How sensitive are your probes?

Annelie Pernthaler, Jakob Pernthaler, and Rudolf Amann Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria, Appl. Envir. Microbiol., Jun 2002; 68: 3094 - 3101.

Fluorescence in situ hybridization (FISH) of microbes was a break-through well over a decade ago. However initial researchers found it difficult to apply the technique to anything other than eutrophic systems. As we know most bacteria in the oceans are slow growing, vastly small and starving, which results in the intensities of signal hybridization fluorescence getting lost in background fluorescence. This is attributed to low levels of 16S ribosomal RNA being present, thus causing low complementary base pairs and probe binding. Due to the lack of sensitivity of early FISH methods, they were not ideal for application to marine oligotrophic microbes. Therefore it was a key interest of marine microbial researchers to develop more sensitive techniques (2002).

Researchers from the Max Plank Research Institute, Bremen, Germany further developed an established more sensitive method (which used FISH combined with horseradish (HRP)-labelled oligonucleotides probes and tyramide signal amplification, known as catalysed reporter deposition (CARD)), they advanced the protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. The main problem for them to overcome was high sp.-selective cell loss, as a result of cell wall degradation from HRP penetration following permeabilization. This was observable through decreases in cell counts (using DAPI) following permeabilization, rendering the technique unreliable as a tool for the detection of O.T.U. and for the enumeration of marine microbes.

These Max Plank Institute researchers developed a method where concentrated sample filters were embedded in low-gelling agarose, which resulted in no observable decreases in bacterial cell counts during a 90 min incubation of lysozyme. These modifications in preparation and permeabilization procedures, as well as improvement to staining protocols for CARD-FISH, for the quantification of marine microbes, led to a significant improvement in detection rates compared to previous protocol which involved mono-labelled probes (such as EUB338 mono, compared with probe EUB338-HRP). These researchers, when using their improved method (multi-labelled), reported an improvement of 46% based on the differences between mean values of mono-labelled probes and multi-labelled probes in detection rates of coastal North Sea bacterioplankton. They also reported that the modified technique detects clades e.g. SAR86 which were previously undetectable by mono-labelled probes.

These discoveries led to the authors concluding that oligonucleotide probes are more superior for the staining of bacteria with low rRNA content in the marine environment, provided cell disintegration is minimised during the permeabilization stage of the technique.

In the discussion the authors discuss the potential and limitations to their advancements to the CARD-FISH technique for environmental microbiology. They acknowledge that there are limitations to the technique e.g they are not confident that the technique they developed is suitable to be used on Archea. However they do highlight the potential of the CARD-FISH approach, and suggest it is not just limited to staining of rRNA’s. They suggest a wide range of methods used in histology and cytology could also be used in marine environmental microbiology e.g. the detection of mRNA. The works of these authors demonstrate minor advancements in techniques of molecular methods result in a widening of disciplines for the application of its use. This progress in specialized areas of microbial ecology provides future marine microbial scientists with far more powerful and sensitive tools, resulting in more confidence in the accuracy of researchers work.

On a more negative note the authors failed to produce vital unpublished data which supports their argument for the evidence of the improvement of their method. These omissions cast doubt on the confidence of readers,( such as myself) have in what seems to be such a pertinent development in molecular techniques.

To conclude advancements in molecular methods provided by researchers like Pernthaler et al. result in further superior research being produced provided their protocol is adopted and is as good as stated on the tin.

The Baltic Sea: A Brackish Environment

Many different environmental factors influence the composition of communities in marine environments, and this paper expands on previous research which has shown that salinity has a particularly important effect on the distribution of both pelagic and benthic organisms. Although research has been carried out to investigate the effect of salinity on micro-organisms, this study is the first to investigate the distribution and community composition of micro-organisms found in brackish environments with the use of 454 pyrosequencing of partial 16S rRNA genes.

Brackish environments occur where freshwater mixes with saltwater, as happens in estuaries. Typically a reduction in diversity is seen in these environments due to the adaptations of multicellular organisms to either freshwater or saltwater conditions, but less is known about the diversity of micro-organisms within these environments. Previous studies of microbial communities have been carried out in areas such as river outlets where conditions change rapidly and communities of micro-organisms adapted to brackish environments are not able to establish.

The Baltic Sea differs from these types of sites because it is comparatively more stable, with a water retention time between 3 and 30 years. The Baltic Sea is one of the largest brackish environments in the world, with salinity gradients maintained by the discharge of freshwater and a limited exchange with the North Sea.

A total of 213 samples were collected from 60 sampling stations along the salinity gradient of the Baltic Sea during June and July 2008. DNA was extracted, amplified and sequenced, to produce a ‘bacterial inventory’ of the sea.

Results revealed three clusters of samples at salinities of 0-3, 5-8, and 10-31, providing evidence of three bacterial communities, which were described as freshwater-brackish, brackish and brackish-marine. A shift in community composition was observed across the salinity gradient. It was predicted that at intermediate salinities there would be minimum species richness but results showed no significant difference in diversity between the three salinity clusters. The authors conclude that freshwater and marine clades that have apparently adapted to brackish conditions exist within the Baltic Sea and maintain this diversity at intermediate salinities.

This paper details the first taxonomic study of a brackish microbiome, and highlights the differences between bacteria and multicellular organisms in their ability to adapt to these conditions. Further research is needed to understand more about the micro-organisms within these communities and how they contribute to the brackish environment, and their functioning within ecosystems.

A review of: Herlemann, D.P.R., Labrenz, M., Jürgens, K., Bertilsson, S., Waniek, J.J. and Andersson, A.F. (2011) Transitions in bacterial communities along the 2000km salinity gradient of the Baltic Sea, The ISME Journal 5, 1571-1579.

Direct Effect of CO2 Concentration on Phytoplankton Bloom.

Engel, A., Zondervan, I., Aerts, K., et al. (2005) Testing the direct effect of CO2 concentration on a bloom of the coccolithophorid Emiliania huxleyi in mesocosm experiments. Limnol. Oceanogr., 50(2): 493–507.

An extensive paper which investigates the direct effect of CO2 and the associated alterations in carbonate chemistry in sea water on marine planktonic organisms. The authors give a synopsis on the biogeochemistry and development of a bloom of E. huxleyi.

During the last 250 years, a rapid increase in CO2 concentration has been observed with estimates proposing a further increase up until 2100. It is unclear how the steady increase of CO2 concentration affects the fundamental cycling and surface ocean chemical equilibrium that maintains the marine environment. Environmental parameters are suggested to affect marine organism physiology by means of species composition, competition and the cycling of key biogeochemical elements (C, N and P.).

Changes in carbonate chemistry are suggested to effect calcification rates of marine phytoplankton through a change in the ratio of particulate organic carbon (POC) to particulate inorganic carbon (PIC).The quantity of biological calcification and rate of organic assimilation has the potential to manipulate the efficiency of global biological carbon pumps.

An experiment was designed enabling the manipulation of seawater CO2 concentration and maintaining the most natural conditions possible. Nine enclosures were created. The atmospheric and seawater pCO2 were manipulated to create three different CO2 concentrations in triplicate.
1. High pCO2 – Similar to CO2 concentrations expected in 2100.
2. Present pCO2 - Similar to CO2 concentrations to present day.
3. Low pCO2 - Similar to CO2 concentrations of glacial atmosphere.

Nutrient induced blooms of E. huxleyi were monitored over a 19 day period. An overview of all 28 variables that were determined during the study is described, as well as details of their individual methods. Phytoplankton counts were performed and measurements of size and weight were recorded.

Bloom of E. Huxleyi occurred concurrently in all nine enclosures showing exponential growth until total assimilation of nitrate and phosphate was complete. The characteristics of the comparative bloom associated with CO2 related effects were hidden by a variable concentration of particulates observed in the replicate mesocosms. E. huxleyi was affected by CO2 related treatments in the formation of calcite. This decreased as CO2 concentration increased.

Nonetheless, results obtained should be treated with vigilance when determining differences between the enclosures. It must be noted that, any environmental parameters acting upon on the E. huxleyi bloom were kept as constant as possible. This is not representative of the development of the oceanographic and chemical condition of the ocean between glacial times and the present day.

The changes identified in the calcification of cells, net specific growth rates, and alterations in the processes that maintain the essential chemical balance of the ocean allows the authors to advocate that E. huxleyi is susceptible to changes in CO2 concentration. They conclude that CO2 concentration could directly affect biogeochemical cycling and the carbon chemistry of the ocean, thus leading to the potential manipulation of food web dynamics and carbon export.

Nevertheless, the authors concede that further investigation is needed to determine how the rate and timing of sinking particles, as well as the fundamental composition of exported material affect CO2 exportation rates. They also suggest further examination of heterotrophic processes that could be effected by CO2 concentration.

Microbial Community Dynamic In The English Channel

A review of: Gilbert, JA. Steele, JA. Caporaso, JG. Steinbruck, L. Reeder, J. Temperton, B. Huse, S. McHardy, AC. Knight, R. Joint, I. Somerfield, P. Furhman, JA. Field, D. (2011), Defining seasonal marine microbial community dynamics. International society for Microbial ecology: 1 – 11.

The Western English Channel has been extensively studied for the last 100 years, providing a wealth of data on temporal microbiological complexity. With the recent introduction of molecular techniques, satisfactory descriptions of natural microbial assemblages have been generated.

Previous efforts to determine which factors might affect microbial communities have largely focused on the importance of temperature and nutrient concentrations, understandably so because of the strong effect temperature has upon biological processes and because nutrient concentrations can drive niche structure through resource partitioning. This study however goes beyond the initial 1 year study by Gilbert et al (2009) to test the influence of three competing alternative factors: (1) varying concentration of inorganic nutrients; (2) annual water-temperature cycle; and (3) population structure of phytoplankton and zooplankton and summarizes in this paper a 6-year time series of 16S rRNA tag pyrosequencing taken from a station in the English Channel.

From the observations, the authors divided the different micro-organisms found into three broad categories. The first group was the ‘most abundant’ OTU’s, such as Rickettsiales and Rhodobacterals. The second group was dubbed ‘most common’ OTU’s, containing those organisms that had the highest persistence over the six year sampling time. The third and final group was the ‘most variable’ OTU’s containing organisms that would be rare during certain times and bloom uncontrollably at others.

Overall they concluded that, throughout the 6-year period, the monthly pattern and broad seasonal changes in microbial assemblages indicate that the ‘most abundant’ and the ‘most common’ OTU’s have temporally defined niches. In contrast however, the most variable OTU’s have niches that can be defined temporally as well as by nutrient pulses and changes in currents.

This study highlights the added value of much longer temporal observations of natural communities as subtle changes in certain individual taxa were only detectable because of the long time series. It is quick to point out limitations of studying communities influenced by hydrography and the difficulty it causes in interpreting results. It also calls into question the relative effect of the currents and, by association, wind speeds and directions in the English Channel and its role on microbial community dynamics, admitting that more research needs to be done to fully take into account its potential effect.

Enzyme use by Sulphate-reducing Deltaproteobacteria in environments with archaea that oxidise methane anaerobically

This paper investigates how archaea work in a microbial consortium with Sulphate-reducing Deltaproteobacteria to oxidize methane. I believe this topic to be important as it explores what happens to a lot of the methane that is produced in deep anoxic sediments, as well as how it is able to happen. Specifically, it examines the enzymes that are used in the anaerobic oxidation of methane (AOM) during sulphate reduction. There were three enzymes studied: ATP sulphurylase which activates sulphate to produce adenosine 5′-phosphosulfate (APS) and pyrophosphate (later hydrolysed to phosphate), APS reductase, which reduces APS to bisulphite, and dissimilatory sulphite reductase, which reduces sulphite to sulphide (Dsr).

In the experiment samples of microbial mats were collected from a methane seep in the black sea, these mats were known to have high AOM activity. Sulphate-reducing Deltaproteobacteria reduce Sulphate to Hydrogen Sulphide and when within these mats, when present with archaea and bacteria that can anaerobically oxidize methane, the following reaction occurs:
AOM is performed by archaea that are closely related to methanogens and sulphate reducing bacteria. It is thought that they are able to perform methanogenesis in reverse as they are anaerobic methanogens (ANME) and so they have most of the genes that are required for methanogenesis and the methane purifying enzyme was similar to methyl-coenzyme M reductase, an enzyme used in methanogenesis. In some AOM environments archaeal cells have been found not to have any association with bacterial cells, implying that some archaea must be able to perform both methane oxidation and sulphate reduction.

Fluorescence in situ hybridization (FISH) was used on the selected section of the microbial mat to detect the presence of consortia under a microscope and, as expected, the cells formed dense aggregates. Fluorescent stains with 16s rRNA probes were used, which showed that there was an archaeal core surrounded by either bacterial cells alone or a combination of archaeal and bacterial cells.
The results of this study have shown that ATP sulphurylase and APS reductase are most likely used by the archaea in the reduction of sulphate to sulphide but there is not sufficient evidence to suggest that Dsr is also used in the chain of reactions although this could be due to the experimental technique as much of DsrC, a subunit of Dsr that is important for the enzyme to function properly, was lost during extraction and purification. Changing the way that the enzymes are extracted and purified by experimenting with different saturations of ammonium sulphate precipitation or by reducing the speed of the centrifuge may help to reduce the amount of DsrC that is lost in the process and give more accurate results.

Reference: Basen, M et al. (2011), Bacterial enzymes for dissimilatory sulfate reduction in a marine microbial mat (Black Sea) mediating anaerobic oxidation of methane. Environmental Microbiology, Vol 13, Issue 5, pg 1370–1379

How microbial biofilm communities respond to ocean acidification ?

As a result of massive CO2 release due to industrial activities, pH oceans is rapidly decreasing in last decades. This process is called "ocean acidification" and is becoming a rising problem that scientific community need to investigate. Bacterial biofilm communities play a key role in understanding environmental disturbance by reduced pH in sea water. In this paper authors investigated the effects of ocean acidification on the activity ( expressed as O2 fluxes) and community composition of the tropical coral reef-associated biofilms from the Australian Great Barrier Reef.

Glass slides were immersed into a flow-through tank with natural seawater from the lagoon for 24 d. Natural biofilms grown on glass slides were subsequently exposed for 11 days to four controlled pCO2 conditions, representing four scenarios ( increasing pCO2 ) :

A) Pre-industrial: ~ 300 ppm (pH 8.1-8.2)

B) Present day: ~ 400 ppm (pH 8)

C) Projected Mid century: ~ 560 ppm (pH 7.9)

D) Projected Late century: ~ 1140 ppm (pH 7.6)

Samples collected were processed to analyze O2 fluxes, C:N ratios, bacterial phylogenetic analysis, determination of macro communities.

Results obtained show that frequency of phototrophic flora components significantly changed among the four treatments: at high pCO2 phototrophic community members exclusively comprised diatoms, green filamentous and green algae, while all red algae types were completely absent.

Most relevant bacteria found belong to Alphaproteobacteria, Bacteroidetes and Gammaproteobacteria; bacterial T-RFLP analysis shows different profiles of initial community from those at the end of the experiment: Bacteroidetes increase with rising pCO2, Alphaproteobacteria show a opposite trend (decreasing). Further, bacterial assemblages at low pCO2 had the highest variability, while high CO2 treatments showed less community variability.16S rRNA sequence analysis demonstrates that sequence affiliated with the Alphaproteobacteria were most abundant in all treatments, except in the control ( B - present day ), where Bacteroidetes affiliated sequences were most frequent. Statistically significant differences were detected between A and B compared to D scenarios, while the bacterial communities from C and D were statistically indistinguishable. Diatoms plastid affiliated sequences was the only group showing a treatment related trend and increasing with rising pCO2. Cyanobacterial sequences belonged to three different orders ( Chroococcales, Oscillatoriales and Nostocales ) in A treatment, but in D Chroococcales sequences were found exclusively.

Findings suggest that exposure during the early stages of biofilm development to short-term high CO2 levels significantly decreases algal diversity and promotes a shift towards diatom and filamentous green algae dominated biofilm communities. This confirms results of previous similar studies.
The average respiration and production rates suggests that investigated communities were net autotrophic, indicating a dominant phototrophic component in the biofilms.
Assumption of algal dominated community is supported by the fact that C:N ratios are slightly higher than the Redfield ratio. Authors hypothesize that EPS production under stress due to nutrient limitation or to CO2 bubbling treatment could elevate C and N contents, leading to exceeding the Redfield ratio, but they suggest that further research is required to investigate this hypothesis, as well as the possibility of enhanced EPS production as a protection against harmful UV- radiation, an important factor to consider in Australia.
Regarding bacterial community composition, authors suggest increasing Bacteroidetes in the biofilm may be due to the fact that many members of this group excrete exoenzymes to decompose high molecular weight organic material from detritus, which is a process accelerated by high pCO2; a potentially higher EPS production under higher pCO2 may give them a selective advantage due to the more effective acquisition of catabolic substrates. This leads to less community variability found at high pCO2.

Hence, high pCO2 affects community variability so that communities become more specialized and adapted to lower seawater pH. This could have relevant implications for ecological processes, because bacteria play a key role in biogeochemistry, especially regarding larval settlement and development.

Although many results remain not clear and further investigations are necessary to draw conclusions for future ecological scenarios, this study represents a good starting point for future research and a good multidisciplinary approach example, which lead us consider the whole issue we want to investigate, taking into account all possible related factors and so all the consequences on global scale.

Reference: Witt, V., Wild, C., Anthony, K. R. N., Diaz-Pulido, G. and Uthicke, S. (2011), Effects of ocean acidification on microbial community composition of, and oxygen fluxes through, biofilms from the Great Barrier Reef. Environmental Microbiology. doi: 10.1111/j.1462-2920.2011.02571.x

Recent findings on the viable but nonculturable state in pathogenic bacteria

This paper is a review of what is currently known about the viable but nonculturable state (VNBC) in pathogenic bacteria, the paper outlines what is the VNBC state? The factors regulating it, gene expression, and probably the most relevant in medical terms the virulence of cells in the VNBC state.
The viable but nonculturable state, or VNBC state, refers to when bacteria are unable to be cultured on laboratory mediums in which they would usually grow, however these cells still remain viable and alive. RT-PCR (reverse transcriptase) is one of the most important methods in not only showing that the cells are alive but also in showing that these cells retain their “pathogenic potential” while in the VNBC state. The half-life of bacterial mRNA is 3-5 minutes, so if RT-PCR shows continuing gene expression in cells which are unculturable, these cells must remain viable. Furthermore it has been shown that genes for the 16S rRNA synthesis continued to be expressed in non culturable E.coli, the author concluded that this was strong evidence that these cells remained viable.
The review states that cells in the VNBC state are unlikely to cause infection, however that they retain their virulence and once resurrected can cause reinfection in the host organism. The review uses Mycobacterium tuberculosis as an example of this, the dormant stage in these bacteria is in fact the VNBC state, and if reinfection occurs many years after the primary infection it is because the bacteria have been resurrected from the VNBC state. In the review there are several examples of studies which have found cells which are non culturable, but continue to produce known toxins and also express virulence gene. For example cells of V.cholerae O1, V.parahaemolyticus O3:K6 and V.vulnificus expressed known toxins (ctxAB, rtxA, hlyA, tl, tdh and vvhA), while also expressing the virulence genes (tcpA and TTSS) therefore retaining the ability to infect the host organism when not in the VNBC state.
The exact role the VNBC state plays in bacteria is still not fully understood; however it is thought to be survival strategy and has been shown to be induced by various environmental factors, such as nutrient starvation, incubation outside the normal temperature range and changes in the osmotic concentrations. The VNBC state can only be a means of survival if the cells can be resurrected, resurrection has been most extensively studied in V.vulnificus, like many vibrio species the VNBC state can be induced due to a temperature downshift, V.vulnificus enters the state when temperatures drop below 10 0c. It has been shown both in situ, in vivo and in vitro in V.vulnificus that reversing the stressor, e.g. a temperature upshift will allow the cells to be resurrected.
Perhaps one of the most important consequences to humans of cells into the VNBC state is the effect that this has on antibiotic resistance. As cells in the VNBC state have such low metabolic activity, they effectively become resistant to antibiotics, and then are able to resurrect and reinitiate the infection. Furthermore it was found that, only when the concentration of Vancomycin was 500 times the MIC was it effective against cells of E.faecalis in the VNBC state. The author suggests that the antibiotic resistance of cells in the VNBC state is a reason why many bacterial infection seem to clear with antibiotics, however the course of antibiotics is finished, cells revert to their metabolically active phase and cause reinfection in the host organism. The author outlines two examples of this, H.pylori which are responsible for gastric and duodenal ulcers, and E.coli which can be responsible for urinary tract infections, the antibiotic resistance in the cells which are in the VNBC state likely accounts for high amount of recurrent infections.
The author concludes the review by stating that a large number of bacteria, including those which are pathogenic to humans enter the VNBC state, maintaining cellular structure and biology and continuing gene expression even when they are not able to be cultured by standard laboratory methods. He says that the VNBC state plays a critical role in the survival of pathogens and their ability to produce disease.
A review of: Oliver, J.D. 2010. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol Rev. 34, 415-425.