allivibrio fischeri), that colonise the light organ of the squid post hatch. However in a world saturated with an array of microorganisms how are the correct bacteria enticed and selected to colonise the juvenile squid light organ?

A ‘winnowing’ effect in recruitment has been observed: initially gram negative bacteria are selected, followed by A. fischeri then subsequently motile A. fischeri which form tight clusters at the entrance of the light organ pore. Aggregation of A. fisheri symbionts occurs in squid-derived mucus, induced by a two-component relay signalling mechanism in the bacteria. In this experiment, derivatives of wild type A. fischeri labelled with green fluorescent protein (GFP) could be seen aggregated in the mucus field just outside the pore within 2 hours of exposure.

The underlying mechanism of colonization of the light-organ and the related specificity challenges has been shown to be achieved by chemical communication. Using fluorescein isothiocyanate (FITC), a chitin-binding protein, and confocal microscopy, chitin was found to be present within the light organ ducts, indicating the presence of light-organ derived chitin. Chitin is thought to be a chemoattractact and important in symbiont colonization.

Using soft-agar swim plates inoculated with N-acetylglucosamime (GlcNAc or (GlcNAc)₂, a mono and disaccharide of chitin respectively), a positive chemoattraction was observed in A. fischeri.  However when added to water containing squid, the chemoattraction to squid-produced chitin was disrupted.  Exogenous addition of the monosaccharide GlcNAc had no effect on squid colonization efficiency, however addition of disaccharide (GlcNAc)₂ diluted the endogenous gradient produced by the squid, and resulted in <10% of squid becoming colonized (fig. 1).

Figure 1. (j) Efficiency of squid colonisation upon addition of chemoattractants at 0.625% (wt/vol) and (k) effect of dose of (GlcNAc)₂.

Newly hatched squid were exposed to A. fischeri in combination with an inoculum of 0.625% (wt/vol) of either GlcNAc or (GlcNAc)₂ (fig. 1), squid were anaesthetised at various intervals and examined under a microscope. By staining host tissues with molecular probes, and using GFP properties, the location and aggregation of bacteria relative to the light-organ, ducts and pores was observed. After 4 hours, bacterial aggregates in 90% of squid (GlcNAc treated) had begun travelling down ducts to the crypts of light organ, whereas in (GlcNAc)₂ treated water, the effect was only apparent around the pore, with only 7% of bacterial aggregates entering the ducts. This suggests that chemotaxis to chitin oligosaccharides derived from the light-organ are important in inducing the bacteria to swim towards and into the light organ from the pore entrance. As an aside, it would be interesting to find out if A. fischeri use flick motility (see Stocker, 2012) to increase their efficiency in swimming against the host ciliary mucus current through the pores and into the light organ.

The authors suggest that squid-derived chitin oligosaccharides act as a synomone “a compound that is produced by one species and invokes a behavioural response in another that is beneficial to them both”.  It is now hypothesized that encoded chitinases degrade chitin to chitin oligosaccharides that A. fischeri chemotactically use to colonise the crypts of the light organ. Whilst chitin oligosaccharides are ubiquitous in the marine environment and also found in the tissues of the host, the increased concentrations coming from the light organ induce the chemotactic response. This migration is dependent upon this chemical gradient which, if disrupted, impairs the colonisation of the squid.  

By Marie Dale and Rachel Coppock

Mandel M., Schaefer A., Brennan C., Heath-Heckman E., DeLoney-Marino C., McFall-Ngai M. and Ruby E. (2012) Squid-derived chitin oligosaccharides are a chemotactic signal during colonisation by Vibrio fischeri. Applied and Environmental Microbiology, 78(13), 4620-4626

PHAGE THERAPY FOR TREATING A CORAL DISEASE

          Phage therapy means using bacteriophage/phages capable of killing the bacterial pathogens, for treating the disease caused by that bacterial pathogen. Using this method of disease treatment has a number of benefits such as particular phage specifically target bacterial pathogen and other bacteria (including beneficial associated microbes) remain unaffected, as density of bacterial pathogen decreases, consequently phage concentration also declines. The same authors have previously reported isolation and characterisation of lytic phages for two bacterial pathogens of corals. This study particularly looked at treatment of white plague-like (WPL) disease in a coral species Favia favus, caused by a bacterium Thalassomonas loyana.

          

        Field experiments were conducted in the Red sea of Eilat, Israel. The study was conducted by injecting phage BA3 (capable of infecting specifically T. loyana) to the treatment which was compared to controls which were without the dose of phage. The experiments were conducted for two times, firstly in 2009 and then in 2011. Corals from these treatments were transferred in aquaria and divided as per their healthy/diseases status. Phage concentration in these aquaria waters was measured by soft agar overlay technique. Coral samples were prepared and used for identification of T. loyana by 16S rRNA gene sequencing.

          

      The authors noted little reduction in the living tissue in the phage treated corals whereas in the non-treated controls, living tissue was significantly reduced along with the mortality of few diseased corals. They also noted that phage treatment also significantly reduced transmission of WPL disease.

If phage therapy is to be operated on a large scale for treating coral diseases, developing a technique for delivering phages to infected corals over a large area, production of sufficient phages for treating large area of a reef and convincing local authorities for safety of this therapy by providing necessary evidence, would be required. Regarding concentration of phages required for treating the disease, authors showed that 10³ phage per ml (which is a thousand times higher than their natural abundance in Eilat seawater) is enough to prevent the spread of WPL. Extrapolating these figures, the authors suggested that production of sufficient phages to treat a large area of a reef is possible. Nevertheless, authors did not address issues such as feasibility of phage therapy on reefs, in terms of its costs and effectiveness. Similarly, they did not address any limitations of it. A limitation which I can think of is its applicability to only few bacterial diseases which are caused by a single bacterial pathogen, but what about diseases like black band disease which is caused by a consortium of pathogens?

This study proves that phage BA3 can be effective against progression and transmission of WPL disease. Interestingly, as BA3 phage can only infect T. loyana; this study also confirms that T. loyana is the causative agent of WPL disease. This discovery led authors to suggest that in nature, phages may be playing a role in making few of the corals naturally resistant to certain bacterial diseases, which I found thought provoking. As authors suggested, perhaps, this is why many times healthy corals are surrounded by diseased corals. Could the phages of Vibrio shiloi have played any role in making the coral Oculina patagonica resistant against V. shiloi? The authors could have discussed this idea of “can phages confer resistance against bacterial pathogens?” in the context of coral probiotic hypothesis and hologenome theory of evolution. The discussion of this paper is very short and I think it is incomplete. I think this is a revolutionary idea that phages could play a role in providing natural resistance to organisms against bacterial diseases. How widely applicable this idea would be? Could we link it to natural resistance of few individuals to a bacterial disease epidemic of humans and other animals?

Atad, I., Zvuloni, A., Loya, Y., & Rosenberg, E. (2012). Phage therapy of the white plague-like disease of Favia favus in the Red Sea. Coral reefs, 31(3), 665-670.

                            

Evidence for a persistent microbial seed bank throughout the global ocean

This paper attempts to answer the question, “Do bacterial taxa demonstrate clear endemism?”, are they restricted to specific geographical ranges to the extent that macroorganisms are, or can one sample site contain the total phylogenetic diversity of the world’s oceans.

The microbial community within an environment at any given time is determined by current environmental and biological factors, so it changes across space and time as niches open and close. Different species bloom and then decrease in certain conditions, but they must all be present at low levels for these blooms to occur, in what is known as the “microbial seed bank”.

For this study, across a 6 year period, around 10 million 16S rRNA sequences were recorded at a deep water sampling site in the West English Channel, known as the L4-DeepSeq dataset. Their first finding was that all operational taxanomic units (OTUs) identified throughout the 6 years were present at any time point within the 6 years. All taxa were present at all times, but their abundance varied dramatically. These results contrast the widely accepted model that it is presence/absence of taxa that controls community structure, suggesting instead that all taxa persist and it is their abundances that control the community composition.

From this they suggest that sufficient sequencing would allow the testing of the null hypothesis “all bacteria are found in any particular environment because of an immense and persistent microbial seed bank”, this would be falsified by evidence of clear endemism, the lack of certain taxa in some environments.

In order to begin investigating this, they compared the L4-DeepSeq dataset with the global International Census of Marine Microbes (ICoMM), which contains 356 datasets of bacterial 16S rRNA sequences ranging from pelagic and sediment samples to mangrove and sponge environments.
They found that the L4-DeepSeq dataset overlapped significantly with ICoMM datasets, containing 31.7-66.2% of taxa in any given ICoMM biome. They also discovered the relationship that the greater the sequencing depth in L4 samples, the greater their overlap with global ICoMM data. Sequencing depth here does not mean depth in the ocean but the number of sequences within a sample. By extrapolating the relationship, they suggested that 100% overlap between L4-DeepSeq data and ICoMM global data would be achieved when a 16S rRNA sequencing depth of 1.93x10¹¹ reads was reached. Achieving this would require over 200L of seawater to be filtered, rather than the 2L per sample used in this study, in order to collect enough cells to allow a sequencing depth that great. It would also cost an estimated $1.34million in sequencing costs. Regardless of the feasibility of such a study, it seems incredibly implausible that 100% overlap would ever truly be found, I think it far more likely that the relationship between sampling depth and phylgenetic overlap would at some point ‘level off’, where increased sequencing effort would cease to produce additional operational taxomic units. Though it seems the opinion of the authors that every taxa will be able to be found in every environment, if the sequencing depth is great enough.

The main value of this paper is not so much that it proves the existence of microbial endemism, as such a thing seems obvious given the existence of highly specialised extremophiles. It is valuable because it shows that an arbitrary sample of a marine microbial community overlaps on average 44% with other samples from varying marine environments around the world, with the suggestion that increased detection resolutions may show even greater overlap. So rather than 100% of taxa being present in all environments, I think they provide evidence for a global seedbank for a large number of cosmopolitan taxa.

One weakness of the study in my opinion is that comparing one sample point in Britain against many sample points from around the world, and generating the overlap percentages is not the best method for investigating global taxa overlap of communities. I think that comparing each sample point in the ICoMM to every other point in the ICoMM would give a higher average overlap, rather than using L4-DeepSeq as the central reference point. The average overlap between all samples is likely greater than the average overlap between L4-DeepSeq with each other sample.
The use of extreme habitats such as hydrothermal vents, cold seeps and estuaries likely pulled the average down due to their large number of specialised and endemic taxa. Excluding them from the dataset may produce stronger evidence for a global microbial seedbank for the general ocean.

Gibbons, S. M., Caporaso, J. G., Pirrung, M., Field, D., Knight, R., & Gilbert, J. A. (2013). Evidence for a persistent microbial seed bank throughout the global ocean. Proceedings of the National Academy of Sciences, 110(12), 4651-4655.