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

Abstract

r

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!

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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

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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

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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

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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

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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

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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).

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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

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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

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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.

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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.+C B.c. TSB B.c.+C

Treatment

The pooled standard deviation was used to calculate the intervals.

B.a. TSB

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.

Treatment 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 3 02923 0.0181 ( 0.2489,0.3358)

B.c.+C 3 0.2090 0.0624 ( 0.1655, 02525)

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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