Food Control 20 (2009) 1141–1144

Contents lists available at ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Effect of weak acids on Listeria monocytogenes survival: Evidence for a viable

but nonculturable state in response to low pH
Eilís Cunningham a, Conor O’Byrne a, James D. Oliver b,*
a
National University of Ireland, Galway, Ireland
b
University of North Carolina at Charlotte, Department of Biology, 9201 University City Blvd, Charlotte, NC 28223, USA

a r t i c l e i n f o a b s t r a c t

Article history: Listeria monocytogenes is capable of surviving environmental conditions that are normally fatal to many
Received 21 January 2009 other bacteria, enabling this pathogen to remain on foods and eventually establish infection after con-
Received in revised form 3 March 2009 sumption. This study investigated the extent to which L. monocytogenes is killed by several weak acids,
Accepted 18 March 2009
with particular emphasis on the commonly used food preservative, potassium sorbate. The effects of
potassium sorbate were found to be highly pH dependant, with significant killing only observed at a
pH of 4.0. At this pH, the order of effectiveness of the acids tested was Na benzoate > Na propionate > ace-
Keywords:
tic acid > K sorbate. Temperature had a significant impact on sensitivity, with K sorbate being effective at
Listeria
pH
50 mM and elevated (37 °C) temperature, but failing to affect survival when cells were at 4 °C or 21 °C.
Culturability Finally, we found that cells grown in the presence of K sorbate at pH 4.0 entered a viable but noncultur-
VBNC able (‘‘VBNC”) state, but became nonviable by 24 h. In contrast, cells incubated under these conditions in
Acid the absence of K sorbate entered the VBNC state and maintained viability throughout the 24 h study
Benzoate period.
Sorbate Ó 2009 Elsevier Ltd. All rights reserved.
Propionate
Acetic

1. Introduction
Listeria monocytogenes is a robust and resistant Gram-positive
pathogen which is capable of causing life-threatening diseases
including listeriosis, meningitis, septicaemia, and cervical or in-
tra-uterine infections in pregnant women, which result in sponta-
neous abortion or stillbirth (for a review of this pathogen, see
Cossart, 2007). The mortality rate amongst infected individuals is
typically 30%, making it the second most deadly food-borne path-
ogen in the US, with similar rates in Ireland and throughout Europe
(Lorber, 1997; Rocourt & Bille, 1997). Persons most at-risk of infec-
tion include pregnant women, immunocompromised individuals
such as AIDS sufferers, the elderly and cancer patients, although
in Europe a significant increase in listeriosis in persons P 60 years
of age has recently been reported (Goulet, Hedberg, Le Monnier, &
de Valk, 2008). Foods most commonly infected with L. monocytog-
enes, and thus of greatest concern to at-risk persons, are raw
milk, cheeses (especially soft-ripened varieties), raw vegetables,
raw-meat and fermented raw-meat sausages (Swaminathan,
Cabans, Zhang, & Cossart, 2007).
L. monocytogenes is able to survive a variety of environmental
stresses, such as high salt, low pH and low temperature, attributes
* Corresponding author. Tel.: +1 704 687 8516; fax: +1 26 327 2083.
E-mail address: jdoliver@uncc.edu (J.D. Oliver).
that allow the pathogen to survive in foods which have been trea-
ted to enhance preservation. Because of these traits, and its signif-
icant public health significance, the presence of this organism in
foods is of critical concern to the food industry. The studies pre-
sented here focused on the response of L. monocytogenes to several
of the weak acids that are commonly used in food preservation to
inhibit bacterial growth. Weak acids can be associated (uncharged)
or dissociated (charged), depending on the pKa of their acidic
group and the pH of the environment surrounding it. At low pH,
weak acids are more likely to be in the undissociated (uncharged)
state, allowing them to pass freely across the lipid membrane into
the cytoplasm where their inhibitory effects are manifested (Fos-
ter, 1999; Hirshfield, Terzullik, & O’Byrne, 2003). At pH values
above an acid’s pKa, weak acids remain in the dissociated (charged)
form. In this state, these acids are not lipid permeable and thus
have significantly less effect on microbial survival. The food indus-
try typically uses such weak acids as acetic, propionic, sorbic, lactic
and benzoic acids as preservatives (Naidu, 2000). For example,
cheese and cheese products generally contain a sorbic acid content
typically in the 1–10 mM range to prevent microbial growth
(Tfouni & Toledo, 2002).
Another aspect of our studies was whether the decreases in
culturability generally reported when this pathogen is exposed to
weak acids may not be due to cell death but to entry of L. monocyt-
ogenes into a ‘‘viable but non culturable” (VBNC) state. Many

0956-7135/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2009.03.005
1142 E. Cunningham et al. / Food Control 20 (2009) 1141–1144

bacteria are known to respond to stresses by entering this physio- 100

logical state, in which they still exhibit metabolic activity but can 10
no longer be cultured on routine media (Oliver, 2005a). The pres-
1
ence of pathogens in foods while in the VBNC state is of obvious
concern to the food industry as such cells are not detected when 0.1

% Survival
routine plating methods to determine food contamination are em-
0.01
ployed (Oliver, 2005b).
0.001

2. Methods 0.0001

0.00001
2.1. Bacteria and culture conditions
0.000001
Potassium Sodium Sodium Acetic Acid Contol
L. monocytogenes 10403S, serotype 1/2a, was routinely grown Sorbate Proprionate Benzoate
overnight in brain heart infusion (BHI, Difco) broth at 37 °C with
gentle agitation. Fig. 1. Survival of L. monocytogenes in the presence of different acids. Shown are the
percent survival of cells following treatment with selected weak acids at a final
concentration of 25 mM at pH 4.0. Percent survival after 5 h was calculated based
2.2. Effects of weak acids on culturability on the culturability of cells compared to control cells to which no weak acid was
added, set at 100%) incubated at pH 4.0.
Log phase cells were diluted into fresh BHI at 37 °C that had
been adjusted to pH 4.0 with HCl and contained the weak acid to
be tested. The effects on survival of L. monocytogenes of 0, 5, 10, ogenes following 5 h of exposure to the four acids at 25 mM and a
25, 50 and 100 mM concentrations (final concentration, v/v) of ace- pH of 4.0. At this concentration, the order of effectiveness was Na
tic acid (BDH), potassium sorbate, sodium propionate, and sodium benzoate > Na propionate > acetic acid > K sorbate. If killing was
benzoate (Sigma–Aldrich) were examined at this same tempera- exclusively a function of the amount of undissociated weak acid
ture. Aliquots were removed at 0, 1, 5, and 24 h and examined in the medium at pH 4.0 then the order of killing would be ex-
for culturability on BHI agar. pected to be PA > SA > AA > BA, based on the known pKa values
for these weak acids (4.9, 4.8, 4.7 and 4.2, respectively). The finding
2.3. Effect of pH on potassium sorbate killing that BA was the most lethal weak acid in this test suggests that its
lethal effect is probably due to effects other than simply reducing
Separate samples of log phase cells were diluted into BHI broth the intracellular pH.
containing 25 mM K sorbate and adjusted to pH 4, 5, 6, 7 or 8. Cells Fig. 2 shows the response of L. monocytogenes to 50 mM K sor-
were incubated at 37 °C and aliquots removed and their culturabil- bate and different pH levels. It is clear that, at a pH of 4.0, cells lost
ity assayed at intervals. significant culturability over a 24 h period. However, at pH 5.0 the
effect of K sorbate was negligible, and at higher pH values, the cells
2.4. Effect of temperature on potassium sorbate killing were capable of growth in the presence of this additive. Thus, the
effect of K sorbate on the survival of L. monocytogenes appears to
Log phase cells were diluted in BHI and examined for sensitivity be highly pH dependant.
to K sorbate (50 mM) at temperatures of 4 °C, 21 °C and 37 °C.
Culturability was assayed at 0, 2, 4, 7, and 24 h.
pH 4 pH7
2.5. Entrance of sorbate-treated cells into the VBNC state
pH5 pH8
Log phase cells in BHI broth at pH 4.0 and 37 °C were treated
pH6
with K sorbate (0, 5, 10, 25, 50, and 100 mM). At 2, 4, 7, and
24 h, cells were monitored for culturability and examined for via-
bility using a direct microscopic assay (BacLight Live/Dead; Invitro- 10
10
gen Paisley, UK). This assay utilizes SYTO 9 and propidium iodide 9
to discriminate between live cells with intact membranes (green 10
fluorescence) and dead cells with compromised membranes (red 8
10
fluorescence). Stained samples were filtered onto black carbonate
7
filters and viewed using epifluorescence microscopy. A minimum 10
CFU/ml

of 300 cells or 30 fields was counted, and total viable cell numbers 6
10
were determined as we have previously described (Smith & Oliver,
5
2006). 10
4
10
3. Results
3
10
In order to know when the cells had entered into log phase, at 2
10
which time we wished to examine the effects of the various acids,
1
we first conducted a growth study for this strain of L. monocytoge- 10
nes in BHI at 37 °C. Based on these results, cells for the acid sensi- 0 5 10 15 20 25
tivity studies were routinely removed at log phase (OD600 0.5, ca.
2  109 CFU/ml), and inoculated into fresh BHI containing the acid
Incubation time (h)
and pH to be tested. Fig. 2. Effect of pH on K sorbate killing. Cells were grown in BHI broth adjusted to
We examined four different weak acid preservatives at concen- pH values of 4–8, in the presence of 50 mM K sorbate. Culturability following
trations from 5 to 100 mM. Fig. 1 shows the survival of L. monocyt- treatment for various periods was monitored by growth on BHI agar.
 E. Cunningham et al. / Food Control 20 (2009) 1141–1144 1143

was present or not. At 37 °C, however, incubation at a pH of 4.0

4 ° C 0mM 21 ° C 50mM
caused loss of culturability, which was accentuated by the pres-
4 ° C 50mm 37 ° C 0mm ence of K sorbate, and no surviving cells were detectable by 24 h.
21 ° C 0mm This result suggests that survival of L. monocytogenes at low pH val-
37 ° C 50mM
ues is enhanced at reduced temperatures, regardless of whether or
8 not sorbate is present in the medium.
10 When cells were treated with 50 mM K sorbate at pH 4.0 and
7 37 °C, culturability decreased >99.9% within 7 h (Fig. 4A). However,
10
the cells remained fully viable during this time, as determined by
6 the Live/Dead direct viability assay, indicating that L. monocytoge-
10
nes had entered a VBNC state. However, by 24 h culturability was
CFU/ml

5
10 lost (6102 CFU/ml), and viability counts also decreased to unde-
4 tectable levels, indicating that K sorbate had killed the bacterium.
10 At pH 4.0 but in the absence of K sorbate (Fig. 4B), L. monocytogenes
3 cells underwent a fairly linear decrease in culturability, to a final
10
6102 CFU/ml at 24 h, but remained fully viable, indicating that
2 while the bacterium appeared to have been killed based on plate
10
counts, they were still viable at >106 cells/ml.
1
10
0 5 10 15 20 25 4. Discussion
Incubation time (h)
The culturability of L. monocytogenes was affected differently by
Fig. 3. Effect of temperature on the killing efficiency K sorbate. Cells were grown in the different weak acids we examined (Fig. 1), with Na benzoate
BHI broth, pH 4.0, at 4 °C, 21 °C, and 37 °C, and in the presence or absence of being the most effective preservative under the conditions em-
50 mM K sorbate. Culturability following treatment for various periods was
ployed (pH 4.0 and 37 °C). We selected K sorbate for further exam-
monitored by growth on BHI agar. Down arrow indicates culturability below the
level of detection. ination, however, due to its widespread use in the food industry
(Sofos, 2000; Sofos & Busta, 1981).
The effect of K sorbate on L. monocytogenes was found to be
highly pH dependant (Fig. 2), with cell inhibition only observed
A 10 8
at low pH. Indeed, this weak acid had little effect on the cells at
10 7 pH 5, and above this pH the bacterium was capable of normal
growth. This is a direct result of the pKa (dissociation constant)
10 6
Cell number/ml

of K sorbate, which is 4.76 [pKa is a pH-independent property]

10 5 but 0.17 at a pH of 4.0. This finding has relevance to the food indus-
try as many weak acids are used as preservatives, but may totally
10 4 lose effectiveness depending on the environmental (e.g. pH)
conditions.
10 3 CFU
As shown in Fig. 3, the potentially lethal effects of incubation at
total
10 2 viable
pH 4.0 were negated at 4 °C and 21 °C. Further, at these tempera-
tures the addition of 50 mM K sorbate had no apparent detrimental
10 1 effects. It is well documented (Cole, Jones, & Holyoak, 1990; Walk-
0 5 10 15 20 25 er, Archer, & Banks, 1990) that L. monocytogenes is capable of grow-
Incubation time (h) ing at refrigeration temperatures, but our results suggest K sorbate
may have little to no preservative value under these conditions. K
B 10 8 sorbate at a concentration of 50 mM was found to be bactericidal
for L. monocytogenes at 37 °C. However most household foods are
10 7 stored at refrigerator or room temperatures at which this preserva-
tive is not likely to cause death of this pathogen. The mechanisms
Cell number/ml

10 6
for this observation are not known, but might result from a change
CFU
10 5 in membrane lipid composition at reduced temperatures (Farkas,
total 2007; Phadtare, Yamanaka, & Inouye, 2000) that might influence
10 4 viable diffusion rates into the cell. This could also be a consequence of
10 3 the cold-shock response that results in some degree of cross-pro-
tection against weak acid and/or low pH stress (Price, 2000;
10 2 Rosche, Bates, Smith, Parker, & Oliver, 2005). Given the induction
of rB by cold stress in L. monocytogenes (Becker, Cetin, Hutkins, &
10 1 Benson, 1998; Chan, Boor, & Wiedmann, 2007), the potential role
0 5 10 15 20 25 30
of this alternate sigma factor in surviving organic acid stress might
Incubation time (h) be especially interesting to examine. Indeed, it is already known
that rB is required by L. monocytogenes for efficient survival of
Fig. 4. Induction of L. monocytogenes into the VBNC state by K sorbate. Cells were
treated at pH 4.0 and 37 °C with K sorbate at 0 mM (A) or 50 mM (B). Culturability
strong acid stress (Ferreira, Sue, O’ Byrne, & Boor, 2003; Wied-
following treatment for various periods was monitored by growth on BHI agar. Cells mann, Arvic, Hurley, & Boor, 1998).
were also examined for total number and for viability (see Section 2). In agreement with our findings, Zuliani et al. (2007) observed
significant decreases in culturability when L. monocytogenes (pres-
As shown in Fig. 3, this pathogen remained fully culturable at a ent in ground pork) was exposed to acetate, lactate, or sorbate at
pH of 4.0 and temperatures of 4 °C and 21 °C, whether K sorbate 20–66 mMol and pH 5.6. These authors did not consider the
1144 E. Cunningham et al. / Food Control 20 (2009) 1141–1144
possibility, however, that the cells may have entered a VBNC state.
In contrast, one of our most interesting findings was that, while
ultimately lethal, L. monocytogenes cells treated with K sorbate
were capable of existing in a VBNC state for several hours. This is
significant in that foods may be presumed to be pathogen free
based on routine culturability assays, but subsequently cause
infection in susceptible individuals consuming the contaminated
foods. In this context it may be an especially important finding that
cells of L. monocytogenes incubated at pH 4.0 remained fully viable,
even when standard plate counts indicated the complete absence
of this pathogen. Very few studies have been reported on the VBNC
state in L. monocytogenes. Dreux and Cossart (2007) demonstrated
entry of this pathogen into the VBNC state when incubated on the
surface of parsley leaves under low relative humidity, and Besnard,
Federighi, Declerq, Jugiau, and Cappelier (2002) found that higher
salt concentrations and incubation temperatures both decreased
the time necessary to enter the VBNC state. Both groups concluded
that the presence of VBNC cells of this pathogen in food could
pose a major public health hazard. While we did not attempt in
this study to resuscitate these cells, several investigators have
reported the successful resuscitation of bacteria from the VBNC
state back to the fully culturable state (reviewed in Oliver,
2005a,b). A determination of conditions permitting resuscitation
of L. monocytogenes in foods should prove especially valuable in
clarifying the potential role of this physiological state in this
important human pathogen.
Acknowledgements
JDO would like to acknowledge the generous support of the
Society for General Microbiology for providing an International Re-
search Grant to permit this collaboration. We also acknowledge the
contribution of Sinéad Heavin for reading drafts of this paper.
References
Becker, L. A., Cetin, M. S., Hutkins, R. W., & Benson, A. K. (1998). Identification of the
gene encoding the alternative sigma factor rB from Listeria monocytogenes and
its role in osmotolerance. Journal of Bacteriology, 180, 4547–4554.
Besnard, V., Federighi, M., Declerq, E., Jugiau, F., & Cappelier, J.-M. (2002).
Environmental and physio-chemical factors induce VBNC state in Listeria
monocytogenes. Veterinary Research, 33, 359–370.
Chan, Y. C., Boor, K. J., & Wiedmann, M. (2007). rB-dependent and rB-independent
mechanisms contribute to transcription of L. monocytogenes cold stress genes
during cold shock and cold growth. Applied and Environmental Microbiology, 73,
6019–6029.
Cole, M. B., Jones, M. V., & Holyoak, C. (1990). The effect of pH, salt concentration
and temperature on the survival and growth of Listeria monocytogenes. Journal
of Applied Bacteriology, 69, 63–72.
Cossart, P. (2007). Listeriology (1926–2007): The rise of a model pathogen. Microbes
and Infection, 9, 1143–1146.
Dreux, N. C., & Cossart, P. (2007). Listeriology (1926–2007): The rise of a model
pathogen. Microbes and Infection, 9, 1143–1146.
Farkas, J. (2007). Physical methods of food preservation. In M. P. Doyle & L. R.
Beuchat (Eds.), Food microbiology fundamentals and frontiers (3rd ed.).
Washington, DC: American Society for Microbiology Press.
Ferreira, A., Sue, D., O’ Byrne, C. P., & Boor, K. J. (2003). Role of Listeria monocytogenes
rB in survival of lethal acidic conditions and in the acquired acid tolerance
response. Applied and Environmental Microbiology, 69, 2692–2698.
Foster, J. W. (1999). When protons attack: Microbial strategies of acid adaptation.
Current Opinion in Microbiology, 2, 170–174.
Goulet, V., Hedberg, C., Le Monnier, A., & de Valk, H. (2008). Increasing incidence of
listeriosis in France and other European countries. Emerging Infectious Diseases,
14, 734–740.
Hirshfield, I. N., Terzullik, S., & O’Byrne, C. (2003). Weak organic acids: A panoply of
effects on bacteria. Science Progress, 86, 245–269.
Lorber, B. (1997). Listeriosis. Clinical Infectious Diseases, 24, 1–11.
Naidu, A. S. (2000). Natural food antimicrobial systems. Boca Raton, FL: CRC Press.
Oliver, J. D. (2005a). The viable but nonculturable state in bacteria. Journal of
Microbiology, 43, 93–100.
Oliver, J. D. (2005b). Viable but nonculturable bacteria in food environments. In P.
M. Fratamico, A. K. Bhunia, & J. L. Smith (Eds.), Food-borne pathogens:
Microbiology and molecular biology (pp. 99–112). Norfolk, UK: Caister
Academic Press.
Phadtare, S., Yamanaka, K., & Inouye, M. (2000). The cold shock response. In G. Storz
& R. Hengge-Aronis (Eds.). Bacterial stress responses (pp. 33–46). Washington,
DC: American Society for Microbiology Press.
Price, C. W. (2000). Protective function and regulation of the general stress response
in Bacillus subtilis and related gram-positive bacteria. In G. Storz & R. Hengge-
Aronis (Eds.), Bacterial stress responses (pp. 179–197). Washington, DC:
American Society for Microbiology Press.
Rocourt, J., & Bille, J. (1997). Foodborne listeriosis. World Health Statistics Quarterly,
50, 67–73.
Rosche, T. M., Bates, T. C., Smith, D. J., Parker, E. E., & Oliver, J. D. (2005). RpoS
involvement in osmotically-induced cross protection in Vibrio vulnificus. FEMS
Microbiology Ecology, 53, 455–462.
Smith, B. E., & Oliver, J. D. (2006). In situ and in vitro gene expression by Vibrio
vulnificus during entry into, persistence within, and resuscitation from the
viable but nonculturable state. Applied and Environmental Microbiology, 72,
1445–1451.
Sofos, J. N. (2000). Sorbic acid. In A. S. Naidu (Ed.), Natural food antimicrobial systems
(pp. 637). Boca Raton, FL: CRC Press.
Sofos, J. N., & Busta, F. F. (1981). Antimicrobial activity of sorbate. Journal of Food
Protection, 44, 614–617.
Swaminathan, B., Cabans, D., Zhang, W., & Cossart, P. (2007). Listeria monocytogenes.
In M. P. Doyle & L. R. Beuchat (Eds.), Food microbiology fundamentals and frontiers
(3rd ed., pp. 457–497). Washington, DC: American Society for Microbiology
Press.
Tfouni, S. A. V., & Toledo, M. C. F. (2002). Determination of benzoic and sorbic acids
in Brazilian food. Food Control, 13, 117–123.
Walker, S. J., Archer, P., & Banks, J. G. (1990). Growth of Listeria monocytogenes at
refrigeration temperatures. Journal of Applied Bacteriology, 68, 15–162.
Wiedmann, M., Arvic, T. J., Hurley, R. J., & Boor, K. J. (1998). General stress
transcription rB factor and its role in acid tolerance and virulence of Listeria
monocytogenes. Journal of Bacteriology, 180, 3650–3656.
Zuliani, V., Lebert, I., Augustin, J.-C., Garry, P., Veneuvre, J.-L., & Lebert, A. (2007).
Modelling the behaviour of Listeria monocytogenes in ground pork as a function
of pH, water activity, nature and concentration of organic acid salts. Journal of
Applied Microbiology, 103, 536–550.