Food Microbiology 46 (2015) 154e160

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Effects of osmotic pressure, acid, or cold stresses on antibiotic susceptibility of Listeria monocytogenes Anas A. Al-Nabulsi a, *, Tareq M. Osaili a, Reyad R. Shaker a, Amin N. Olaimat b, Ziad W. Jaradat c, Noor A. Zain Elabedeen a, Richard A. Holley b a b c

Department of Nutrition and Food Technology, Jordan University of Science and Technology, Irbid, Jordan Department of Food Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada Department of Biological and Genetic Engineering, Jordan University of Science and Technology, Irbid, Jordan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 March 2014 Received in revised form 12 July 2014 Accepted 18 July 2014 Available online 7 August 2014

Prevalence of antibiotic resistance of Listeria monocytogenes isolated from a variety of foods has increased in many countries. L. monocytogenes has many physiological adaptations that enable survival under a wide range of environmental stresses. The objective of this study was to evaluate effects of osmotic (2, 4, 6, 12% NaC), pH (6, 5.5, 5.0) and cold (4
C) stresses on susceptibility of three isolates of L. monocytogenes towards different antibiotics. The minimal inhibitory concentrations (MICs) of tested antibiotics against unstressed (control), stressed or post-stressed L. monocytogenes isolates (an ATCC strain and a meat and dairy isolate) were determined using the broth microdilution method. Unstressed cells of L. monocytogenes were sensitive to all tested antibiotics. In general, when L. monocytogenes cells were exposed to salt, cold and pH stresses, their antibiotic resistance increased as salt concentration increased to 6 or 12%, as pH was reduced to pH 5 or as temperature was decreased to 10
C. Results showed that both meat and dairy isolates were more resistant than the ATCC reference strain. Use of sub-lethal stresses in food preservation systems may stimulate antibiotic resistance responses in L. monocytogenes strains. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Listeria monocytogenes Antibiotic susceptibility Osmotic pressure Acid stress Cold stress Stress adaptation

1. Introduction Listeria monocytogenes is a Gram-positive organism and one of 5 five species including Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria ivanovii, and Listeria grayi which comprise this genus (Farber and Peterkin et al., 1991). Recently, two newly identified species (Listeria marthii and Listeria rocourtiae) were reported (Graves et al., 2010; Leclercq et al., 2009). L. monocytogenes is widely distributed and has been isolated from a variety of readyto-eat and raw dairy, meat and meat products, sea foods and fresh produce (Bell and Kyriakides, 2005). L. monocytogenes is a major concern for food producers, health regulatory officials, and consumers since it is considered one of the more virulent foodborne pathogens (McLauchlin et al. 2004). Although the incidence of listeriosis is rare compared to illness caused by other foodborne pathogens such as Escherichia coli O157:H7, Campylobacter jejuni or Salmonella spp., L. monocytogenes

* Corresponding author. Tel.: þ962 02 7201000; fax: þ962 02 7201078. E-mail address: [email protected] (A.A. Al-Nabulsi). http://dx.doi.org/10.1016/j.fm.2014.07.015 0740-0020/© 2014 Elsevier Ltd. All rights reserved.

has been extensively studied during the past several decades because of its high (20%) case-fatality and high hospitalization (90%) rates (Lianou and Sofos, 2007). Furthermore, L. monocytogenes is a versatile organism that has the ability to survive for long periods under adverse conditions including cold storage, high NaCl concentrations and acidic pH (Rhoades et al., 2009). Antibiotic treatment of infectious listeriosis is indicated and has proven to be successful. These treatments usually rely upon the use of b-lactam antibiotics such ampicillin or penicillin, alone or in combination with an aminoglycoside (gentamicin). However, with patients allergic to b-lactams, trimethoprim and a sulfonamide have been used as an alternative with success (Conter et al., 2009). Even though it has been experimentally shown that L. monocytogenes strains are generally susceptible to a wide range of antibiotics, occasionally antibiotic resistant L. monocytogenes strains have been observed (Depardieu et al. 2007). The first multidrug resistant strain of L. monocytogenes was reported in 1988 by Poyart-Salmeron et al. (1990). Since then, L. monocytogenes strains have been isolated from food, environmental and human clinical samples which have shown resistance to one or more antibiotics (Morvan et al., 2010).

A.A. Al-Nabulsi et al. / Food Microbiology 46 (2015) 154e160

During food processing bacteria may encounter a variety of conditions that may cause chemical (acids, ethanol, alkaline, chlorine and salts) or physical (heat, radiation and pressure) stresses (Yousef and Courtney, 2003). When bacteria are exposed to mild forms of these stresses, opportunity is provided for them to improve their ability to adapt and become resistant to subsequent more extreme exposures through physiological adjustment, enabling reproduction (Depardieu et al. 2007; Hill et al. 2002). Furthermore, the adaptive responses to these stresses may enhance resistance to others such as exposure to antibiotics and lead to what is termed “cross-protection” (Doyle et al. 2006; McMahon et al. 2007). L. monocytogenes may develop an adaptive response during exposure to sublethal food processing conditions (Hill et al. 2002). Lou and Yousef (1997) confirmed that L. monocytogenes adapted to pH 4.5e5.0 had increased resistance to lethal concentrations of H2O2, ethanol, and acid. In addition, L. monocytogenes is able to transfer antibiotic resistance genes to other strains of Listeria spp. as well as other pathogens, like Staphylococcus aureus, Enterococcus spp., and non-pathogenic bacteria (Courvalin, 1994; Walsh et al. 2001). Therefore, understanding the effects of stress during food processing on the antibiotic susceptibility of L. monocytogenes is important in developing strategies to facilitate effort to achieve clinical control over pathogens that originate from food. The objectives of the current study were to investigate the effect of osmotic, cold and acid stresses on the susceptibility of L. monocytogenes toward different antibiotics. 2. Materials and methods 2.1. Preparation of bacterial cultures Three strains of Listeria moncytogenes were used in the current study; L. moncytogenes ATCC 7644 and two L. moncytogenes isolates from processed meat and dairy products, respectively, in Jordan. These isolates were obtained from the Department of Nutrition and Food Technology at Jordan University of Science and Technology and were kept frozen in glycerol. They were maintained on Typtic Soy Agar (TSA, Oxoid Ltd., Basingstoke, UK) slants at 4
C and transferred bi-weekly to maintain viability. Working cultures were prepared by growth in 10 ml Tryptic Soy Broth (TSB, Oxoid) incubated at 37
C for 20 h. For tests, 100 ml was transferred to 100 ml TSB and incubated at 37
C for 20 h. The cultures were diluted in Mueller Hinton Broth (MHB, Oxoid) to give a final concentration of approximately 6 log10 CFU/ml in the reaction mixtures. 2.2. Antibiotics The three L moncytogenes isolates were challenged with 9 antibiotics (Table 1), chosen according to their mode of action and use in clinical therapy. Those that inhibit cell wall synthesis included vancomycin 98%, ampicillin 84.5% (Bio Basic Inc., Markham, ON, Canada) and penicillin G sodium salt 98% (Biochemika Int., Hangzhou, China). Those that inhibit protein synthesis included tetracycline hydrochloride 90%, gentamicin sulfate 59%, streptomycin sulfate 65% (Bio Basic Inc.), and doxycycline HCL 99.5% (Tocelo Chemicals B.V., Hertogenbosch, Netherlands). Those that inhibit nucleic acid synthesis included enrofloxacin 99.39% (Tocelo Chemicals B.V.) and ciprofloxacin 98% (Biochemika Int.) (Yanoeyama and Katsumata, 2006; Al-Nabulsi et al. 2011). 2.3. Preparation of antibiotic stock solutions Antibiotic stock solutions were prepared following the manufacturers' recommendations. Vancomycin, ampicillin, penicillin,

155

Table 1 Minimal inhibition concentration (MIC) breakpoints used to determine antibiotic susceptibility.a Antibiotics

MICb breakpoint (mg/ml) Sc

Aminoglycosides antibiotics Streptomycin 8 Gentamycin 4 Tetracycline antibiotics Tetracycline 4 Doxycycline 4 Aminopenicillin antibiotics Ampicillin 4 Penicillin 0.25 Glycopeptide antibiotics Vancomycin 4 Fluoroquinolone antibiotics Ciprofloxacin 1 Enrofloxacin 1

I

R

>8, 16 >4, 8

>16 >8

>4, 8 >4, 8

>8 >8

>4, 16 >0.25, 16

>16 >16

>4, 16

>16

>1, 2 >1, 2

>2 >2

a
de I'Antibiogramme de la Socie
te
Française de As proposed by the Comite Microbiologie (CA-SFM) in France (Acar et al., 1992). b MIC: (Minimal Inhibitory Concentration) the lowest concentration of the antibiotics required to inhibit the visible growth of the microorganism. c S: Susceptible, I: Intermediate, R: Resistant.

tetracycline, gentamicin and streptomycin were dissolved directly in 10 ml distilled water. Doxycycline and ciprofloxacin were dissolved in sterile distilled water with a drop (0.05 ml) of 1 N HCL. Enrofloxacin was first dissolved in a few drops of 0.1 N NaOH and then diluted with sterile distilled water. All stock solutions were sterilized by using 0.20 mm disposable syringe filter units (Toyo Roshi Kaisha, Ltd., Tokyo, Japan) and serially diluted to provide the desired concentrations (0.01e1000 mg/ml) in the reaction mixtures. 2.4. Preparation of stressed L. moncytogenes cultures Preliminary tests showed that the mild stresses to be used in the current study would decrease the initial number of cells by approximately 1.0 log10 CFU/ml except for osmotic stress at 12% NaCl, where the initial number of cells was decreased by approximately 2 log10 CFU/ml. The number of survivors was determined by plating on TSA before and after exposure to stress. 2.4.1. Osmotic stress Four different levels of NaCl were used; 2%, 4%, 8%, and 12% (wt/ vol) in TSB. The NaCl-supplemented suspensions were inoculated with L. monocytogenes and incubated at 37
C for 24 h. L. monocytogenes cells were harvested by centrifugation (Herolab, UniCen M, Wiesloch, Germany) at 4000 g for 20 min and the pellet was resuspended and diluted to give a final concentration of approximately 6 log10 CFU/ml in the reaction mixtures for antibiotic challenge. 2.4.2. Acid stress Three different levels of pH were used in the current study; 6.0, 5.5 and 5.0. The acid-stressed cells were prepared as described by Francois et al. (2006) and Osaili et al. (2008). Briefly, 10 ml of an overnight culture of L. monocytogenes (pH 6.3) was harvested by centrifugation at 4000 g for 20 min and the pellet was resuspended with 10 ml 0.1M potassium phosphate buffer previously adjusted to pH 6.0, 5.5 or 5.0 with 85% lactic acid (Sigma, St. Louis, MO, USA) and then held at room temperature for 30 min. The culture was diluted to give a final concentration of approximately 6 log10 CFU/ml in the reaction mixtures for antibiotic challenge.

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2.4.3. Cold stress Cold-stressed cultures were prepared as described by AlNabulsi et al. (2011). One ml of each freshly prepared L. moncytogenes cell suspension was added to 9 ml of sterile 0.1 M potassium phosphate buffer (pH 6.8) in 15 ml screw capped test tubes, mixed thoroughly for 1 min and stored 24 h at 10
C. Then, the culture was diluted to give a final concentration of approximately 6 log10 CFU/ml in the reaction mixtures for antibiotic challenge. 2.5. Preparation of post-stressed L. moncytogenes cultures To examine the effect of post-stress conditions on the susceptibility of L. monocytogenes toward tested antibiotics, cells stressed by osmotic, acid, and cold exposure were inoculated into TSB and incubated at 37
C for 24 h. Then the cells were harvested by centrifugation at 4000 g for 20 min, the pellets were resuspended and diluted to give a final concentration of about 6 log10 CFU/ml in the reaction mixtures. Cell suspensions were challenged by antibiotic exposure as described below. 2.6. Antimicrobial assays (broth microdilution method) The minimal inhibitory concentration (MIC), defined as the lowest concentration of antibiotic required to inhibit visible growth of the test microorganisms, was determined using the broth microdilution method according to the procedure described by Langfield et al. (2004) and Klare et al. (2005). Ninety-six well microtiter plates (CELLSTAR®, Greiner Bio-One, Frickenhausen, Germany) with a 300 ml well capacity were used to evaluate the effects of tested antibiotics on the viability of unstressed and stressed L. monocytogenes cells. In each well of the microtiter plate a total volume of 250 ml was used. First, 100 ml of a test strain previously diluted in MHB was added to give a final inoculum of 6 log10 CFU/ml with an optical density (OD) at 620 nm of 0.1. Then 100 ml of MHB plus 50 ml of the test antibiotic solution were added. Control wells contained 100 ml culture, 100 ml MHB and 50 ml of sterile distilled water. After incubation at 37
C for 24 h, the microtiter plates were shaken using a Titer plate shaker (Barnstead International, Dubuque, IA, USA) at 400 rpm for 2 min before the absorbance at 620 nm was measured using a microplate reader (Dynatech MR5000, Mount Holly NJ, USA). The lowest concentration of antibiotic at which the OD reading was 0.1, was considered the MIC of the tested antibiotic. To confirm the MIC, a 100 ml sample was taken at the end of incubation and viable cell numbers in visibly clear wells were assessed by plating on TSA. The MIC was confirmed if the viable cell numbers after incubation were not significantly different from the numbers at initial inoculation. Each antibiotic sensitivity trial was performed in triplicate.

3. Results 3.1. The susceptibility of unstressed L. monocytogenes to antibiotics All unstressed L. monocytogenes isolates were sensitive toward the tested antibiotics (Table 2). However, some variation in the susceptibility of unstressed isolates was noted, for example, the meat isolate was more susceptible to streptomycin than the ATCC strain or the dairy isolate. The latter was the most susceptible to tetracycline, ciprofloxacin and enrofloxacin while the ATCC strain was more susceptible to gentamicin, penicillin and vancomycin. All isolates showed the same susceptibility to ampicillin and doxycycline. 3.2. The susceptibility of stressed and post-stressed L. monocytogenes cells to antibiotics 3.2.1. Effect of osmotic stress on L. monocytogenes antibiotic susceptibility Stressed L. monocytogenes isolates at NaCl concentrations up to 12% showed increased resistance to the tested antibiotics. Osmotic stress at 2, 4, 6, and 12% NaCl for 24 h reduced the susceptibility of L. monocytogenes isolates when challenged with ampicillin, tetracycline, doxycycline and vancomycin. With increases in NaCl concentrations, bacterial antibiotic resistance increased to the extent that susceptible isolates became moderately resistant or resistant. Meat and dairy isolates previously exposed to 4, 6, and 12% NaCl were resistant to streptomycin and ciprofloxacin, although the meat isolate previously exposed to 4% was moderately resistant to ciprofloxcacin. In addition, after 12% NaCl exposure only the dairy isolate was resistant to gentamycin and enrofloxacin. The development of antibiotic resistance by meat and dairy isolates during NaCl exposure was greater than that shown by the ATCC reference strain. Further, this resistance increased or remained constant after removal of the stress (Table 2). 3.2.2. Effect of acid stress on L. monocytogenes antibiotic susceptibility It was notable that acid stress at pH 5.5 to 6.0 for 30 min tended to increase the resistance of L. monocytogenes isolates to most antibiotics, although with some isolateeantibiotic pairs, resistance development was not as pronounced as it was with osmotic stress. When L. monocytogenes cells were exposed to pH 5.0, all isolates were found to be moderately resistant to penicillin, however, the ATCC strain and the dairy isolate became resistant to streptomycin while the meat isolate became only moderately resistant. In contrast, the meat isolate became resistant to ciprofloxacin while the other two isolates became moderately resistant. The antibiotic resistance of post-acid-stressed L. monocytogenes isolates increased or remained the same as the acid-stressed cells (Table 3).

2.7. Interpretation of results The breakpoint MICs of tested antibiotics against L. monocytogenes were determined using reference values recom
de I'Antibiogramme de la Socie
te
Française mended by the Comite de Microbiologie (CA-SFM), France (Acar et al. 1992) (Table 1). 2.8. Statistical analysis Data obtained were evaluated using ManneWhitney U analysis as recommended by McMahon et al. (2007) using the Statistical Package for the Social Sciences (SPSS), version 11.0.

3.2.3. Effect of cold stress on L. monocytogenes antibiotic susceptibility Cold stress (10
C for 24 h) consistently increased the resistance of L. monocytogenes toward the tested antibiotics, although there were differences in the extent of the increases which were both strain and antibiotic dependent. Changes noted did not appear to occur more extensively with one specific strain or antibiotic; however, the meat and dairy strains tended to show enhanced resistance more often than the ATCC strain. Cold stress also increased the antibiotic resistance of L. monocytogenes isolates even when the stress was removed (Table 4).

þþþ þþþ þþþ þþþ

þ þþ þþþ þþþ

0 þ þþ þþþ

Post

þþþ þþþ þþþ þþþ

þþ þþ þþþ þþþ

þþþ þþþ þþþ þþþ

0.25 (S) 0 þ þþ þþþ 0.125 (S) þ þþ þþþ þþþ 0.06 (S) þþþ þþþ þþþ þþþ 0 þ þþ þþ

þþþ þþþ þþþ þþþ

þþþ þþþ þþþ þþþ

þþþ þþþ þþþ þþþ

þþ þþþ þþþ þþþ

þþ þþþ þþþ þþþ

þþþ þþþ þþþ þþþ

þ þþ þþþ þþþ

þ þþ þþþ þþþ

0 þ þþ þþþ

þ þþ þþþ þþþ

þ þþ þþþ þþþ

0 þ þþ þþþ

þ þþ þþþ þþþ

0 þþ þþþ þþþ

0.06 (S) þ þþ þþþ þþþ 0.125 (S) þ þþ þþþ þþþ 0.125 (S) þ þ þþ þþþ þ þþ þþ þþþ Dairy

Meat

þ e 0.5e2-fold increase in MIC (P < 0.05). þþ e 2.1e4-fold increase in MIC (P < 0.05). þþþ e greater than 4-fold increase in MIC (P < 0.05). 0 e no change (P > 0.05). S: Susceptible; I: Intermediate resistance.

þþþ þþþ þþþ þþþ

þ þþ þþ þþþ

0 þ þþ þþþ 0 þ þþ þþþ

þþ þþþ þþþ þþþ

Stress Post

0.5 (S) þ þ þþ þþþ 0.5 (I) þþ þþ þþþ þþþ 0.25 (S) þþþ þþþ þþþ þþþ

Stress Post

0.125 (S) þþþ þþþ þþþ þþþ 0.25(S) þþþ þþþ þþþ þþþ 1 (S) 0 þþ þþ þþþ

Stress Post

0.06 (S) þþ þþ þþþ þþþ 0.06 (S) þþ þþþ þþþ þþþ 0.06 (S) þþþ þþþ þþþ þþþ

Stress Post

0.25 (S) þ þ þþ þþþ 0.25 (S) þ þþ þþ þþþ 0.06 (S) þþþ þþþ þþþ þþþ

Stress

157

4. Discussion

0.125 (S) þ þþ þþþ þþþ 0.125(S) þ þþ þþþ þþþ 0.125 (S) þ þ þ þþþ 0.125 (S) 0 þ þ þþþ 1 (S) þ þ þþ þþþ 0.25 (S) þþþ þþþ þþþ þþþ

Stress

0% 2% 4% 6% 12% 0% 2% 4% 6% 12% 0% 2% 4% 6% 12% ATCC

8 (S) 0 þ þ þþ 4 (S) þþ þþþ þþþ þþþ 8 (S) þ þþ þþ þþþ

Post Stress

Post

Stress

Post

Stress

Post

Tetracycline Penicillin Ampicillin Gentamycin Streptomycin

MICs of antibiotic (mg/ml) against osmotically-stressed or post-osmotically-stressed L. monocytogenes Salt % Isolate

Table 2 Changes in MIC of antibiotics against osmotically-stressed or post-osmotically-stressed L. monocytogenes strains.

Doxycycline

Vancomycin

Ciprofloxacin

Enrofloxacin

A.A. Al-Nabulsi et al. / Food Microbiology 46 (2015) 154e160

Historically, most L. monocytogenes strains isolated from clinical, food and environmental samples have been found susceptible to antibiotics active against Gram-positive bacteria; however, antibiotic resistant isolates of L. monocytogenes have been observed over the last two decades (Zhang et al., 2007; White et al., 2002). In the current study, the three L. monocytogenes isolates examined were initially sensitive toward the antibiotics tested, but when they were exposed to osmotic, acid or cold stresses, their antibiotic resistance increased. If this represents a broader phenomenon, it may in part explain the emergence of antibiotic resistance among L. monocytogenes isolates from food products. Environmental stresses such as osmotic, acid and cold shockinduced mutation in bacterial cells has been reported to be associated with the development of antibiotic resistance (Foster, 2000; Jolivet-Gougeon et al. 2000). L. monocytogenes is osmotically tolerant and can grow at > 10% NaCl (McClure et al. 1989). The organism is able to adapt to elevated osmolarity by accumulating compatible solutes or osmolytes (Ko and Smith, 1999). In the present study, bacterial resistance toward ampicillin, tetracycline, doxycycline and vancomycin was consistently increased in response to increased osmotic stress from higher salt concentrations. With the other antibiotics, increased salt also increased bacterial resistance, but responses varied according to NaCl concentration. For example, isolates stressed at 12% NaCl generally showed increased antibiotic MICs greater than 4-fold, except for that of streptomycin against the ATCC strain which increased slightly less (2.1e4-fold). These results were similar to those of Alonso-Hernando et al. (2009) who found that the resistance of L. monocytogenes strains to various antibiotics increased after exposure to acidified sodium chlorite. In contrast, Faezi-Ghasemi and Kazemi (2015) reported that L. monocytogenes cells osmotically-stressed at 7% NaCl had decreased resistance to selected antibiotics including tetracycline, rifampicin, gentamycin, penicillin, ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. However, in that work L. monocytogenes PTCC 1297 (serotype 4a) was exposed to 7% NaCl for only 1 h before antibiotic challenge compared to the 24 h used in the present work. In further support of the present observations, McMahon et al. (2007) found that > 4.5% NaCl increased the antibiotic resistance of E. coli, Salmonella Typhimurium and S. aureus. In addition, Ganjian et al. (2012) found that NaCl concentrations up to 35% increased the antibiotic resistance of S. aureus cells. It is also important to note that the exposure of L. monocytogenes to NaCl stress can lead to cross-protection against other stresses such as heat and acid (Skandamis et al., 2008). In the presence of mild concentrations of weak acid preservatives, organisms have been shown to adapt by regulation of outer cell membrane protein synthesis (Beales, 2004), and by making changes in their cell membrane fatty acid composition to alter membrane permeability and fluidity (Diakogiannis et al., 2013). In the present study, acid stress consistently increased bacterial resistance toward ampicillin, tetracycline, doxycycline, gentamicin, vancomycin and enrofloxacin. It also increased resistance toward the remaining 3 antibiotics (streptomycin, penicillin, ciprofloxacin), but the extent of the increase varied with the bacterial strain. Nonetheless, increased antibiotic resistance was observed as the pH decreased. The MIC values of most (5e7) of the 9 antibiotics tested were increased when L. monocytogenes isolates were exposed to acid stress at pH 5.0. Similarly, Alonso-Hernando et al. (2009) found that L. monocytogenes cells exposed to citric acid were more resistant to a number of antibiotics than were unexposed cells. Al-Nabulsi et al. (2011) found that acid-stressed Cronobacter sakazakii cells were more resistant toward

158

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Table 3 Changes in MIC of antibiotics against acid-stressed or post-acid-stressed L. monocytogenes strains. Strain

ATCC

Meat

Dairy

pH

7 6 5.5 5 7 6 5.5 5 7 6 5.5 5

MICs of antibiotic (mg/ml) against acid-stressed or post-acid-stressed L. monocytogenes Streptomycin

Gentamycin

Ampicillin

Penicillin

Tetracycline

Doxycycline

Vancomycin

Ciprofloxacin

Enrofloxacin

Stress

Stress

Stress

Stress

Stress

Stress

Stress

Stress

Stress

8 (S) þ þ þþ 4 (S) þ þþ þþ 8 (S) 0 þ þþ

Post þ þ þþ þþ þþþ þþþ þ þ þþ

Post

0.125 (S) þþ þþþ þþþ þþþ þþþ þþþ 1 (S) þ þþ þþ þþþ þþþ þþþ 0.25 (S) þþþ þþþ þþþ þþþ þþþ þþþ

Post

0.125 (S) þ þ þþ þþ þþþ þþþ 0.125 (S) þ þ þþþ þþþ þþþ þþþ 0.125 (S) 0 þ þ þþ þ þþþ

Post

0.06 (S) þþ þþþ þþþ þþþ þþþ þþþ 0.125 (S) þþ þþþ þþþ þþþ þþþ þþþ 0.125 (S) 0 0 þ þþ þþ þþþ

0.25(S) þþþ þþþ þþþ 0.25 (S) þþ þþþ þþþ 0.06 (S) þ þþþ þþþ

Post þþþ þþþ þþþ þþþ þþþ þþþ þþ þþþ þþþ

0.06 (S) þ þþþ þþþ 0.06 (S) þþþ þþþ þþþ 0.06 (S) þ þþþ þþþ

Post þþ þþ þþþ þþþ þþþ þþþ þþ þþþ þþþ

Post

0.125 (S) 0 þ þþ þþ þþþ þþþ 0.25 (S) 0 þ þ þþ þþ þþ 1 (S) þ þ þ þþ þþ þþ

0.5 (S) 0 þ þþ 0.5 (S) þ þþ þþþ 0.25 (S) þ þþ þþþ

Post þ þ þþþ þþ þþ þþþ þþ þþ þþþ

Post

0.25 (S) 0 þ þ þþ þ þþ 0.125 (S) þþ þþ þþþ þþþ þþþ þþþ 0.06 (S) þ þþþ þþ þþþ þþþ þþþ

þ e 0.5e2-fold increase in MIC. þþ e 2.1e4-fold increase in MIC. þþþ e greater than 4-fold increase in MIC. 0 e no change. S: Susceptible.

tetracycline, tilmicosin, florfenicol, amoxicillin, ampicillin, vancomycin and enrofloxacin. McMahon et al. (2007) also found that E. coli, Salmonella Typhimurium and S. aureus stressed at pH 4.0 to 5.0 were more resistant to antibiotics than unstressed cells. Results from a number of studies showed that exposure of L. monocytogenes to acid stress increased its resistance to osmotic, ethanol, and oxidative stresses (Lou and Yousef, 1997; Phan-Thanh et al., 2000; Faleiro et al., 2003). In contrast with the present findings, FaeziGhasemi and Kazemi (2015) reported that L. monocytogenes exposed to pH 5.0 was more susceptible to tetracycline, rifampicin, gentamycin, penicillin, ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. Since the lengths of acid stress exposure in the two studies were similar (1 h), differences may have occurred because the L. monocytogenes cells used by Faezi-Ghasemi and Kazemi (2015) were in the exponential rather than the stationary phase as used in the present study. Storage temperature is one of the most important parameters regulating the activities of microorganisms in food systems. L. monocytogenes is a psychrotrophic microorganism which has the capacity to grow at temperatures as low as 4
C. In the current study, cold stress consistently increased resistance of tested strains toward tetracycline, doxycycline, vancomycin and enrofloxacin, and increased the resistance of the meat and dairy isolates, in particular, toward the other antibiotics. In other work with C. sakazakii, it was similarly found that exposure of cells to 4
C for

24 h increased bacterial resistance to kanamycin, neomycin, tetracycline, florfenicol, ampicillin, amoxicillin, vancomycin and enrofloxacin (Al-Nabulsi et al., 2011). However, McMahon et al. (2007) found that cold stress had the opposite effect; it reduced the antibiotic resistance of E. coli, S. Typhimurium and S. aureus. Nonetheless, alternative sigma (s) factors can produce dramatic changes in bacterial gene expression after exposure to environmental stress. The genome of L. monocytogenes encodes four alternative s factors including sB, sC, sH and sL (Glaser et al., 2001); however, sB which is encoded by sigB plays the major role in the survival of L. monocytogenes under stressful conditions (Gray et al., 2006). Sigma factor B was found to regulate >170 dependent genes in L. monocytogenes including gbu, betL, and opuC during osmotic stress; gadD1, gadD2, gadD3, gadT1 and gadT2 during acid stress, and betL, ltrC, fri and opuC during cold stress (Raengpradub et al., 2008). These genes encode cold shock proteins and compatible solutes (betaine and carnitine) that enable the bacterium to be more resistant to environmental and nutritional stresses (O'Byrne and Karatzas, 2008). The increase in antibiotic resistance observed in the present work may be related to one or more of the following mechanisms: reduction of cell wall antibiotic binding sites, amplification of genes responsible for efflux pump synthesis and operation, and induction of stress shock proteins (McMahon et al., 2007). Bacteria can respond to adverse environmental challenges involving osmotic,

Table 4 Changes in MIC of antibiotics against cold-stressed or post-cold-stressed L. monocytogenes strains. Isolate

ATCC Meat Dairy

Temp
C

37 10 37 10 37 10

MICs of antibiotic (mg/ml) against cold-stressed or post-cold-stressed L. monocytogenes Streptomycin

Gentamycin

Ampicillin

Penicillin

Tetracycline

Doxycycline

Vancomycin

Ciprofloxacin

Enrofloxacin

Stress

Stress

Stress

Stress

Stress

Stress

Stress

Stress

Stress

8 (S) þþ 4 (S) þþþ 8 (S) þþþ

Post þþ þþþ þþþ

Post

0.125 (S) þþþ þþþ 1 (S) þþþ þþþ 0.25 (S) þþþ þþþ

þ e 0.5e2-fold increase in MIC (P < 0.05). þþ e 2.1e4-fold increase in MIC (P < 0.05). þþþ e greater than 4-fold increase in MIC (P < 0.05). 0 e no change (P > 0.05). S: Susceptible.

Post

0.125 (S) þþþ þþþ 0.125 (S) þþþ þþþ 0.125 (S) þþþ þþþ

Post

0.06 (S) þþ þþþ 0.125 (S) þþ þþ 0.125 (S) þ þþ

Post

0.25 (S) þ 0 0.25 (S) þþ þþþ 0.06 (S) þþþ þþþ

Post

0.06 (S) þþ þþ 0.06 (S) þþþ þþþ 0.06 (S) þþþ þþþ

Post

0.125 (S) þþ þþþ 0.25 (S) þþþ þ 1 (S) þ þ

Post

0.5 (S) þ þþ 0.5 (S) þþþ þþþ 0.25 (S) þþþ þþþ

Post

0.25 (S) þ þ 0.125 (S) þþþ þþþ 0.06 (S) þþþ þþþ

A.A. Al-Nabulsi et al. / Food Microbiology 46 (2015) 154e160

acid and cold stress by down-regulation of penicillin binding proteins in the cell wall, can enhance their ability to reduce cytoplasmic concentrations of antibiotics by enhanced efflux pump operation and can improve survival by synthesis of chaperone proteins to maintain protein functionality during stress or antibiotic challenge (Bremer and Kr€ amer, 2000; Rahmati-Bahram and Magee, 1997). It was observed in the present work that L. monocytogenes continued to show higher levels of antibiotic resistance after removal of each of the three types of stress. Similar results were reported by McMahon et al., 2007 who found that NaCl or acidstressed E. coli and S. aureus cells were more resistant (4 times) to the antibiotics tested than control cells. On the other hand, postNaCl-stressed or post-acid-stressed S. Typhimurium cells were similarly or less resistant than unstressed cells. Since retention of enhanced antibiotic resistance was also observed with the three strains of L. monocytogenes used in the present study for >1 d (at least 10 generations) after removal of the stress, it is possible that mild stress may contribute to antibiotic resistance, at least temporarily or for longer sustained periods.

5. Conclusions L. monocytogenes is exposed to a variety of environmental stresses during food processing and appears able to adapt to many of these stresses. It was found that cold, acid and osmotic stresses increased the resistance of this pathogen to 9 currently used antibiotics. Increases in antibiotic resistance observed were maintained for at least a day after the stress was removed. It was significant that cold (10
C), acid (pH 5) and osmotically (12% NaCl)-stressed cells were more resistant to streptomycin, gentamycin, ampicillin, penicillin, ciprofloxacin and enrofloxacin. Exposure to milder acid or NaCl stress elicited levels of antibiotic resistance that were lower. Results suggest that the increased use of bacteriostatic (sub-lethal) stress, rather than bactericidal treatments in food preservation systems has the potential to increase the antibiotic resistance among L. monocytogenes if present.

Acknowledgment This project was financially supported by the Deanship of Research at Jordan University of Science and Technology (grant # 162/2011), Irbid, Jordan.

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