Effects of Subinhibitory Concentration of Carvacrol on BiofiJm Formation in Bacillus cereus and Bacillus amyloliquefaciens Isolated From Ultra High Temperature Treated Commercial Milk An Honors Thesis (HONR 499)
by
Alexander J Thomas
Thesis Advisor Dr. John McKillip Ball State University Muncie, Indiana April 2015 Expected Date of Graduation May 2015 1
r
Abstract
Biofilms arise in many everyday settings, including natural environments such as
streams, soils, and caves, or home and commercial environments such as restrooms, kitchens,
and industries. Food safety and sanitation is a constant concern for the growing population as
bacterial biofilms transmitted through the food industry are implicated in an increasing variety of
human diseases. Bacteria cultured from ultra high temperature (URT) treated milk are of interest,
showing the ability of some bacteria to survive processes meant to eliminate all potentially
harmful microbes. It has become important to discover new methods of biofilm control .
Carvacrol, an essential oil isolated from oregano (Origanum vulgare), has been shown to inhibit
the biofilm formation of Bacillus species of bacteria cultured from commercial milk, including
the flagship species Bacillus cereus. Bacillus amyloliquefaciens and several other Bacillus
species has also been cultured from commercial milk and may be inhibited by carvacrol
treatment. Carvacrol has a pleasant odor, is safe for human consumption, and may be a
potentially useful tool for food industry biofilm control.
2
Acknowledgments
Dr. John McKillip - Ball State University Department of Biology
I would like to thank Dr. McKillip for making this thesis project possible and for all of his help
and direction through this experience. His teachings in other Ball State courses, including
microbiology, pathogenic bacteriology, and Fine Focus has been essential. Working with Dr.
McKillip has been the highlight of my college career!
3
Biofilm Background and Composition
Biofilms are potentially harmful accumulations of bacteria, fungi, viruses, and/or
protozoa trapped in products secreted by bacterial cells that merge together in a film-like
aggregation with a sticky appearance and texture. In general, biofilms are associated with a rigid
surface on which they adhere and grow. In aqueous and food environments nutrients tend to
accumulate near solid surfaces of containers, rather than float freely. This becomes advantageous
for surface-adhered bacteria, as nutrients are more readily available compared to free-floating
planktonic bacteria. As a result, bacteria within a single species may behave differently when in
the planktonic stage, producing different extracellular products compared to bacteria in the
biofilm stage (Jay, 2000; Wilson et al. 2011). Bacterial species in the biofilm stage produce and
secrete large amounts of protein, and chains of sugar monomers called polysaccharides, creating
a glycocalyx. These components are extracellular - they are produced within the cell, or along
the cell membrane, and are then secreted as a matrix surrounding the cell, which allows a large
number of bacteria to bind together producing a film layer (Reece et al. 2011). Within the
glycocalyx of a multi-organism biofilm, water channels form allowing a vascular-like flow of
nutrients and toxins in and out of the system. This mechanism ajds in cell growth and persistence
of the bacteria.
The production of a biofilm creates a protective barrier between bacteria and
antimicrobial agents, such as sanitizers and cleansers used in the food industry, or even the
macrophages and lymphocytes of the human immune system. Not all strains within the same
genus and species may produce biofilms, but some bacteria are more likely to produce a
glycocalyx, including species of the Bacillus, Psuedomonas, and Streptococcus genera. Biofilm
4
production may be attributed to a virulent response to environmental stimuli, as producing large
amounts of extracellular products requires significant energy and resources.
The Bacillus subspecies B. amyloliquefaciens and B. cereus are of particular interest
because ultra high temperature (UHT) pasteurization and general pasteurization processes are
meant to eliminate pathogens, including biofilm-producing bacteria living in milk and other food
products. Both of these species have been cultured from UHT milk, making them significant for
study. UHT processing involves heating the milk product to 131°C (268 oF) for 2 seconds. This
process is expensive, adding to the cost of UHT milk (often marketed as organic milk), but it
greatly increases the shelf life of the product. UHT milk can be stored unopened for several
months compared to the average lO-day shelf life of high-temperature short-time (HTST) milk,
which is processed between 72-75 °C for 15-30 seconds (Lorenzen et at. 2011). However, hardy
endospores from Bacillus subspecies, along with several other spore-forming genera, have been
cultured from UHT treated milk and are capable of producing biofilms.
Biofilms can be very harmful to human populations being indicated in as many as 80% of
microbial infections, and more than 65% of healthcare-related infections, making the study of
this virulence factor very important for our understanding of how bacteria can survive UHT
processing and cause disease (Percival et al. 2014). This raises the question of whether or not a
more pathogenic species of UHT resistant bacteria may arise. B. amyloliquefaciens and B. cereus
can cause disease in humans, but strains isolated from spores retrieved from dairy products have
not yet proven to be any more pathogenic. Bacillus has proven to be a valuable model for study.
Justification for Study
The objective of this overall Bacillus project is to gather data on the bio-chemical
composition of biofilms produced by UHT isolates. Carvacrol has been found to inhibit bacterial
5
growth and may be a potential agent for biofilm control in Bacillus. Data on differences in
biofilm formation between carvacrol treated Bacillus and non-stressed Bacillus subspecies is
necessary for determining if carvacrol is a plausible agent for microbial control. The results of
preliminary data may be helpful for further investigation and will improve our understanding of
how a bacterial species can survive high temperature treatment. The data collected may also
become useful for future research if a more pathogenic (disease causing) species should arise in
the future.
This thesis project is for a target audience interested in UHT resistant bacteria and other
organisms that may survive the pasteurization process. This may be other researchers, milk
product producers, veterinarians, dairy farmers, and the FDA. The implication that UHT
pasteurization does not completely eliminate bacteria from milk products may be of concern
because of the potential health risks to humans and the fact that a new pasteurization process
would need to be developed to fend off new strains of biofilm-producing bacteria. The audience
is expected to learn the attributes of these Bacillus species and the expected circumstances under
which these specific species grows. The audience will also learn about health risks posed by
biofilms and potential alternatives to UHT pasteurization.
Implications of Biofilms in the Dairy Industry
Several authors have identified a variety of mesophilic Bacillus subspecies capable of
surviving ultra high temperature pasteurization via endospore formation (Sutyak et al. 2008;
Araujo et al. 2009; Scheldeman et al. 2006; Lindsey et al. 2002). Using bacterial cultures
sampled from dairies, 16s rRNA, and peR amplification, some of the most prevalent and
potentially problematic species in regards to biofilm production have been characterized. These
6
species include, but are not limited to B. sporotherrnodurans, B. cereus, and B.
arnyloliquefaciens. In particular, UHT treatment using indirect heating methods has proven to be
more problematic with greater bacterial densities measured in CFU mL· 1 relative to direct UHT
heating methods. Indirect heating methods are generally used as a means of limiting chemical
changes in the product, which can affect the milks taste and overall quality. Although vegetative
cells may be eliminated via UHT pasteurization, spores are capable of surviving treatment and
later producing new vegetative cells once conditions become more favorable for growth
(Montanari et al. 2004).
Unfortunately, UHT techniques used to limit biofilm growth present negative side effects
lowering the quality of commercially sold milk. Heat treatment administered to milk can
potentially degrade enzymes, and proteins altering the chemical make-up of the product. This
alteration can affect quality (flavor) and shelf life (Cattaneo et al. 2008). Dairies strive to
administer the minimal amount of heat processing, eliminating harmful microbes, while still
retaining a suitable quality product. This balance may be difficult to obtain based on the rate of
mutations or adaptations in bacterial species.
Overtime, continued polysaccharide secretion increases the thickness and overall
hardiness of biofilms. Other species of bacteria and some viruses can also adhere as the biofilm
continues to cement more toughly to the surface. This cementing process is problematic in
dairies because biofilms eventually become a tough plastic sheet that is not easily removed from
production surfaces, such as stainless steel and packing materials including glass and paper
cartons (Marriott, 1999). The addition of other bacterial species and viruses in a mixed-culture
biofilm may aid in attachment to specific surface types and in resilience against sanitizers.
Mixed-culture biofilms found in the dairy industry are particularly problematic from an
7
economic standpoint, costing dairies in sanitations costs, lost product quality, and potential
product recall (Jay, 2000; Lorenzen et a1. 2011).
Extracellular bacterial products such as proteases and lipases cause dairy products to
spoil more quickly. Protease production in biofilms results in the "sweet-curdling" of milk.
Proteases, sometimes referred to as peptidases or proteinases, are enzymes that catalyze the
hydrolysis of peptide bonds between amino acids. The dissolution of amino acid linkages in a
polypeptide chain ultimately results in the complete degradation of the primary structure of a
protein. This structure is the basis for the final tertiary or quaternary folded protein products that
are capable of facilitating cellular activity. Non-linked, free-floating amino acids cause an
increased rate of milk spoilage and curdling. B. cereus, in particular, is responsible for this form
of spoilage in UHT milk. Similarly, lipases catalyze the hydrolysis of fats (lipids), which varies
in content based on the type of dairy product. In commercial milk, product labeling differs based
on the percentage of milk fat. Extensive degradation of lipids will also cause a quicker rate of
spoilage. Other various Bacillus subspecies are also known to be effective lipase producers.
Adherence of microbial biofilms to dairy production surfaces makes sanitization more
difficult, and increases cost via labor and chemical usage along with lost production time. FDA
involvement and subsequent product recalls can also occur causing further financial problems for
dairies. Araujo et al. (2009) have proposed a basic mechanism for biofilm adhesion based on six
general stages. First, the biofiJm surface must be primed for adhesion with the existence of food
deposits. The biofilm-producing microorganism must then come into contact with the primed
surface. Positive and negative biochemical forces including van der Waals forces and other
electrostatic forces then allow the biofilm to make a non-permanent attachment to the surface
when microorganism are between 20 and 50 nm away. Irreversible adhesion results within 1.5
8
run when extracellular polysaccharide production, ionic bonds, and hydrophobic forces occur.
The forth stage is described by the multiplication of bacterial cells and an increase in secreted
polysaccharides and the fifth stage involves strong metabolism in the biofilm. Lastly,
microorganisms begin to be released from the biofilm during the sixth stage.
UHT resistant strains have altogether been characterized as hazardous to human
populations. However, Sutyak et a1. (2008) have reported the hOlizontal gene transfer of the
antimicrobial protein subtilosin A from B. subtilis into B. amyloliquefaciens in milk products.
Subtilosin is a known bacteriocin, which promotes activity against vaginosis-related pathogens
and was originally thought to only be produced by B. subtilis bacteria. The presence of this
antimicrobial peptide and its isolation from B. amyloliquefaciens demonstrates the theoretical use
of this species as a study model.
Problematic Bacterial Species
Bacillus sporothermodurans has been a known spore-forming bacterium present in
commercial milk since the 1980s. The concentration of this species in commercial UHT milk is
not allowed to exceed 10 CFU mL- 1 in European UHT milk, a standard set in 1985 (Council
Directive 85/ 397/EEC, 1985). This species is the most common found among UHT milk
samples, while B. cereus is the most widely isolated bacteria in raw milk (Aouadhi et al. 2014).
B. sporothermodurans has also presented increased heat resistance under sublethal stress
conditions, suggesting that inadequate pasteurization can result in more durable bacterial spores.
Additionally, hydrogen peroxide, used as a sanitizer in some dairies, has been linked with
increased thermotolerance in laboratory-grown B. sporothermodurans strains (Scheldeman et ai.
2006; Montonari et ai. 2004).
9
Bacillus cereus is one of the most well studied Bacillus subspecies and is recognized as
the flagship species for this genus. It is described as a thermotolerant, mesophilic, Gram-positive,
spore-forming bacteria. Some species of Bacillus are capable of growing at normal refrigeration
temperatures below 7°C, but ideal normal growth temperatures for B. cereus range from 25-37
°C giving this species the mesophilic classification. The production of heat resistant spores
allows B. cereus to survive high temperatures making this species thermo tolerant even though
the vegetative cells are not. Overall, the variety of subspecies within the Bacillus genus have
given a possible growth temperature range between 3 °C and 50°C. In humans, B. cereus causes
several illnesses via toxin production. This includes enterotoxin production, which causes
diarrheal syndrome and emetic syndrome. B. cereus infections can also manifest as
endophthalmitis, causing severe inflammation of the eyes. Some strains have been shown to
present psychrotrophic properties. (Montanhini et al. 2014).
Bacillus cereus is particularly problematic in the food industry due to the production of
both lipase and protease enzymes. These extracellular enzyme secretions decreases the yield and
quality of dairy products and causes the "sweet-curdling" of milk (Jay, 2000). Proteolytic
organisms cause destabilization of casein micelles causing the curdling effect. Mesophilic and
psychrotrophic gene signatures have been found to exist simultaneously in certain B. cereus
strains showing the diversity of bacteria even within a single genus or species. This contributes
to the overall stability of this species across differing environments.
A variety of other spore-forming UHT resistant Bacillus species have also been
identified, but appear in lower percentages of tested milk compared to B. Cereus, which is
present in as much as 22.5% ofUHT milk. These include, B. pumilis, B. subtilis, Brevibacillus
brevis, B. licheniformis, and B. sphaericus. Each of these species is present in fewer than 12% of
10
milk samples (Aouadhi et al. 2014). Paenibacillus lactis and Geobacillus stearothermophilus are
two more bacteria found in UHT milk, but are less concerning as they have only been measured
at low CFUs in fewer than 10% of dairy samples. Of the species shown to be problematic, the
majority are mesophilic, not psychrotrophic, which would be suspected for bacteria growing in
cold-stored foods. The International Dairy Federation designates a psychrotrophic species
classification to bacteria that grow at or below 7 °C regardless of optimum growth temp. B.
cereus for example does not grow at this temperature therefore considered mesophilic. Typically,
mesophilic bacteria are more prevalent and problematic to food borne illness and food spoilage
compared to other bacterial classifications.
Biofilm Control
Biofilms have grown in significance since their discovery and are linked to as many as 10
million nosocomial infections from implants and intrusive surgery each year in the United States
alone. The growing concern over biofilm-related illnesses acquired from hospitals and the food
industry has caused biofilm control to become "big business." The dairy industry, along with
other food industries, uses sizeable financial resources to prevent biofilm growth. Transportation
equipment, such as piping and containers, and productions surfaces made from stainless steel are
treated with antimicrobials capable of eliminating existing biofilms and preventing the adherence
of new growth. New antimicrobials are in research and development stages and show promising
results as control agents (Madigan et al. 2012).
A class of chemicals called furanones has demonstrated antimicrobial properties that
have successfully reduced or eliminated biofilm growth on rigid surfaces. F202, a synthetically
produced brominated furanone, was shown to effectively inhibit biofilm growth in E. coli
0103 :H2 and Salmonella serovar Agona, both of which are recognized as human pathogens
11
found in contaminated food products. This specific furanone inhibits flagellar function, resulting
in bacteria remaining in a planktonic stage, rather than adhered to a surface in a biofilm stage.
For an agent such as F202 to be useful as a food industry antimicrobial it must be able to inhibit
bacterial species under normal food preparation and storage conditions. F202 is capable of
causing flagellar inhibition under these conditions, but more studies must be conducted before
this chemical can be marketed as a safe and effective method of biofilm control (Vestby et al.
2013).
Certain genes have been linked to biofilm formation and are possible targets for biofilm
control. As stated, in order for biofilms to form bacteria must use extracell ular appendages to
adhere to a surface. These appendages are encoded in bacterial DNA and their usage can be
regulated via operons or other regulation factors triggered by environmental conditions. For
example, the cupD gene located on a pathogenicity isJand PAPI-l in Pseudomonas aeruginosa
facilities fimbriae production, a major factor in biofilm attachment. Targeting and altering
(turning off) specific genes, such as cupD, can indirectly inhibit biofilm growth. The difficulty in
this method of biofilm control is finding chemical agents capable of substantially inhibiting
virulence factors, while remaining safe for human consumption (Mikkelsen et al. 2013).
Operons in Methicillin-resistant Staphylococcus aureus (MRSA) have also been
identified as components of virulence regulation that can be targeted to inhibit biofilm growth as
biofilm control. Operons in MRSA and many other species control the expression of virulence
genes functioning as an on-off mechanism. Biofilm development is shown to be affected by the
msaC gene - a part of the four-gene operon msaABCR. By targeting the msaC gene and
inhibiting its functionality, the operon ceases to function and biofilm production stops. This
12
method demonstrates another potential approach for biofilm control by dismantling operons
directly used in virulence and extracellular secretion (sahukhal et at. 2014).
Although some control methods have shown to significantly decrease biofilm growth
very few have been accepted as safe and approved for use in the food industry. Currently, well­
studied sanitization methods incorporating known cleansers and sanitizers are used while more
modem control methods continue to be studied and tested for approval. Further research is
needed in order for the food industry to keep up with continuously evolving pathogens.
Materials and Methods: Quantifying the Effects of SIC of Carvacrol on Biofilm Production
5 mL tryptic soy broth (TSB) cultures of B. amyloliquefaciens, B. amyloliquefaciens
stressed by SIC of carvacrol (1 millimolar), B. cereus, and B. cereus stressed by SIC of
carvacrol were grown and subcultured every 72 hours and incubated on a shaker at 150 rpm and
37 DC for 10 weeks. Fresh TSB media originating from a single batch was used to maintain
consistency in each subculture.
Crystal Violet - Microtiter Plate Assay Protocol for Bacillus ssp.
Growth:
1. Grow a culture of the Bacillus subspecies overnight in a rich medium, 5ml TSB.
2. Standardize subcultures by optical density using spectrophotometry at 600nm - TSB as a
blank.
3. Add 100 ilL of each standardized culture to the first wells of 4 designated rows on a 96 well
microtiter plate - one row for each test group.
4. Add 50
!!L of sterile saJine to wells 2 through
12 in each row.
13
5. Serially dilute the OD standardized wells, taking 50 /-LL from the first well and adding it to the
next and so forth (1:2 dilution).
6. Cover the microtiter plate in paraffin paper.
7. Incubate the microtiter plate for 7+ days at 25°C.
8. Check the plate regularly, adding sterile saline as needed.
Stain:
1. After incubation, dump out well contents by inverting the plate and shaking.
2. To reduce background staining rinse wells with water. Remove well
contents again via shaking. Repeat the washing step at least once more to prevent
background staining.
3. Add 125 /-LL of 0.1 % CV solution in water to each well of the microtiter plate.
4. Incubate the plate for 15 min at 25°C.
5. Again rinse the plate at least twice using the same method previously described. Blot dry
on paper towel.
6. Turn the microtiter plate upside-down and dry for several hours at 25°C.
Qualitative Analysis:
1. After the plate has dried overnight cut between wells.
2. Different dilutions can be compared qualitatively based on the strength/depth of CV
staining.
Note - Different species may form biofilms at the bottom of the microtiter wells or along
the air-medium boundary. uantitative Anal sis: 1. To solubilize the CV, add 125 /-LL of 30% acetic acid in water to each well of the microtiter
14
plate.
2. Incubate the microtiter plate at room temperature for 15 min .
3. Measure absorbance in a plate reader at 595 nm using 30% acetic acid in water as the
blank.
4. The concentration of CV per well is proportional to the number of cells in the biofilm
(O'Tool 2011; Yadav et al. 2013; Darouiche et al. 2010; Mizrahi et al. 2014; Burm¢lle et al.
2007; Leboffe et al. 2005)
Results and Discussion
Interval Plot of A595 vs Treatment
95% C I for the Mean
0.35
0.30
0.25
0.20
LI\
en
LI\
c(
0.15
0.10
0.05
0.00
B.a. TSB
B.a.+C
B.c. TSB
B.c.+C
Treatment
The pooled standard deviation was used to calculate the intervals.
FIj!.UJ"1: I: An intcnal plot of mean absorbllDce lit 595nm ror each treatment group -lion-stressed B. amylo/iquejaclem in
TS8,. Ie of can'acro\ B. amy/oilqllejocien ,non-stre. sed B. Cerel/.f ill T. 0, lind 81 flf CIIM'lIcrol B. cerew'.
15
It was hypothesized that under SIC of carvacrol, stressed Bacillus ssp. would exhibit
increased biofilm production as a result of the up-regulation of virulence genes. However, the
results of this experiment are inconsistent with this hypothesis and suggest that genes for biofilm
production are down-regulated under SIC of carvacrol stressed conditions. It has been shown in
previous experiments that genes for capsule production, PIeR, are up-regulated under these
conditions, which would suggest the possibility that these two known virulence factors are
contained on two separate regulons within both B. cereus, and B. amyloliquefaciens genomes .
These findings are important as they support the use of carvacrol as an agent used for limiting
biofilm production in the dairy industry.
A one-way ANOVA was used to determine the significance of this data . The ANOVA
showed a value of p
< 0.001 -
there was a significant difference in the means for each treatment
group (Table 1). This demonstrates the substantial effect of carvacrol on biofilm production in
both species. Biofilm formation in B. amyloliquefaciens was most clearly inhibited by carvacrol
treatment, but B. cereus shows little change.
N
Mean
StDev
95% CI
P-Value
Pooled StDev
B.a. TSB
3
0 .2763
0.0055
( 0.2329, 0.3198)
0.000
0.03263
B.a.+C
3
0.0430
0.0020
( -0 .0005, 0.0865)
B.c. TSB
B.c.+C
3
3
02923
0.2090
0.0181
0.0624
( 0.2489,0.3358)
( 0.1655, 02525)
Treatment
TlIllle I: Slatio,ticaillnal)~i, rrom a onc-wll~ A:-.IUVA cumparing the mean ab,orbllncc of Bacillul' treatment group, - Don­
,tre. sed B. amy/v/iqlJe/acien in TS8. SIC or carvacrol B. omy/o/iqlJe/acielll, non-. tre... ~ 'd B. cerell.1 in TSB, .md SIC or
canacrnl D. cereus.
16
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