Listeria monocytogenes is problematic for the manufacturing, storage, and consumption of ready-to-eat foods. This bacterium may cause listeriosis upon consumption often with deadly complications. Queso Fresco (QF), a pasteurized Hispanic-style fresh cheese (HSFC), has been shown to support the growth of L. monocytogenes. QF is the most widely produced and implicated HSFC in the U.S., representing a significant health hazard to at risk populations such as infants, pregnant women, elderly, and the immunocompromised. The objective of this research project is to evaluate the efficacy of ten novel antimicrobial endolysins in combatting L. monocytogenes contamination in a miniaturized lab-scale queso fresco model over a 28 day shelf life

Holle, Maxwell J.

Miller, Dr. Michael

Smith, Daniella

English

Scholarship at Parkland

ObjectiveTo evaluate the efficacy of ten novel antimicrobial endolysins in combatting L. monocytogenescontamination in a miniaturized lab-scale queso fresco model over a 28 day shelf life. Daniella Smith1, Maxwell J. Holle2, and Dr. Michael Miller2Pikes Peak Community College, Colorado Springs, Colorado1Department of Food Science & Human Nutrition, College of ACES, University of Illinois at Urbana-Champaign2Comparative Efficacy of Novel Endolysins in Queso FrescoConclusionThis research suggests that P118 was the most successful at inhibiting the growth of L. monocytogenes in QF.  Although all of the cheeses were plated in duplicate, the accuracy for days 3 and 7 is limited due to there being only a single biological replicate as apposed to day 14 which contains two. In addition, PlyP PSA is shown to have inhibited growth on day 3, followed by a lack of inhibition at days 7 and promotion of bacterial growth on day 14. This result may be caused by an outlier in one of the biological replicates on day 14 possibly indicating an excessive inoculation of L. monocytogenes, deficient amount of PlyP PSA, or user error in spiral plating. The differences observed among the endolysins could be explained by differences in their respective lytic spectrums.Future WorkFurther research needs to be performed to determine the efficacy of the proposed endolysins in QF.  Additionally, optimization of the endolysin concentrations should be determined for the most economical and industrial application. This may include combining endolysins to be used in tandem to evaluate synergy. These endolysins may also further lend themselves as antimicrobials to combat L. monocytogenes in other foods while maintaining quality structure and flavor.ProblemListeria monocytogenes is problematic for the manufacturing, storage, and consumption of ready-to-eat foods. This bacterium may cause listeriosis upon consumption often with deadly complications. QuesoFresco (QF), a pasteurized Hispanic-style fresh cheese (HSFC), has been shown to support the growth of L. monocytogenes. QF is the most widely produced and implicated HSFC in the U.S., representing a significant health hazard to at risk populations such as infants, pregnant women, elderly, and the immunocompromised. Approximately 16 to 27% of all L. monocytogenes infections occur in pregnant women, making them 18 times more likely to contract listeriosis, often resulting in lethal complications for the mother and/or child.1Methods1. Production of Queso Fresco2. Application of experimental treatmentsEach cheese was enumerated by spiral plating.Rennet and CaCl2  were added to warm milk (35ºC).After incubation, the curds were cut with sterile needle and cooked again. The milk was divided into 1 mL batches.The whey was poured off of each curd, NaCl was added, and the batches were cooked a final time. The curds were inoculated with L. monocytogenes and 2.5 U/mg of each endolysin was added. PlyP100 showed the greatest percent reduction on day 3 at 81.90%±5.87 P118 demonstrated the highest percentage reduction at 14 days, with a 97.28%±9.52 reduction on day 7 and a 95.34%±11.41reduction on day 14.IntroductionListeria monocytogenes is a gram-positive foodborne pathogen that may form biofilms within food processing facilities and in domestic refrigerators. L. monocytogenescan survive for years under refrigerated storage and high salt contents regardless of frequent sanitation. The FDA has declared L. monocytogenes a zero-tolerance organism—completely unfit for human consumption, that cannot be present, at any level, in ready-to-eat foods.2Queso Fresco is a soft HSFC most commonly associated with foodborne disease outbreaks. QF is particularly susceptible to L. monocytogenescontamination due to a relatively low acid content, high water activity, and near neutral pH. Typically, pasteurization is an effective process for eliminating L. monocytogenes from the raw ingredients prior to cheesemaking. However, despite advances in food processing technology no suitable solution for post processing contamination has been discovered.3Endolysins are enzymes derived from bacteriophages that hydrolyze bacterial cell walls leading to bacterial cell death.3  Endolysins maintain their lytic activity when applied exogenously and have a specific lytic spectrum that only target bacteria within a certain genus. Unlike traditional antimicrobials, bacteria are incapable of developing antimicrobial resistance to endolysins.4 In addition to the aforementioned properties, endolysins are desirable antimicrobials since they will not inadvertently affect the food or consumer’s microbiome. AcknowledgmentsFinancial support was provided by the National Science Foundation under grant #NSF REU 1559908/1559929, as part of the Phenotypic Plasticity Research Experience for Community College Students, through the University of Illinois at Urbana-Champaign Institute for Genomic Biology and Parkland College. http://precs.igb.illinois.edu/I would like to thank my research mentor Maxwell Holle and faculty mentor Dr. Michael Miller for volunteering their time and energy to mentor me over the summer. Thank you to the Miller Lab for their patience and support. Thank you to the project PIs Dr. Nathan Schroeder and Dr. C. Britt Carlson and to support staff at the Institute for Genomic Biology for providing me with this outstanding opportunity.  Finally, thank you to my colleges and friends in the program for the encouragement and seeing me through to success. ResultsHighest Percent ReductionLowest Percent Reduction P35 demonstrated the lowest percent reduction on day 3 at 35.15%±5.87.  PlyP PSA showed the lowest percent reduction on both day 3 at 8.42%±5.87, and day 14 at -14.78%±11.41.References1. Lamont RF, Sobel J, Mazaki-Tovi S, et al. Listeriosis in human pregnancy: a 2018). Microbrial analysis of commercially available US Queso Fresco. Josystematic review. J Perinat Med 2011;39:227-2362. FDA Committee Calls for Policy Changes Regarding Listeria Monocytogenes. (2015, December 16). Retrieved June/July, 2019, from https://www.idfa.org/news-views/headline-news/article/2015/12/16/fda-committee-calls-for-policy-changes-regarding-listeria-monocytogenes3. Holle, M. J. et al.  (urnal of Dairy Science,101(9). doi:https://doi.org/10.3168/jds.2017-140374. Fischetti, V.A. 2005. Bacteriophage lytic enzymes: Novel anti-infectives. Trends Micribial. 13:491-496-30.00-10.0010.0030.0050.0070.0090.00110.00P118 P35 P40 P500 P70 PlyP BO25 PlyP100 PlyP PSAPercent ReductionEndolysinsDay 3 Day 7 Day 14Figure 1. L.monocytogenes cell counts were determined in Queso Fresco treated with 10 different endolysins, stored at 4°C, after days 3, 7, and 14 . The percent reduction was calculated by comparing the resulting colonies/mL to the control cheese.  Days 3 & 7 were done in duplicate while day 14 results were measured in duplicate across two biological replicates. Results means with ± SE

Comparative Efficacy of Novel Endolysins in Queso Fresco

SPARK: Scholarship at Parkland | Parkland College

Objective
To evaluate the efficacy of ten novel antimicrobial 
endolysins in combatting L. monocytogenes
contamination in a miniaturized lab-scale queso fresco 
model over a 28 day shelf life. 
Daniella Smith1, Maxwell J. Holle2, and Dr. Michael Miller2
Pikes Peak Community College, Colorado Springs, Colorado1
Department of Food Science & Human Nutrition, College of ACES, University of Illinois at Urbana-Champaign2
Comparative Efficacy of Novel Endolysins in Queso Fresco
Conclusion
This research suggests that P118 was the most 
successful at inhibiting the growth of L. monocytogenes in 
QF.  Although all of the cheeses were plated in duplicate, 
the accuracy for days 3 and 7 is limited due to there 
being only a single biological replicate as apposed to day 
14 which contains two. In addition, PlyP PSA is shown to 
have inhibited growth on day 3, followed by a lack of 
inhibition at days 7 and promotion of bacterial growth on 
day 14. This result may be caused by an outlier in one of 
the biological replicates on day 14 possibly indicating an 
excessive inoculation of L. monocytogenes, deficient 
amount of PlyP PSA, or user error in spiral plating. The 
differences observed among the endolysins could be 
explained by differences in their respective lytic 
spectrums.
Future Work
Further research needs to be performed to determine the 
efficacy of the proposed endolysins in QF.  Additionally, 
optimization of the endolysin concentrations should be 
determined for the most economical and industrial 
application. This may include combining endolysins to be 
used in tandem to evaluate synergy. These endolysins 
may also further lend themselves as antimicrobials to 
combat L. monocytogenes in other foods while 
maintaining quality structure and flavor.
Problem
Listeria monocytogenes is problematic for the 
manufacturing, storage, and consumption of ready-to-eat 
foods. This bacterium may cause listeriosis upon 
consumption often with deadly complications. Queso
Fresco (QF), a pasteurized Hispanic-style fresh cheese 
(HSFC), has been shown to support the growth of L. 
monocytogenes. QF is the most widely produced and 
implicated HSFC in the U.S., representing a significant 
health hazard to at risk populations such as infants, 
pregnant women, elderly, and the immunocompromised. 
Approximately 16 to 27% of all L. monocytogenes 
infections occur in pregnant women, making them 18 
times more likely to contract listeriosis, often resulting in 
lethal complications for the mother and/or child.1
Methods
1. Production of Queso Fresco
2. Application of experimental treatments
Each cheese was enumerated by 
spiral plating.
Rennet and CaCl2  were 
added to warm milk 
(35ºC).
After incubation, the curds were 
cut with sterile needle and 
cooked again. 
The milk was divided into 1 mL 
batches.
The whey was poured off of 
each curd, NaCl was added, 
and the batches were 
cooked a final time. 
The curds were inoculated with L. 
monocytogenes and 2.5 U/mg of 
each endolysin was added.
 PlyP100 showed the greatest 
percent reduction on day 3 at 
81.90%±5.87
 P118 demonstrated the 
highest percentage reduction 
at 14 days, with a 
97.28%±9.52 reduction on 
day 7 and a 95.34%±11.41
reduction on day 14.
Introduction
Listeria monocytogenes is a gram-positive foodborne 
pathogen that may form biofilms within food processing 
facilities and in domestic refrigerators. L. monocytogenes
can survive for years under refrigerated storage and high 
salt contents regardless of frequent sanitation. The FDA 
has declared L. monocytogenes a zero-tolerance 
organism—completely unfit for human consumption, that 
cannot be present, at any level, in ready-to-eat foods.2
Queso Fresco is a soft HSFC most commonly 
associated with foodborne disease outbreaks. QF is 
particularly susceptible to L. monocytogenes
contamination due to a relatively low acid content, high 
water activity, and near neutral pH. Typically, 
pasteurization is an effective process for eliminating L. 
monocytogenes from the raw ingredients prior to 
cheesemaking. However, despite advances in food 
processing technology no suitable solution for post 
processing contamination has been discovered.3
Endolysins are enzymes derived from bacteriophages 
that hydrolyze bacterial cell walls leading to bacterial cell 
death.3  Endolysins maintain their lytic activity when 
applied exogenously and have a specific lytic spectrum 
that only target bacteria within a certain genus. Unlike 
traditional antimicrobials, bacteria are incapable of 
developing antimicrobial resistance to endolysins.4 In 
addition to the aforementioned properties, endolysins are 
desirable antimicrobials since they will not inadvertently 
affect the food or consumer’s microbiome. 
Acknowledgments
Financial support was provided by the National Science Foundation 
under grant #NSF REU 1559908/1559929, as part of the Phenotypic 
Plasticity Research Experience for Community College Students, through 
the University of Illinois at Urbana-Champaign Institute for Genomic 
Biology and Parkland College. http://precs.igb.illinois.edu/
I would like to thank my research mentor Maxwell Holle and faculty 
mentor Dr. Michael Miller for volunteering their time and energy to mentor 
me over the summer. Thank you to the Miller Lab for their patience and 
support. Thank you to the project PIs Dr. Nathan Schroeder and Dr. C. 
Britt Carlson and to support staff at the Institute for Genomic Biology for 
providing me with this outstanding opportunity.  Finally, thank 
you to my colleges and friends in the program for the 
encouragement and seeing me through to success. 
Results
Highest Percent Reduction
Lowest Percent Reduction
 P35 demonstrated the lowest 
percent reduction on day 3 at 
35.15%±5.87. 
 PlyP PSA showed the lowest 
percent reduction on both day 3 
at 8.42%±5.87, and day 14 at 
-14.78%±11.41.
References
1. Lamont RF, Sobel J, Mazaki-Tovi S, et al. Listeriosis in human 
pregnancy: a 2018). Microbrial analysis of commercially available US 
Queso Fresco. Josystematic review. J Perinat Med 2011;39:227-236
2. FDA Committee Calls for Policy Changes Regarding Listeria 
Monocytogenes. (2015, December 16). Retrieved June/July, 2019, 
from https://www.idfa.org/news-views/headline-
news/article/2015/12/16/fda-committee-calls-for-policy-changes-
regarding-listeria-monocytogenes
3. Holle, M. J. et al.  (urnal of Dairy Science,101(9). 
doi:https://doi.org/10.3168/jds.2017-14037
4. Fischetti, V.A. 2005. Bacteriophage lytic enzymes: Novel anti-
infectives. Trends Micribial. 13:491-496
-30.00
-10.00
10.00
30.00
50.00
70.00
90.00
110.00
P118 P35 P40 P500 P70 PlyP BO25 PlyP100 PlyP PSA
P
e
rc
e
n
t 
R
e
d
u
c
ti
o
n
Endolysins
Day 3 Day 7 Day 14
Figure 1. L.monocytogenes cell counts were determined in Queso Fresco treated with 10 different endolysins, stored 
at 4°C, after days 3, 7, and 14 . The percent reduction was calculated by comparing the resulting colonies/mL to 
the control cheese.  Days 3 & 7 were done in duplicate while day 14 results were measured in duplicate across two 
biological replicates. Results means with ± SE


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Comparative Efficacy of Novel Endolysins in Queso Fresco

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