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Unique response characteristics in persistent strains of Listeria monocytogenes exposed to low pH
Food Microbiology 86 (2020) 103312
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Unique response characteristics in persistent strains of Listeria monocytogenes exposed to low pH
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Yoshitsugu Ochiaia,∗, Yuko Yoshikawaa, Mariko Mochizukib, Takashi Takanoa, Fukiko Uedaa a b
Division of Veterinary Public Health, Departments of Veterinary Science, Nippon Veterinary and Life Sciences University, Tokyo, 180-8602, Japan Departments of Veterinary Nursing and Technology, Nippon Veterinary and Life Sciences University, Tokyo, 180-8602, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Listeria monocytogenes Persistence Acid response Glucose Glutamate
Some Listeria monocytogenes strains are persistent in food processing environments, where this pathogen may be subjected to various stresses. This study aimed to elucidate the response of persistent strains of L. monocytogenes to low pH and H2O2 exposure. Almost all of the persistent strains examined were highly susceptible to low pH, whereas H2O2 susceptibility was comparable to that of control strains. Two persistent strains isolated from the same sample, however, exhibited lower susceptibility to low pH. These findings suggest an acid-susceptible phenotype predominates in the habitat, indicating that environmental conditions contribute to the establishment of persistence. Representative strains exhibiting acid-susceptible and less acid-susceptible phenotypes were further investigated regarding acid response characteristics. Less acid-susceptible strains exhibited increased survival in acidified brain heart infusion (BHI) broth compared with acidified phosphate-buffered saline (PBS). These strains also exhibited increased survival in acidified PBS containing glucose and glutamate, which are involved in acid response mechanisms, compared with acidified PBS alone. However, neither acidified BHI broth nor exogenous glucose and glutamate increased survival of acid-susceptible strains. An adaptive acid tolerance response of the acid-susceptible phenotype was observed, but this was limited compared with that of the less acid-susceptible phenotype.
1. Introduction Listeria monocytogenes is a food-borne pathogen that causes severe invasive diseases, including neurologic infections and septicemia, in immunocompromised individuals (Buchanan et al., 2017). The presence of this pathogen in food processing facilities is thought to be a leading cause of post-processing contamination of finished food products (Buchanan et al., 2017). Some L. monocytogenes strains isolated repeatedly from food processing environments or foods over extended periods of time have been designated persistent strains (Ferreira et al., 2014). The persistent nature of these L. monocytogenes strains in food processing environments increases the chance of food product exposure, which is consistent with a report indicating that this pathogen is associated with the greatest number of food product recalls in the United States (Gorton and Stasiewicz, 2017). It has been suggested that several characteristics of persistent strains facilitate their colonization and long-term persistence in certain environments, including biofilm formation, acquisition of antimicrobial resistance, and stress response mechanisms (Elhanafi et al., 2010; Ortiz
et al., 2016). Comparative studies of L. monocytogenes strains have identified several specific characteristics of persistent strains, including enhanced biofilm formation (Borucki et al., 2003; Latorre et al., 2011; Nakamura et al., 2013; Nowak et al., 2017) and preferential growth under unfavorable conditions (Magalhães et al., 2016). In contrast, comparative analyses of disinfectant susceptibility showed no correlation between tolerance to benzalkonium chloride and persistence (Heir et al., 2004). Furthermore, no difference was observed in biofilm formation between persistent and non-persistent strains (Djordjevic et al., 2002; Nilsson et al., 2011). Therefore, an alternative explanation may be that food processing facilities and equipment characterized by inappropriate cleaning and poor hygienic conditions promote the persistence of L. monocytogenes (Abee et al., 2016; Carpentier and Cerf, 2011). We repeatedly isolated a series of serologically and genetically identical L. monocytogenes strains from chicken over a 6-month period and found no difference in biofilm formation between these and nonpersistent strains (Ochiai et al., 2014). Further investigation of stress response characteristics of the persistent strains was undertaken, under
∗ Corresponding author. Division of Veterinary Public Health, Departments of Veterinary Science, Nippon Veterinary and Life Sciences University, Kyonan 1-7-1, Musashino, Tokyo, 180-8602, Japan. E-mail address: [email protected] (Y. Ochiai).
https://doi.org/10.1016/j.fm.2019.103312 Received 27 February 2019; Received in revised form 8 August 2019; Accepted 24 August 2019 Available online 26 August 2019 0740-0020/ © 2019 Elsevier Ltd. All rights reserved.
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strains assigned to the same genotype as 72C2 were only isolated on two sequential days (Ochiai et al., 2014). The other control strains were isolated from meat samples, as reported previously (Ochiai et al., 2010). With the exception of strain 72C2, the persistent and control strains were isolated from different retail shops from which meat samples were obtained for bacterial isolation. The control strains included three of serotype 1/2a, two of 1/2b, one of 1/2c and one of 4b. All strains were stored and propagated as described previously (Ochiai et al., 2017), except that cultivation was performed at 30 °C.
the assumption that stress response mechanisms facilitate persistence. Low pH is a stress that L. monocytogenes encounters frequently in food processing environments (NicAogáin and O'Byrne, 2016). In addition, comparative studies have shown large variations in acid tolerance among L. monocytogenes strains (Adrião et al., 2008; Faleiro et al., 2003; Karatzas et al., 2012; Lundén et al., 2008). Based on these reports, we hypothesized that unique properties that promote acid tolerance are shared among persistent strains of this pathogen. A number of acid response mechanisms, including the adaptive acid tolerance response (ATR) (Davis et al., 1996), the glutamate decarboxylase (GAD) system (Cotter et al., 2001), F0F1-ATPase (Cotter et al., 2000), and the arginine deaminase system (Ryan et al., 2009), have been reported. The variety of different acid response mechanisms may contribute to observed variations in the acid tolerance of L. monocytogenes strains. The aim of the present study was to investigate the response to acid exposure in previously isolated persistent strains of L. monocytogenes. A comparative study was conducted in persistent and control strains. As L. monocytogenes is subjected to H2O2 in foods, food processing facilities, and intracellular environments (Ochiai et al., 2017), we also characterized the H2O2 response, to determine whether susceptibility to low pH and H2O2 are correlated in the persistent strains. Representative persistent and control strains were examined further to elucidate the acid response mechanisms involved.
2.2. Survival of L. monocytogenes strains following H2O2 exposure Survival of L. monocytogenes strains exposed to H2O2 was addressed as described previously (Ochiai et al., 2017), with some modifications. Aliquots (0.5-ml) of cultures in the mid-logarithmic growth phase (optical density at 660 nm [OD660] of ~0.3) at 30 °C were used in the present study. The prepared cells were exposed to 60 mM H2O2 at 20 °C for 150 min or 37 °C for 30 min, and surviving bacteria were plated on tryptic soy agar supplemented with 0.6% yeast extract (TSAYE) containing 1% sodium pyruvate and cultured at 37 °C for 5 days. These exposure conditions were determined based on survival curves of strain EGD-e after H2O2 exposure, as reported previously (Ochiai et al., 2017). 2.3. Survival of L. monocytogenes strains following low-pH exposure
2. Materials and methods
Aliquots (0.5-ml) of cultures in the mid-logarithmic growth phase (OD660 of ~0.3) at 30 °C were harvested and washed in phosphatebuffered saline (PBS). The concentration of L. monocytogenes cells was adjusted to ~3 × 107 CFU/ml, and then the bacteria were pelleted and suspended in 5 ml of brain heart infusion (BHI) broth or PBS adjusted to pH 3.0 with hydrochloric acid, which rapidly inactivates this pathogen (Davis et al., 1996). The bacterial suspension was agitated (160 rpm) at 20 °C or 37 °C for different time periods up to 120 min. Bacterial survival was quantitatively assessed by plating serial dilutions onto TSAYE at 37 °C. Counts of surviving bacteria were logarithmically transformed as follows: log(N/N0), where N represents the bacterial density at each time point of acid exposure and N0 represents the initial bacterial density (Komora et al., 2017). The detection limit for bacterial cell recovery (5 CFU/ml) was used as the final bacterial density for calculating log(N/N0) when no colonies were obtained from plates inoculated with stress-exposed L. monocytogenes strains. In order to evaluate the role of the GAD system (Cotter et al., 2001) or energy-dependent mechanisms (Shabala et al., 2002) in the acid response of L. monocytogenes, the strains were suspended in PBS adjusted to pH 3.0 and supplemented with 1 mM glutamate (Sigma-Aldrich, Tokyo, Japan) or 1 mM glucose (Wako Pure Chemical Industries, Osaka, Japan), respectively, and cultured at 20 °C for different time periods up to 120 min. The ATR was evaluated as described elsewhere (Davis et al., 1996), with some modifications. Harvested and washed bacterial cells were first suspended in BHI broth adjusted to pH 5.1 or 7.0 and cultured at 30 °C for 60 min with agitation (160 rpm). The cells were then cultured in BHI broth adjusted to pH 3.0 at 20 °C with agitation (160 rpm) for different time periods up to 240 min. A total of six biological replicates were analyzed for each data point. Namely, two or three individual colonies were analyzed per experiment, and each experiment was performed independently three or two times, respectively.
2.1. Bacterial strains and culture conditions The L. monocytogenes strains used in the present study are listed in Table 1. A group of 11 persistent strains, which were isolated repeatedly from retail chicken over a 6-month period, as reported previously (Ochiai et al., 2014), were examined. In that study, we showed that a series of strains of serotype 1/2b and the same genotype determined by multi-virulence-locus sequence typing (MVLST) (Zhang et al., 2004) were repeatedly isolated from 16 of 21 (76.2%) chicken samples. These chickens were processed in the same processing plant and sold in the same retail shop. Two persistent strains, 72C3 and 72C5, both of which were simultaneously isolated from the same sample, were used in the present study. Of the seven control strains, EGD-e is used as a reference in many laboratories. Although strain 72C2 was obtained from the same sample as strains 72C3 and 72C5, it was assigned to a different genotype than the persistent strains based on MVLST results (Ochiai et al., 2014). Strain 72C2 was considered to be transient, as Table 1 L. monocytogenes strains used in the present study. Strain
Isolation date
Persistent strains 69C3 12/14/1997 72C3 02/09/1998 72C5 02/09/1998 77C1 02/11/1998 87C4 03/10/1998 91C3 03/12/1998 97C5 04/13/1998 99C4 04/15/1998 103C4 04/17/1998 109C1 05/20/1998 116C1 06/10/1998 Cotrol strains EGD-e 72C2 02/09/1998 76P2 02/10/1998 179B3 06/13/1999 183P3 06/12/1999 227BP1 06/21/1999 229C2 10/06/1997
Origin
Serotype
Reference
Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken
1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b
Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai
Guiniea pig Chicken Pork Beef Pork Minced beef-pork Chicken
1/2a 1/2b 1/2a 1/2c 1/2a 1/2b 4b
Glaser et al. (2001) Ochiai et al. (2014) Ochiai et al. (2010) Ochiai et al. (2010) Ochiai et al. (2010) Ochiai et al. (2010) Ochiai et al. (2010)
et et et et et et et et et et et
al. al. al. al. al. al. al. al. al. al. al.
(2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014)
2.4. Statistical analyses All statistical analyses were performed using Excel Statistics 2010 for Windows(R) (SSRI, Tokyo, Japan). Differences between two groups were analyzed using the Student's t-test, while Welch's t-test was used to determine the significance of differences between groups of different sample size. Significant differences were accepted at a P-value < 0.05. 2
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Fig. 1. Logarithmic reduction in survival of persistent and control L. monocytogenes strains following exposure to 60 mM H2O2 exposure at 20 °C for 150 min. Each data point represents the mean ± standard error of the mean based on a total of six biological replicates.
Fig. 2. Survival of persistent and control L. monocytogenes strains following culture in BHI broth adjusted to pH3.0 at 20 °C for 120 min. Each data point represents the mean ± standard error of the mean based on a total of six biological replicates.
3. Results
3.3. Survival of L. monocytogenes strains following low-pH exposure under nutrient-poor and nutrient-rich nutrient conditions
3.1. Survival of L. monocytogenes strains following H2O2 exposure In order to evaluate the effect of nutrients on the acid response, survival curves for L. monocytogenes strains in PBS or BHI broth adjusted to pH 3.0 were generated at 20 °C or 37 °C. Four strains were examined, including the two control strains (EGD-e and 72C2) and two persistent strains (72C3 and 77C1). As shown above, strains EGD-e and 77C1 exhibited an acid-susceptible phenotype, whereas strains 72C2 and 72C3 exhibited a less acid-susceptible phenotype. Among persistent strains exhibiting the acid-susceptible phenotype, strain 77C1 (isolated following 72C3 two days later) was selected (Table 2). Among control strains exhibiting the less acid-susceptible phenotype, strain 72C2 (isolated simultaneously with 72C3) was selected. Survival curves obtained for all strains following exposure to acidic conditions at 20 °C exhibited an initial plateau phase, followed by an inactivation phase (Fig. 3). In contrast, no plateau phase was observed in survival curves obtained following exposure to acidic conditions at 37 °C a(Fig. S3), though we cannot exclude the possibility of a plateau phase of < 15 min in duration. A comparison of the two curves for strain EGD-e revealed a higher survival rate in acidified PBS than acidified BHI broth at both temperatures (Fig. 3A and S3A). These findings were confirmed by the significant difference in log(N/N0) values for this strain between culture in acidified BHI broth and PBS at 20 °C for 60 min (Student's ttest, P < 0.01) and 37 °C for 15 min (Student's t-test, P < 0.01). These results suggest a negative effect of nutrients on the acid response in strain EGD-e. Survival curves for strain 77C1 were similar to those for strain EGD-e (Fig. 3B and S3B). However, no significant differences were observed in log(N/N0) for this strain between culture in acidified BHI broth and PBS at 20 °C for 60 min (Student's t-test, P > 0.05) and 37 °C for 15 min (Student's t-test, P > 0.05), suggesting that nutrients had no effect on the acid response in strain 77C1. In contrast, strains 72C2 and 72C3 exhibited increased survival in acidified BHI broth, which was characterized by an extended duration of the survival curve plateau phase and slow decline in the inactivation phase (Fig. 3C and D, S3C, and S3D). These findings suggest that the response to exposure to acidic conditions is nutrient dependent in the less acid-susceptible strains.
Listeria monocytogenes strains were exposed to H2O2 in PBS at 20 °C or 37 °C, as temperature-dependent survival was observed in the reference strain EGD-e following stress exposure (Ochiai et al., 2017). A temperature-dependent response to H2O2 exposure was also observed for all strains examined in the present study, and therefore strains were exposed to H2O2 at 20 °C for 150 min or 37 °C for 30 min. Values of log (N/N0) for each strain following H2O2 exposure at 20 °C or 37 °C are shown in Figs. 1 and S1, respectively. The mean log(N/N0) values obtained for the persistent strains following stress exposure at 20 °C ranged from −1.20 to −0.01, whereas those for the control strains ranged from −3.71 to −0.36 (Fig. 1). The mean log(N/N0) values obtained for the persistent strains exposed to H2O2 at 37 °C ranged from −0.64 to −0.14, whereas those for the control strains ranged from −2.51 to −0.24 (Fig. S1). Based on the log(N/N0) values for the persistent and control strains, a comparative study was conducted to investigate whether the persistent strains have a unique H2O2 response mechanism. However, no significant differences in log(N/N0) at 20 °C or 37 °C were observed between the persistent and control strains (Table 2 and S1).
3.2. Survival of L. monocytogenes strains following low pH exposure Listeria monocytogenes strains were suspended in BHI broth adjusted to pH 3.0 and cultured at 20 °C or 37 °C. A temperature-dependent response to the acidic condition was observed for all strains examined; exposure of strains to low pH was performed at 20 °C for 120 min or 37 °C for 15 min. Except for two strains, almost no survival of persistent strains was observed, resulting in mean log(N/N0) values of less than −6 at both temperatures (Fig. 2 and S2). Acid-susceptible strain EGD-e (Karatzas et al., 2012) exhibited almost no survival following exposure to low pH. In contrast, surviving cells of two persistent strains, 72C3 and 72C5, were observed following low-pH exposure, with these strains exhibiting > 3 log higher survival than the other persistent strains. The mean log(N/N0) of the control strains (except for strain EGD-e) ranged from −1.35 to −0.22 at 20 °C (Fig. 2) and from −2.33 to −0.73 at 37 °C (Fig. S2). The log(N/N0) values of the persistent strains cultured in BHI broth adjusted to pH 3.0 were significantly lower than those of the control strains at both 20 °C and 37 °C (Table 2 and S1), confirming the high susceptibility of persistent strains to low pH.
3.4. Survival of L. monocytogenes strains following low-pH exposure in the presence of glucose or glutamate The effects of exogenous glucose and glutamate on the acid response of the four strains were investigated because glucose and glutamate are 3
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Table 2 Comparison of log (N/N0) between persistent and control strains. Stress
Exposure temperature
Exposure duration
Mean log (N/N0) of persistent strans
Mean log (N/N0) of control strans
P valuea
H2O2 BHI (pH3.0)
20 °C 20 °C
150 min 120 min
−0.66 ± 0.39 −5.85 ± 1.34
−1.47 ± 1.03 −1.65 ± 2.29
0.071 0.003b
a b
Analyse by the Welch's t-test were performed. Siginificant differences between persistent and control strains.
mechanisms.
involved in the acid-tolerance mechanism of the GAD system (Cotter et al., 2001) and energy-dependent mechanisms, including F0F1-ATPase (Shabala et al., 2002), respectively. Survival curves were generated following culture in PBS supplemented with glucose or glutamate and adjusted to pH 3.0 at 20 °C, and comparative analyses were performed for stress exposure in BHI broth, PBS alone, and PBS containing glucose or glutamate (Fig. 3). Almost no difference in survival was observed in strains EGD-e and 77C1 subjected to acidic conditions in PBS supplemented with glucose or glutamate in comparison with the strains cultured in BHI broth or PBS alone (Fig. 3A and B). These findings suggest that glucose and glutamate have no effect on the acid response in acid-susceptible strains. In contrast, increased survival in the presence of exogenous glucose and glutamate was observed in strains 72C2 and 72C3 when compared with cells cultured in PBS alone (Fig. 3C and D). The survival curves for the two strains following exposure to acidic conditions in PBS with glucose or glutamate were comparable to those for cells cultured in BHI broth. These findings suggest that the acid response of the less acid-susceptible phenotype of strains 72C2 and 72C3 involves the GAD system and energy-dependent
3.5. The ATR of L. monocytogenes strains The ATR of the four L. monocytogenes strains was also investigated. Induction of the ATR was performed in BHI broth adjusted to pH 5.1, whereas control cells were cultured in BHI broth adjusted to pH 7.0. Survival curves were then generated following secondary culture in BHI broth adjusted to pH 3.0 at 20 °C. The survival curves appeared to show enhanced survival of EGD-e and 77C1 cells in which the ATR had been induced in comparison with cells in which the ATR was not induced (Fig. 4A and B). Significant differences were observed between the log (N/N0) values for strain EGD-e with and without induction of the ATR following exposure to low pH for 45, 60, and 90 min (Student's t-test, P < 0.01) and between log(N/N0) values for strain 77C1 with and without induction of the ATR following exposure to low-pH stress for 45, 60, 90, and 120 min (Student's t-test, P < 0.01). These findings suggest the presence of a functional ATR in the acid-susceptible strains. In contrast, survival curves for strains 72C2 and 72C3 in which the ATR
Fig. 3. Survival curves of L. monocytogenes strains EGD-e (A), 77C1 (B), 72C2 (C), and 72C3 (D) following exposure to acidic conditions adjusted to pH3.0 at 20 °C. The strains used were subjected to acid stress in PBS (squares), BHI broth (diamonds), PBS supplemented with glucose (triangles) and PBS supplemented with glutamate (crossbars). Each data point represents the mean ± standard error of the mean based on a total of six biological replicates. 4
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Fig. 4. Survival curves of L. monocytogenes strains EGD-e (A), 77C1 (B), 72C2 (C), and 72C3 (D) with induction of the ATR (squares) or without induction of the ATR (diamonds) following culture in BHI broth adjusted to pH3.0 at 20 °C. Each data point represents the mean ± standard error of the mean based on a total of six biological replicates.
tolerant of low-pH conditions than non-persistent strains. Faleiro et al. (2003) showed that L. monocytogenes strains isolated from cheese tend to be more tolerant of low-pH conditions than strains isolated from meat and fish, suggesting that differences in the frequency of exposure to low pH promote higher acid tolerance in strains commonly found in cheese. The low frequency of exposure to acidic conditions may allow the acid-susceptible L. monocytogenes strains to become persistent in meat processing facilities. Although our findings were inconsistent with those reported by Lundén et al. (2008), the demonstrated persistence of both the less acid-susceptible and acid-susceptible strains supports the suggestion that almost any L. monocytogenes strains can become persistent if it is introduced into a suitable niche that provides both protection from lethal stress and growth-permissive conditions, as described by Ferreira et al. (2014). Therefore, the persistence of strains exhibiting the acid-susceptible phenotype suggest that environmental conditions are the primary contributor to the establishment of persistence by L. monocytogenes strains (Carpentier and Cerf, 2011; Ferreira et al., 2014). The persistent strains 72C3 and 72C5 exhibited decreased susceptibility to low pH when compared with the other persistent strains. Both of these strains, which were isolated from the same sample, responded similarly to acidic conditions, and this response appeared to remain stable during storage. It should be noted that these persistent strains were isolated simultaneously with strains assigned to a different genotype, including strain 72C2. The strains assigned to a different genotype than the persistent strains were considered transient, because they
was induced exhibited no apparent inactivation phase up to 240 min, whereas the survival curves for cells of these strains in which the ATR had not been induced exhibited an initial plateau-phase followed by an inactivation phase (Fig. 4C and D). 4. Discussion No significant difference in survival following H2O2 exposure was observed between persistent and control strains, indicating that the persistent strains investigated in the present study have no unique H2O2 response characteristics. In contrast, all persistent strains except 72C3 and 72C5 were highly susceptible to low pH. The susceptibility of these persistent strains to acidic conditions was comparable to that of the known acid-susceptible control strain EGD-e (Karatzas et al., 2012). This reference strain was shown to be one of the most sensitive to gastric fluid (pH 3.5) among 50 L. monocytogenes strains examined (Olier et al., 2004). In addition, the GAD system was shown to be nonfunctional in strain EGD-e (Karatzas et al., 2012; Olier et al., 2004). Strain 77C1, a persistent strain exhibiting acid-susceptibility, did not exhibit increased survival in the presence of exogenous glutamate in the present study. Such common characteristics between strain EGD-e and the persistent strains examined in the present study suggest these strains possess unique acid responses, as reported by Karatzas et al. (2012). Lundén et al. (2008) reported that persistent strains isolated from meat products or meat processing facilities are significantly more 5
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Conflicts of interest
were only isolated on two sequential days (Ochiai et al., 2014). The simultaneous isolation of persistent and transient strains suggests that the transient strains have an effect on the isolation of persistent strains that are less acid susceptible. However, how the transient strains are involved in the emergence of the persistent strains exhibiting less acidsusceptibility remains unclear. A nutrient-dependent response to low pH was observed in strains exhibiting the less acid-susceptible phenotype but not in strains exhibiting the acid-susceptible phenotype. Increased survival of strain EGD-e following low-pH exposure was observed under low-nutrient conditions, but the biological significance of these findings remains unclear. Further analyses of survival under acidic conditions were performed in PBS supplemented with glutamate or glucose. Survival curves for strains EGD-e and 77C1 cultured under both conditions were similar to those of these strains cultured in acidified PBS alone. These findings showed that not only the GAD system, which was reported elsewhere (Karatzas et al., 2012), but also energy-dependent mechanisms, including F0F1-ATPase, were nonfunctional in acid-susceptible strains. In contrast, strain EGD-e was shown to harbor genes encoding proteins involved in the GAD system and F0F1-ATPase responses (Cotter et al., 2000, 2005; Glaser et al., 2001). All survival curves for strains exhibiting the acid-susceptible phenotype following exposure to acidic conditions at 20 °C were characterized by an initial plateau phase that was almost 30 min in duration, as shown in Fig. 3A and B. A plateau phase of comparable duration was observed in the survival curves of strains exhibiting the less acidsusceptible phenotype subjected to the same stress in PBS at 20 °C (Fig. 3C and D). These findings suggest that the acid-susceptible phenotype elicits an initial acid response corresponding to that of the less acid-susceptible phenotype. Such an initial response may include the involvement of endogenous glucose and glutamate or acid tolerance mechanisms independent of glucose, glutamate, and BHI components. In addition, we cannot exclude the possibility that no acid response occurs during the initial phase. In contrast, differences in the inactivation phase were observed between strains exhibiting the acidsusceptible and less acid-susceptible phenotypes. Survival curves indicated more rapid inactivation of the acid-susceptible strains compared with the less acid-susceptible strains. This phenomenon may result from dysfunctions in multiple acid response mechanisms, including the GAD system and energy-dependent mechanisms described above. Both the less acid-susceptible and acid-susceptible strains exhibited an ATR. Survival curves for acid-susceptible strains in which the ATR was induced exhibited two phases, a plateau phase and an inactivation phase, whereas curves for the less acid-susceptible strains in which the ATR was induced appeared to be comprised of only a plateau phase until at least 4 h after the initiation of stress exposure. These findings suggest that acid-susceptible strains in which the ATR was induced did not exhibit a robust response to subsequent exposure to low pH, in contrast to that observed with the less acid-susceptible strains. These results also suggest that the subsequent response to low pH takes precedence over the first response to weak acid exposure.
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Listeria monocytogenes persistence in food-associated environments: epidemiology, strain characteristics, and implications for public health. J. Food Prot. 77, 150–170. Glaser, P., Frangeul, L., Buchrieser, C., Rusniok, C., Amend, A., Baquero, F., Berche, P., Bloecker, H., Brandt, P., Chakraborty, T., Charbit, A., Chetouani, F., Couvé, E., de Daruvar, A., Dehoux, P., Domann, E., Domínguez-Bernal, G., Duchaud, E., Durant, L., Dussurget, O., Entian, K.D., Fsihi, H., García-del, Portillo, F., Garrido, P., Gautier, L., Goebel, W., Gómez-López, N., Hain, T., Hauf, J., Jackson, D., Jones, L.M., Kaerst, U., Kreft, J., Kuhn, M., Kunst, F., Kurapkat, G., Madueno, E., Maitournam, A., Vicente, J.M., Ng, E., Nedjari, H., Nordsiek, G., Novella, S., de Pablos, B., Pérez-Diaz, J.C., Purcell, R., Remmel, B., Rose, M., Schlueter, T., Simoes, N., Tierrez, A., VázquezBoland, J.A., Voss, H., Wehland, J., Cossart, P., 2001. Comparative genomics of Listeria species. Science 294, 849–852. Gorton, A., Stasiewicz, M.J., 2017. Twenty-two years of U.S. meat and poultry products recalls: implications for food safety and food waste. J. Food Prot. 80, 674–684. Heir, E., Lindstedt, B.A., Røtterud, O.J., Vardund, T., Kapperud, G., Nesbakken, T., 2004. Molecular epidemiology and disinfectant susceptibility of Listeria monocytogenes from meat processing plants and human infections. Int. J. Food Microbiol. 96, 85–96. Karatzas, K.A., Suur, L., O'Byrne, C.P., 2012. Characterization of the intracellular glutamate decarboxylase system: analysis of its function, transcription, and role in the acid resistance of various strains of Listeria monocytogenes. Appl. Environ. Microbiol. 78, 3571–3579. Komora, N., Bruschi, C., Magalhães, R., Ferreira, V., Teixeira, P., 2017. Survival of Listeria monocytogenes with different antibiotic resistance patterns to food-associated stresses. Int. J. Food Microbiol. 245, 79–87. Latorre, A.A., Van Kessel, J.A., Karns, J.S., Zurakowski, M.J., Pradhan, A.K., Boor, K.J., Adolph, E., Sukhnanand, S., Schukken, Y.H., 2011. Increased in vitro adherence and on-farm persistence of predominant and persistent Listeria monocytogenes strains in the milking system. Appl. Environ. Microbiol. 77, 3676–3684. Lundén, J., Tolvanen, R., Korkeala, H., 2008. Acid and heat tolerance of persistent and nonpersistent Listeria monocytogenes food plant strains. Lett. Appl. Microbiol. 46,
5. Conclusions This study demonstrated that the persistent L. monocytogenes strains examined were highly susceptible to low pH, but their susceptibility to H2O2 was comparable to that of control strains. However, the emergence of a few persistent strains that were less susceptible to acid was also observed. Our findings indicating a predominance of the acidsusceptible phenotype suggest that environmental conditions contribute to the establishment of persistence by acid-susceptible L. monocytogenes strains. The acid-susceptible phenotype of persistent strains was associated with unique responses to stress resulting from acidic conditions, including a nutrient-independent response. In addition, we found that exogenous glucose and glutamate had no effect on survival under acidic conditions. 6
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formation under different temperature conditions by a single genotype of persistent Listeria monocytogenes strains. J. Food Prot. 77, 133–140. Ochiai, Y., Yamada, F., Yoshikawa, Y., Mochizuki, M., Takano, T., Hondo, R., Ueda, F., 2017. Sequential transition of the injury phenotype, temperature-dependent survival and transcriptional response in Listeria monocytogenes following lethal H2O2 exposure. Int. J. Food Microbiol. 259, 52–58. Olier, M., Rousseaux, S., Piveteau, P., Lemaître, J.P., Rousset, A., Guzzo, J., 2004. Screening of glutamate decarboxylase activity and bile salt resistance of human asymptomatic carriage, clinical, food, and environmental isolates of Listeria monocytogenes. Int. J. Food Microbiol. 93, 87–99. Ortiz, S., Lopez-Alonso, V., Rodriguez, P., Martinez-Suarez, J.V., 2016. The connection between persistent, disinfectant-resistant Listeria monocytogenes strains from two geographically separate Iberian pork processing plants: evidence from comparative genome analysis. Appl. Environ. Microbiol. 82, 308–317. Ryan, S., Begley, M., Gahan, C.G., Hill, C., 2009. Molecular characterization of the arginine deiminase system in Listeria monocytogenes: regulation and role in acid tolerance. Environ. Microbiol. 11, 432–445. Shabala, L., Budde, B., Ross, T., Siegumfeldt, H., McMeekin, T., 2002. Responses of Listeria monocytogenes to acid stress and glucose availability monitored by measurements of intracellular pH and viable counts. Int. J. Food Microbiol. 75, 89–97. Zhang, W., Jayarao, B.M., Knabel, S.J., 2004. Multi-virulence-locus sequence typing of Listeria monocytogenes. Appl. Environ. Microbiol. 70, 913–920.
276–280. Magalhães, R., Ferreira, V., Brandão, T.R., Palencia, R.C., Almeida, G., Teixeira, P., 2016. Persistent and non-persistent strains of Listeria monocytogenes: a focus on growth kinetics under different temperature, salt, and pH conditions and their sensitivity to sanitizers. Food Microbiol. 57, 103–108. Nakamura, H., Takakura, K., Sone, Y., Itano, Y., Nishikawa, Y., 2013. Biofilm formation and resistance to benzalkonium chloride in Listeria monocytogenes isolated from a fish processing plant. J. Food Prot. 76, 1179–1186. NicAogáin, K., O'Byrne, C.P., 2016. The role of stress and stress adaptations in determining the fate of the bacterial pathogen Listeria monocytogenes in the food chain. Front. Microbiol. 7, 1865. Nilsson, R.E., Ross, T., Bowman, J.P., 2011. Variability in biofilm production by Listeria monocytogenes correlated to strain origin and growth conditions. Int. J. Food Microbiol. 150, 14–24. Nowak, J., Cruz, C.J., Tempelaars, M., Abee, T., van Vliet, A.H.M., Fletcher, G.C., Hedderley, D., Palmer, J., Flint, S., 2017. Persistent Listeria monocytogenes strains isolated from mussel production facilities form more biofilm but are not linked to specific genetic markers. Int. J. Food Microbiol. 256, 45–53. Ochiai, Y., Yamada, F., Batmunkh, O., Mochizuki, M., Takano, T., Hondo, R., Ueda, F., 2010. Prevalence of Listeria monocytogenes in retailed meat in the Tokyo metropolitan area. J. Food Prot. 73, 1688–1693. Ochiai, Y., Yamada, F., Mochizuki, M., Takano, T., Hondo, R., Ueda, F., 2014. Biofilm
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Unique response characteristics in persistent strains of Listeria monocytogenes exposed to low pH
T
Yoshitsugu Ochiaia,∗, Yuko Yoshikawaa, Mariko Mochizukib, Takashi Takanoa, Fukiko Uedaa a b
Division of Veterinary Public Health, Departments of Veterinary Science, Nippon Veterinary and Life Sciences University, Tokyo, 180-8602, Japan Departments of Veterinary Nursing and Technology, Nippon Veterinary and Life Sciences University, Tokyo, 180-8602, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Listeria monocytogenes Persistence Acid response Glucose Glutamate
Some Listeria monocytogenes strains are persistent in food processing environments, where this pathogen may be subjected to various stresses. This study aimed to elucidate the response of persistent strains of L. monocytogenes to low pH and H2O2 exposure. Almost all of the persistent strains examined were highly susceptible to low pH, whereas H2O2 susceptibility was comparable to that of control strains. Two persistent strains isolated from the same sample, however, exhibited lower susceptibility to low pH. These findings suggest an acid-susceptible phenotype predominates in the habitat, indicating that environmental conditions contribute to the establishment of persistence. Representative strains exhibiting acid-susceptible and less acid-susceptible phenotypes were further investigated regarding acid response characteristics. Less acid-susceptible strains exhibited increased survival in acidified brain heart infusion (BHI) broth compared with acidified phosphate-buffered saline (PBS). These strains also exhibited increased survival in acidified PBS containing glucose and glutamate, which are involved in acid response mechanisms, compared with acidified PBS alone. However, neither acidified BHI broth nor exogenous glucose and glutamate increased survival of acid-susceptible strains. An adaptive acid tolerance response of the acid-susceptible phenotype was observed, but this was limited compared with that of the less acid-susceptible phenotype.
1. Introduction Listeria monocytogenes is a food-borne pathogen that causes severe invasive diseases, including neurologic infections and septicemia, in immunocompromised individuals (Buchanan et al., 2017). The presence of this pathogen in food processing facilities is thought to be a leading cause of post-processing contamination of finished food products (Buchanan et al., 2017). Some L. monocytogenes strains isolated repeatedly from food processing environments or foods over extended periods of time have been designated persistent strains (Ferreira et al., 2014). The persistent nature of these L. monocytogenes strains in food processing environments increases the chance of food product exposure, which is consistent with a report indicating that this pathogen is associated with the greatest number of food product recalls in the United States (Gorton and Stasiewicz, 2017). It has been suggested that several characteristics of persistent strains facilitate their colonization and long-term persistence in certain environments, including biofilm formation, acquisition of antimicrobial resistance, and stress response mechanisms (Elhanafi et al., 2010; Ortiz
et al., 2016). Comparative studies of L. monocytogenes strains have identified several specific characteristics of persistent strains, including enhanced biofilm formation (Borucki et al., 2003; Latorre et al., 2011; Nakamura et al., 2013; Nowak et al., 2017) and preferential growth under unfavorable conditions (Magalhães et al., 2016). In contrast, comparative analyses of disinfectant susceptibility showed no correlation between tolerance to benzalkonium chloride and persistence (Heir et al., 2004). Furthermore, no difference was observed in biofilm formation between persistent and non-persistent strains (Djordjevic et al., 2002; Nilsson et al., 2011). Therefore, an alternative explanation may be that food processing facilities and equipment characterized by inappropriate cleaning and poor hygienic conditions promote the persistence of L. monocytogenes (Abee et al., 2016; Carpentier and Cerf, 2011). We repeatedly isolated a series of serologically and genetically identical L. monocytogenes strains from chicken over a 6-month period and found no difference in biofilm formation between these and nonpersistent strains (Ochiai et al., 2014). Further investigation of stress response characteristics of the persistent strains was undertaken, under
∗ Corresponding author. Division of Veterinary Public Health, Departments of Veterinary Science, Nippon Veterinary and Life Sciences University, Kyonan 1-7-1, Musashino, Tokyo, 180-8602, Japan. E-mail address: [email protected] (Y. Ochiai).
https://doi.org/10.1016/j.fm.2019.103312 Received 27 February 2019; Received in revised form 8 August 2019; Accepted 24 August 2019 Available online 26 August 2019 0740-0020/ © 2019 Elsevier Ltd. All rights reserved.
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strains assigned to the same genotype as 72C2 were only isolated on two sequential days (Ochiai et al., 2014). The other control strains were isolated from meat samples, as reported previously (Ochiai et al., 2010). With the exception of strain 72C2, the persistent and control strains were isolated from different retail shops from which meat samples were obtained for bacterial isolation. The control strains included three of serotype 1/2a, two of 1/2b, one of 1/2c and one of 4b. All strains were stored and propagated as described previously (Ochiai et al., 2017), except that cultivation was performed at 30 °C.
the assumption that stress response mechanisms facilitate persistence. Low pH is a stress that L. monocytogenes encounters frequently in food processing environments (NicAogáin and O'Byrne, 2016). In addition, comparative studies have shown large variations in acid tolerance among L. monocytogenes strains (Adrião et al., 2008; Faleiro et al., 2003; Karatzas et al., 2012; Lundén et al., 2008). Based on these reports, we hypothesized that unique properties that promote acid tolerance are shared among persistent strains of this pathogen. A number of acid response mechanisms, including the adaptive acid tolerance response (ATR) (Davis et al., 1996), the glutamate decarboxylase (GAD) system (Cotter et al., 2001), F0F1-ATPase (Cotter et al., 2000), and the arginine deaminase system (Ryan et al., 2009), have been reported. The variety of different acid response mechanisms may contribute to observed variations in the acid tolerance of L. monocytogenes strains. The aim of the present study was to investigate the response to acid exposure in previously isolated persistent strains of L. monocytogenes. A comparative study was conducted in persistent and control strains. As L. monocytogenes is subjected to H2O2 in foods, food processing facilities, and intracellular environments (Ochiai et al., 2017), we also characterized the H2O2 response, to determine whether susceptibility to low pH and H2O2 are correlated in the persistent strains. Representative persistent and control strains were examined further to elucidate the acid response mechanisms involved.
2.2. Survival of L. monocytogenes strains following H2O2 exposure Survival of L. monocytogenes strains exposed to H2O2 was addressed as described previously (Ochiai et al., 2017), with some modifications. Aliquots (0.5-ml) of cultures in the mid-logarithmic growth phase (optical density at 660 nm [OD660] of ~0.3) at 30 °C were used in the present study. The prepared cells were exposed to 60 mM H2O2 at 20 °C for 150 min or 37 °C for 30 min, and surviving bacteria were plated on tryptic soy agar supplemented with 0.6% yeast extract (TSAYE) containing 1% sodium pyruvate and cultured at 37 °C for 5 days. These exposure conditions were determined based on survival curves of strain EGD-e after H2O2 exposure, as reported previously (Ochiai et al., 2017). 2.3. Survival of L. monocytogenes strains following low-pH exposure
2. Materials and methods
Aliquots (0.5-ml) of cultures in the mid-logarithmic growth phase (OD660 of ~0.3) at 30 °C were harvested and washed in phosphatebuffered saline (PBS). The concentration of L. monocytogenes cells was adjusted to ~3 × 107 CFU/ml, and then the bacteria were pelleted and suspended in 5 ml of brain heart infusion (BHI) broth or PBS adjusted to pH 3.0 with hydrochloric acid, which rapidly inactivates this pathogen (Davis et al., 1996). The bacterial suspension was agitated (160 rpm) at 20 °C or 37 °C for different time periods up to 120 min. Bacterial survival was quantitatively assessed by plating serial dilutions onto TSAYE at 37 °C. Counts of surviving bacteria were logarithmically transformed as follows: log(N/N0), where N represents the bacterial density at each time point of acid exposure and N0 represents the initial bacterial density (Komora et al., 2017). The detection limit for bacterial cell recovery (5 CFU/ml) was used as the final bacterial density for calculating log(N/N0) when no colonies were obtained from plates inoculated with stress-exposed L. monocytogenes strains. In order to evaluate the role of the GAD system (Cotter et al., 2001) or energy-dependent mechanisms (Shabala et al., 2002) in the acid response of L. monocytogenes, the strains were suspended in PBS adjusted to pH 3.0 and supplemented with 1 mM glutamate (Sigma-Aldrich, Tokyo, Japan) or 1 mM glucose (Wako Pure Chemical Industries, Osaka, Japan), respectively, and cultured at 20 °C for different time periods up to 120 min. The ATR was evaluated as described elsewhere (Davis et al., 1996), with some modifications. Harvested and washed bacterial cells were first suspended in BHI broth adjusted to pH 5.1 or 7.0 and cultured at 30 °C for 60 min with agitation (160 rpm). The cells were then cultured in BHI broth adjusted to pH 3.0 at 20 °C with agitation (160 rpm) for different time periods up to 240 min. A total of six biological replicates were analyzed for each data point. Namely, two or three individual colonies were analyzed per experiment, and each experiment was performed independently three or two times, respectively.
2.1. Bacterial strains and culture conditions The L. monocytogenes strains used in the present study are listed in Table 1. A group of 11 persistent strains, which were isolated repeatedly from retail chicken over a 6-month period, as reported previously (Ochiai et al., 2014), were examined. In that study, we showed that a series of strains of serotype 1/2b and the same genotype determined by multi-virulence-locus sequence typing (MVLST) (Zhang et al., 2004) were repeatedly isolated from 16 of 21 (76.2%) chicken samples. These chickens were processed in the same processing plant and sold in the same retail shop. Two persistent strains, 72C3 and 72C5, both of which were simultaneously isolated from the same sample, were used in the present study. Of the seven control strains, EGD-e is used as a reference in many laboratories. Although strain 72C2 was obtained from the same sample as strains 72C3 and 72C5, it was assigned to a different genotype than the persistent strains based on MVLST results (Ochiai et al., 2014). Strain 72C2 was considered to be transient, as Table 1 L. monocytogenes strains used in the present study. Strain
Isolation date
Persistent strains 69C3 12/14/1997 72C3 02/09/1998 72C5 02/09/1998 77C1 02/11/1998 87C4 03/10/1998 91C3 03/12/1998 97C5 04/13/1998 99C4 04/15/1998 103C4 04/17/1998 109C1 05/20/1998 116C1 06/10/1998 Cotrol strains EGD-e 72C2 02/09/1998 76P2 02/10/1998 179B3 06/13/1999 183P3 06/12/1999 227BP1 06/21/1999 229C2 10/06/1997
Origin
Serotype
Reference
Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken Chicken
1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b 1/2b
Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai Ochiai
Guiniea pig Chicken Pork Beef Pork Minced beef-pork Chicken
1/2a 1/2b 1/2a 1/2c 1/2a 1/2b 4b
Glaser et al. (2001) Ochiai et al. (2014) Ochiai et al. (2010) Ochiai et al. (2010) Ochiai et al. (2010) Ochiai et al. (2010) Ochiai et al. (2010)
et et et et et et et et et et et
al. al. al. al. al. al. al. al. al. al. al.
(2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014) (2014)
2.4. Statistical analyses All statistical analyses were performed using Excel Statistics 2010 for Windows(R) (SSRI, Tokyo, Japan). Differences between two groups were analyzed using the Student's t-test, while Welch's t-test was used to determine the significance of differences between groups of different sample size. Significant differences were accepted at a P-value < 0.05. 2
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Fig. 1. Logarithmic reduction in survival of persistent and control L. monocytogenes strains following exposure to 60 mM H2O2 exposure at 20 °C for 150 min. Each data point represents the mean ± standard error of the mean based on a total of six biological replicates.
Fig. 2. Survival of persistent and control L. monocytogenes strains following culture in BHI broth adjusted to pH3.0 at 20 °C for 120 min. Each data point represents the mean ± standard error of the mean based on a total of six biological replicates.
3. Results
3.3. Survival of L. monocytogenes strains following low-pH exposure under nutrient-poor and nutrient-rich nutrient conditions
3.1. Survival of L. monocytogenes strains following H2O2 exposure In order to evaluate the effect of nutrients on the acid response, survival curves for L. monocytogenes strains in PBS or BHI broth adjusted to pH 3.0 were generated at 20 °C or 37 °C. Four strains were examined, including the two control strains (EGD-e and 72C2) and two persistent strains (72C3 and 77C1). As shown above, strains EGD-e and 77C1 exhibited an acid-susceptible phenotype, whereas strains 72C2 and 72C3 exhibited a less acid-susceptible phenotype. Among persistent strains exhibiting the acid-susceptible phenotype, strain 77C1 (isolated following 72C3 two days later) was selected (Table 2). Among control strains exhibiting the less acid-susceptible phenotype, strain 72C2 (isolated simultaneously with 72C3) was selected. Survival curves obtained for all strains following exposure to acidic conditions at 20 °C exhibited an initial plateau phase, followed by an inactivation phase (Fig. 3). In contrast, no plateau phase was observed in survival curves obtained following exposure to acidic conditions at 37 °C a(Fig. S3), though we cannot exclude the possibility of a plateau phase of < 15 min in duration. A comparison of the two curves for strain EGD-e revealed a higher survival rate in acidified PBS than acidified BHI broth at both temperatures (Fig. 3A and S3A). These findings were confirmed by the significant difference in log(N/N0) values for this strain between culture in acidified BHI broth and PBS at 20 °C for 60 min (Student's ttest, P < 0.01) and 37 °C for 15 min (Student's t-test, P < 0.01). These results suggest a negative effect of nutrients on the acid response in strain EGD-e. Survival curves for strain 77C1 were similar to those for strain EGD-e (Fig. 3B and S3B). However, no significant differences were observed in log(N/N0) for this strain between culture in acidified BHI broth and PBS at 20 °C for 60 min (Student's t-test, P > 0.05) and 37 °C for 15 min (Student's t-test, P > 0.05), suggesting that nutrients had no effect on the acid response in strain 77C1. In contrast, strains 72C2 and 72C3 exhibited increased survival in acidified BHI broth, which was characterized by an extended duration of the survival curve plateau phase and slow decline in the inactivation phase (Fig. 3C and D, S3C, and S3D). These findings suggest that the response to exposure to acidic conditions is nutrient dependent in the less acid-susceptible strains.
Listeria monocytogenes strains were exposed to H2O2 in PBS at 20 °C or 37 °C, as temperature-dependent survival was observed in the reference strain EGD-e following stress exposure (Ochiai et al., 2017). A temperature-dependent response to H2O2 exposure was also observed for all strains examined in the present study, and therefore strains were exposed to H2O2 at 20 °C for 150 min or 37 °C for 30 min. Values of log (N/N0) for each strain following H2O2 exposure at 20 °C or 37 °C are shown in Figs. 1 and S1, respectively. The mean log(N/N0) values obtained for the persistent strains following stress exposure at 20 °C ranged from −1.20 to −0.01, whereas those for the control strains ranged from −3.71 to −0.36 (Fig. 1). The mean log(N/N0) values obtained for the persistent strains exposed to H2O2 at 37 °C ranged from −0.64 to −0.14, whereas those for the control strains ranged from −2.51 to −0.24 (Fig. S1). Based on the log(N/N0) values for the persistent and control strains, a comparative study was conducted to investigate whether the persistent strains have a unique H2O2 response mechanism. However, no significant differences in log(N/N0) at 20 °C or 37 °C were observed between the persistent and control strains (Table 2 and S1).
3.2. Survival of L. monocytogenes strains following low pH exposure Listeria monocytogenes strains were suspended in BHI broth adjusted to pH 3.0 and cultured at 20 °C or 37 °C. A temperature-dependent response to the acidic condition was observed for all strains examined; exposure of strains to low pH was performed at 20 °C for 120 min or 37 °C for 15 min. Except for two strains, almost no survival of persistent strains was observed, resulting in mean log(N/N0) values of less than −6 at both temperatures (Fig. 2 and S2). Acid-susceptible strain EGD-e (Karatzas et al., 2012) exhibited almost no survival following exposure to low pH. In contrast, surviving cells of two persistent strains, 72C3 and 72C5, were observed following low-pH exposure, with these strains exhibiting > 3 log higher survival than the other persistent strains. The mean log(N/N0) of the control strains (except for strain EGD-e) ranged from −1.35 to −0.22 at 20 °C (Fig. 2) and from −2.33 to −0.73 at 37 °C (Fig. S2). The log(N/N0) values of the persistent strains cultured in BHI broth adjusted to pH 3.0 were significantly lower than those of the control strains at both 20 °C and 37 °C (Table 2 and S1), confirming the high susceptibility of persistent strains to low pH.
3.4. Survival of L. monocytogenes strains following low-pH exposure in the presence of glucose or glutamate The effects of exogenous glucose and glutamate on the acid response of the four strains were investigated because glucose and glutamate are 3
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Table 2 Comparison of log (N/N0) between persistent and control strains. Stress
Exposure temperature
Exposure duration
Mean log (N/N0) of persistent strans
Mean log (N/N0) of control strans
P valuea
H2O2 BHI (pH3.0)
20 °C 20 °C
150 min 120 min
−0.66 ± 0.39 −5.85 ± 1.34
−1.47 ± 1.03 −1.65 ± 2.29
0.071 0.003b
a b
Analyse by the Welch's t-test were performed. Siginificant differences between persistent and control strains.
mechanisms.
involved in the acid-tolerance mechanism of the GAD system (Cotter et al., 2001) and energy-dependent mechanisms, including F0F1-ATPase (Shabala et al., 2002), respectively. Survival curves were generated following culture in PBS supplemented with glucose or glutamate and adjusted to pH 3.0 at 20 °C, and comparative analyses were performed for stress exposure in BHI broth, PBS alone, and PBS containing glucose or glutamate (Fig. 3). Almost no difference in survival was observed in strains EGD-e and 77C1 subjected to acidic conditions in PBS supplemented with glucose or glutamate in comparison with the strains cultured in BHI broth or PBS alone (Fig. 3A and B). These findings suggest that glucose and glutamate have no effect on the acid response in acid-susceptible strains. In contrast, increased survival in the presence of exogenous glucose and glutamate was observed in strains 72C2 and 72C3 when compared with cells cultured in PBS alone (Fig. 3C and D). The survival curves for the two strains following exposure to acidic conditions in PBS with glucose or glutamate were comparable to those for cells cultured in BHI broth. These findings suggest that the acid response of the less acid-susceptible phenotype of strains 72C2 and 72C3 involves the GAD system and energy-dependent
3.5. The ATR of L. monocytogenes strains The ATR of the four L. monocytogenes strains was also investigated. Induction of the ATR was performed in BHI broth adjusted to pH 5.1, whereas control cells were cultured in BHI broth adjusted to pH 7.0. Survival curves were then generated following secondary culture in BHI broth adjusted to pH 3.0 at 20 °C. The survival curves appeared to show enhanced survival of EGD-e and 77C1 cells in which the ATR had been induced in comparison with cells in which the ATR was not induced (Fig. 4A and B). Significant differences were observed between the log (N/N0) values for strain EGD-e with and without induction of the ATR following exposure to low pH for 45, 60, and 90 min (Student's t-test, P < 0.01) and between log(N/N0) values for strain 77C1 with and without induction of the ATR following exposure to low-pH stress for 45, 60, 90, and 120 min (Student's t-test, P < 0.01). These findings suggest the presence of a functional ATR in the acid-susceptible strains. In contrast, survival curves for strains 72C2 and 72C3 in which the ATR
Fig. 3. Survival curves of L. monocytogenes strains EGD-e (A), 77C1 (B), 72C2 (C), and 72C3 (D) following exposure to acidic conditions adjusted to pH3.0 at 20 °C. The strains used were subjected to acid stress in PBS (squares), BHI broth (diamonds), PBS supplemented with glucose (triangles) and PBS supplemented with glutamate (crossbars). Each data point represents the mean ± standard error of the mean based on a total of six biological replicates. 4
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Fig. 4. Survival curves of L. monocytogenes strains EGD-e (A), 77C1 (B), 72C2 (C), and 72C3 (D) with induction of the ATR (squares) or without induction of the ATR (diamonds) following culture in BHI broth adjusted to pH3.0 at 20 °C. Each data point represents the mean ± standard error of the mean based on a total of six biological replicates.
tolerant of low-pH conditions than non-persistent strains. Faleiro et al. (2003) showed that L. monocytogenes strains isolated from cheese tend to be more tolerant of low-pH conditions than strains isolated from meat and fish, suggesting that differences in the frequency of exposure to low pH promote higher acid tolerance in strains commonly found in cheese. The low frequency of exposure to acidic conditions may allow the acid-susceptible L. monocytogenes strains to become persistent in meat processing facilities. Although our findings were inconsistent with those reported by Lundén et al. (2008), the demonstrated persistence of both the less acid-susceptible and acid-susceptible strains supports the suggestion that almost any L. monocytogenes strains can become persistent if it is introduced into a suitable niche that provides both protection from lethal stress and growth-permissive conditions, as described by Ferreira et al. (2014). Therefore, the persistence of strains exhibiting the acid-susceptible phenotype suggest that environmental conditions are the primary contributor to the establishment of persistence by L. monocytogenes strains (Carpentier and Cerf, 2011; Ferreira et al., 2014). The persistent strains 72C3 and 72C5 exhibited decreased susceptibility to low pH when compared with the other persistent strains. Both of these strains, which were isolated from the same sample, responded similarly to acidic conditions, and this response appeared to remain stable during storage. It should be noted that these persistent strains were isolated simultaneously with strains assigned to a different genotype, including strain 72C2. The strains assigned to a different genotype than the persistent strains were considered transient, because they
was induced exhibited no apparent inactivation phase up to 240 min, whereas the survival curves for cells of these strains in which the ATR had not been induced exhibited an initial plateau-phase followed by an inactivation phase (Fig. 4C and D). 4. Discussion No significant difference in survival following H2O2 exposure was observed between persistent and control strains, indicating that the persistent strains investigated in the present study have no unique H2O2 response characteristics. In contrast, all persistent strains except 72C3 and 72C5 were highly susceptible to low pH. The susceptibility of these persistent strains to acidic conditions was comparable to that of the known acid-susceptible control strain EGD-e (Karatzas et al., 2012). This reference strain was shown to be one of the most sensitive to gastric fluid (pH 3.5) among 50 L. monocytogenes strains examined (Olier et al., 2004). In addition, the GAD system was shown to be nonfunctional in strain EGD-e (Karatzas et al., 2012; Olier et al., 2004). Strain 77C1, a persistent strain exhibiting acid-susceptibility, did not exhibit increased survival in the presence of exogenous glutamate in the present study. Such common characteristics between strain EGD-e and the persistent strains examined in the present study suggest these strains possess unique acid responses, as reported by Karatzas et al. (2012). Lundén et al. (2008) reported that persistent strains isolated from meat products or meat processing facilities are significantly more 5
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Conflicts of interest
were only isolated on two sequential days (Ochiai et al., 2014). The simultaneous isolation of persistent and transient strains suggests that the transient strains have an effect on the isolation of persistent strains that are less acid susceptible. However, how the transient strains are involved in the emergence of the persistent strains exhibiting less acidsusceptibility remains unclear. A nutrient-dependent response to low pH was observed in strains exhibiting the less acid-susceptible phenotype but not in strains exhibiting the acid-susceptible phenotype. Increased survival of strain EGD-e following low-pH exposure was observed under low-nutrient conditions, but the biological significance of these findings remains unclear. Further analyses of survival under acidic conditions were performed in PBS supplemented with glutamate or glucose. Survival curves for strains EGD-e and 77C1 cultured under both conditions were similar to those of these strains cultured in acidified PBS alone. These findings showed that not only the GAD system, which was reported elsewhere (Karatzas et al., 2012), but also energy-dependent mechanisms, including F0F1-ATPase, were nonfunctional in acid-susceptible strains. In contrast, strain EGD-e was shown to harbor genes encoding proteins involved in the GAD system and F0F1-ATPase responses (Cotter et al., 2000, 2005; Glaser et al., 2001). All survival curves for strains exhibiting the acid-susceptible phenotype following exposure to acidic conditions at 20 °C were characterized by an initial plateau phase that was almost 30 min in duration, as shown in Fig. 3A and B. A plateau phase of comparable duration was observed in the survival curves of strains exhibiting the less acidsusceptible phenotype subjected to the same stress in PBS at 20 °C (Fig. 3C and D). These findings suggest that the acid-susceptible phenotype elicits an initial acid response corresponding to that of the less acid-susceptible phenotype. Such an initial response may include the involvement of endogenous glucose and glutamate or acid tolerance mechanisms independent of glucose, glutamate, and BHI components. In addition, we cannot exclude the possibility that no acid response occurs during the initial phase. In contrast, differences in the inactivation phase were observed between strains exhibiting the acidsusceptible and less acid-susceptible phenotypes. Survival curves indicated more rapid inactivation of the acid-susceptible strains compared with the less acid-susceptible strains. This phenomenon may result from dysfunctions in multiple acid response mechanisms, including the GAD system and energy-dependent mechanisms described above. Both the less acid-susceptible and acid-susceptible strains exhibited an ATR. Survival curves for acid-susceptible strains in which the ATR was induced exhibited two phases, a plateau phase and an inactivation phase, whereas curves for the less acid-susceptible strains in which the ATR was induced appeared to be comprised of only a plateau phase until at least 4 h after the initiation of stress exposure. These findings suggest that acid-susceptible strains in which the ATR was induced did not exhibit a robust response to subsequent exposure to low pH, in contrast to that observed with the less acid-susceptible strains. These results also suggest that the subsequent response to low pH takes precedence over the first response to weak acid exposure.
None. Acknowledgments This study was supported in part by the Ito Foundation. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fm.2019.103312. References Abee, T., Koomen, J., Metselaar, K.I., Zwietering, M.H., den Besten, H.M., 2016. Impact of pathogen population heterogeneity and stress-resistant variants on food safety. Annu. Rev. Food Sci. Technol. 7, 439–456. Adrião, A., Vieira, M., Fernandes, I., Barbosa, M., Sol, M., Tenreiro, R.P.,, Chambel, L., Barata, B., Zilhao, I., Shama, G., Perni, S., Jordan, S.J., Andrew, P.W., Faleiro, M.L., 2008. Marked intra-strain variation in response of Listeria monocytogenes dairy isolates to acid or salt stress and the effect of acid or salt adaptation on adherence to abiotic surfaces. Int. J. Food Microbiol. 123, 142–150. Borucki, M.K., Peppin, J.D., White, D., Loge, F., Call, D.R., 2003. Variation in biofilm formation among strains of Listeria monocytogenes. Appl. Environ. 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5. Conclusions This study demonstrated that the persistent L. monocytogenes strains examined were highly susceptible to low pH, but their susceptibility to H2O2 was comparable to that of control strains. However, the emergence of a few persistent strains that were less susceptible to acid was also observed. Our findings indicating a predominance of the acidsusceptible phenotype suggest that environmental conditions contribute to the establishment of persistence by acid-susceptible L. monocytogenes strains. The acid-susceptible phenotype of persistent strains was associated with unique responses to stress resulting from acidic conditions, including a nutrient-independent response. In addition, we found that exogenous glucose and glutamate had no effect on survival under acidic conditions. 6
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