Extracellular polymeric substances (EPS) provide a strong
and sticky framework that connects cells together; the chemical and physical
properties of EPS vary among bacteria species, concentration of substrates and
environmental conditions. In this paper biofilm formation and EPS production were investigated using; the
gram-negative, non-spore forming, pathogenic bacterium Cronobacter sakazakii. The bacterium Cronobacter
sakazakii has caused disease in all age groups, with infants especially at risk. There has been strains of Cronobacter found in Infant formula milk processing facilities that can produce
inducible (activated in the presence of a specific molecule or substrate) and
non-inducible EPS.
Several strains of Cronobacter
sakazakii (ATCC 12868, ATCC 29004, 42vs and kfris016) were applied to M9
minimum salt medium (MSM) agar, and several supplemented forms at 37˚C for two days.
The
biofilm formation was determined by quantifying the amount of Extracellular
polymeric substances present, the total EPS count was estimated in each sample
using the phenol/sulphuric acid method using glucose as a standard. The results
from the range of media showed EPS production significantly differed among the
various strains and conditions, for instance there was higher cell numbers and
EPS observed on the Tryptic soy agar without dextrose (TSA) media than M9 MSM
for all C.sakazakii strains. Some strains
like C.sakazakii ATCC12868, responded
with higher levels on different media. The authors also observed colony
formations by growing C.sakazakii on
various media; each colony demonstrated a different shape, size and
transparency. A carbohydrate binding dye was used to stain attached cells this
identified EPS, shown in the Light microscopic photographs of C. sakazakii ATCC 12868 (Fig. 2) following staining with
ruthenium red (EPS) and crystal violet (cells). The pink coloured areas indicate the
production of extracellular carbohydrate (Fig.2B, 2D and 2G).
To determine the effects of nutrition conditions on biofilm
formation, EPS production and cell resistance against desiccation, tests using MSM agar with different sucrose
concentrations on stainless steel coupons (SSCs) as well as tests monitoring the survival of C .sakazakii on the surface of SSCs at
various relative humidity levels (23, 43, 68, 85, 100%) were performed. The
addition of sucrose to media could enhance biofilm formation and EPS production
by C. sakazakii on the surface of SSCs;
however it might differ depending on type of C. sakazakii strain. The survival rates of C. sakazakii in biofilm formed on the surface of SSCs in M9 MSM
supplemented with 3% sucrose at 23%, 43%, 68%, and 100% relative humidity, suggested
that the EPS content of a biofilm might affect the survival of C. sakazakii on
the surface of stainless steel under dry conditions at certain relative
humidity levels.
High tolerance to desiccation has been reported in other studies
showing that C .sakazakii has great
resistance at 25-45˚C. EPS may enhance the resistance because it has been
demonstrated that EPS can protect cells in biofilm from environmental stresses.
The authors suggest that EPS production and biofilm formation
by C. sakazakii could be adjusted
depending on nutrient or environmental conditions provided to cells. This is very important
in relation to Infant formula milk processing facilities which have encountered
several strains of this bacterium, by adjusting the nutrient and environmental conditions
it would be a safer way to remove and manage the Cronobacter spp threat without applying
chemicals that may accumulate in the formula and cause more health risks. Further research is still needed to investigate the
correlation between EPS production and resistance of biofilm cells on the
surface against various stressors.
I originally chose this paper to increase my understanding of
EPS in biofilm formation, although this paper does not go into great depth
about extracellular polymeric substances, I have found it useful as it is an example of how the intrinsic and extrinsic
conditions can affect structure and formation of biofilms as well as resistance.
Jung, J.-H., Choi, N.-Y., Lee, S.-Y., (2013), Biofilm formation and exopolysaccharide (EPS)
production by Cronobacter sakazakii depending on environmental conditions, Food
Microbiology, Volume 34, Issue 1, Pages 70- 80
Hey Kathryn,
ReplyDeleteInteresting post! I was just wondering if the authors gave any detail as to the specific factors that caused the different types of colony formations of C.sakazakii?
Thanks, Aimee
ReplyDeleteHi kathryn,
An interesting post, a few quick questions regarding your post. You mention that the addition of sucrose as an extra source of nutrients could influence biofilm production. Did it? There is no mention of the results of this assay.
The idea of changing nutrient and environmental conditions to control Cronobacter sakazaki is interesting, but do you really think this is viable?
The control of Biofilm formation has been extensively linked with quorum sensing (QS) in many gram-negative species. Whether C. sakazaki uses QS to control biofilm formation could be found using a QS reporter, then, to control this, a QS inhibitor (specific to C. sakazaki) could be added. The QS inhibitor would be specific to C. sakazaki and have little effect on other species, I beleive this would be a more viable approach to preventing future C. sakazaki infections. What are your thoughts on this?
Scott.
Hi Aimee,
ReplyDeleteIn terms of colony observations C. sakazakii ATCC 12868 was cultured on various agar media and adjusted to pH 5 with HCl, at 37 ˚C for 24 h.
The various agar media consisted of several supplements:
- tryptic soy agar without dextrose
- M9 minimum salt medium agar,
- M9 MSM agar supplemented with 3% glucose
- M9 MSM agar supplemented with 3% NaCl
- M9 MSM agar supplemented with 3% Tween 80
- M9 MSM agar supplemented with 3% sucrose
Another factor which I’ve mentioned in the post is the various strains, C. sakazakii ATCC 12868 was given as an example.
Is this what you mean by specific factors?
Thanks,
Kathryn
Hi Scott,
ReplyDeleteThe strain C. sakazakii ATCC 12868 produced the largest amount of EPS on M9 MSM supplemented with 3% sucrose compared to any other strain on various media, so the authors used it in an experiment on biofilm formation and EPS production on the surface SSCs. The results of this experiment showed that EPS production by C. sakazakii ATCC 12868 was significantly higher (4.69 mg per coupon) on M9 MSM 5% sucrose compared to all other sucrose concentrations (except for M9 MSM containing 3% sucrose). EPS production by C. sakazakiiATCC 12868 in M9 MSM with 3% sucrose (2.97 mg per coupon) was higher compared to 0 and 1% sucrose (0.71 and 0.98 mg per coupon, respectively). Using these results the authors made the suggestion that the addition of sucrose to media could enhance biofilm formation and EPS production by C. sakazakii on the surface of SSCs.
Adjusting environmental and nutrients was suggested by the authors, however they did not explain further. Interpreting their suggestion I think combining environmental or nutrients changes with sterilising agents would be a worthwhile attempt at reducing cronobacter sp.
Your point about using QS reporters and a QS inhibitor (specific to C. sakazaki) sounds interesting do you recommend any studies that have used this as a method?
Thanks,
Kathryn
ReplyDeleteHi Kathryn,
Thanks for the speedy response. It is interesting that the highest EPS production and therefore, biofilm formation, was measured at 3% sucrose. Did the authors expect this?
A recent study by Hunt et al. (2012) would be a suitable method for ascertaining the presence of QS, though they only look at AHLs, there are other signalling molecules. Hunt and colleagues used 2 AHL reporter strains, one sensitive to long chain AHLs and the other short. This would show the presence of AHLs but also indicate their around about size, you could then further analyse the molecule to find its chemical formula. Now, to find an inhibitor for this QS regulatory pathway depends on which mode of inhibition (degradation of AHL signals or competitive inhibitors of AHL-protein active sites) but a fairly comprehensive method would be the one use in the dobretsov et al. (2011), which I reviewed earlier in the year. They tested 70+ natural products for their ability to inhibit microbial biofouling, without causing toxicity.
Scott.
Dobretsov, S., Teplitski, M., Bayer, M., Gunasekera, S., Proksch, P. and Paul, V.J. (2011) Inhibition of marine biofouling by bacterial quorum sensing inhibitors. Biofouling 27. 893–905.
Hunt, L.R., Smith, S.M., Downum, K.R. and Myldarz, L.D. (2012) Microbial Regulation in Gorgonian Corals. Marine Drugs 10. 1225-1243.
Hi Scott,
ReplyDeleteThe paper explains that the bacterium can enter milk formulas by contaminated ingredients and it has been found in factories that produce cereals, chocolate, potato flour, and pasta. All these foods produced contain a high amount of sucrose however they make no clear hypothesis that it would be higher on the M9 MSM supplemented with 3% sucrose. This could be explain if they based their expectations on previous, for instance Gauri et al. (2009) stated that glucose was the most effective for EPS production.
Thank you for the examples although both address marine environments (coral microbial regulation and biofouling) the use of quorum sensing does seem to be applicable to biofilm formation in food products. In terms of Quorum sensing inhibitors associated with food production, I found a review by Bai and Rai (2011) that explained the implications and possible solutions involved with QS, with AHL detection using biosensors and using inhibitors that can be extracted from common dietary fruits, herbs and spices, and also used as food preservatives. A point I found particularly interesting was that these have been discovered to significantly inhibit QS strains of Chromobacterium violaceum and vanilla has been found to effectively inhibit the strain C. violaceum CV026.
I have struggled to find studies that have used QS to control specifically Cronobacter sakazakii, suggesting more research is needed, possibly to determine a specific inhibitor, or the costs and effectiveness for use in food industries.
Bai, A. J. and Rai, V. R. (2011), Bacterial Quorum Sensing and Food Industry. Comprehensive Reviews in Food Science and Food Safety, volume 10, pages 183–193
Gauri, S.M. Mandal, K.C. Mondal, S. Dey, B.R. Pati (2009) Enhanced production and partial characterization of an extracellular polysaccharide from newly isolated Azotobacter sp. SSB81, Bioresource Technology, volume 100, pages 4240–4243. Cited in Jung et al (2013).
Thanks,
Kathryn
ReplyDeleteHi Kathryn,
Sorry, I didn't realise you were after specific examples of QS in food microbiology, though the two studies I have mentioned would be applicable in terms of the reporter strains and natural products tested. Yes, I have previously found that many terrestrial plants (and marine algae) produce quorum sensing inhibitor compounds. For my research project, I used curry powder as a QS inhibitor, though it soon lost it's novelty after smelling like a curry house for several months!
As you say, it is clear more research is needed to find the specific inhibitor needed, or if Cronobacter sakazakii even uses QS for that matter but I still believe this is a much more viable method of controlling biofilm formation compared to changing environmental and nutrient conditions.
Thanks for the interesting discussion.
Scott.