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Appl Environ Microbiol. Mar 2006; 72(3): 2231–2234.
PMCID: PMC1393204

Tolerance of Listeria monocytogenes to Cell Envelope-Acting Antimicrobial Agents Is Dependent on SigB

Abstract

Mutation of sigB impairs the ability of Listeria monocytogenes to grow in sublethal levels, and to survive in lethal concentrations, of the bacteriocins nisin and lacticin 3147 and the antibiotics ampicillin and penicillin G. SigB may therefore represent an attractive target for the development of new control and treatment strategies for this important pathogen.

Listeria monocytogenes is the etiological agent of listeriosis, an opportunistic infection that affects primarily pregnant and immunocompromised individuals. Food is the major source of infection, and those foods most frequently implicated include soft cheeses, dairy products, salads, and refrigerated ready-to-eat products (6). The β-lactams ampicillin and penicillin G are the antibiotics of choice in the treatment of listeriosis (12); however, despite their therapeutic use, up to one-third of patients die (16). As a result, listeriosis is a significant cause of mortality due to food-borne disease; it is estimated to be responsible for approximately 27.6% of food-related deaths in the United States annually (16). L. monocytogenes is recognized as a serious risk to public health and food safety, and the bacterium was responsible for 71% of all recalls of food products due to bacterial contamination in the United States between 1993 and 1998 (25).

L. monocytogenes must overcome the numerous environmental extremes encountered during food processing, handling, and storage and in vivo following consumption. One important mediator of the bacterium's stress responses is the alternative sigma factor SigB. It has been shown to assist the in vitro survival of cells under a variety of environmental insults, including low pH, high osmolarity, and elevated bile concentrations, and during oxidative stress and carbon starvation (1-3, 7, 8). It is also becoming increasingly evident that SigB regulates stress loci important for intrahost survival, such as bile salt hydrolase (bsh) (3, 22), and also assists in the regulation of dedicated virulence factors such as the principal virulence regulator PrfA (19, 20).

Bioinformatic analysis of the SigB regulon.

The entire L. monocytogenes SigB regulon has not yet been defined; however, initial efforts using a 208-gene microarray identified 55 genes positively regulated by SigB (i.e., their expression is significantly reduced in a sigB mutant) (13). Analysis of the data produced by that study and examination of the sequenced genome of L. monocytogenes strain EGDe with the SigB consensus promoter sequence that was generated (GTTTN13-17GGGWAT was entered as a “Pattern search” in the genome web server ListiList [http://genolist.pasteur.fr/ListiList/], and the search was restricted to patterns located within 350 bp upstream of a predicted open reading frame and showing one mismatch to the consensus) revealed a number of loci which, based on homology searches, may contribute to tolerance of antimicrobial compounds. These loci encode putative efflux pumps, penicillin binding proteins, autolysins, or proteins involved in the modification of the cell envelope (Table (Table1).1). It was therefore decided to investigate whether the SigB regulon contributes to the tolerance of bacteriocins (peptide antimicrobials) and antibiotics (nonpeptide antimicrobials).

TABLE 1.
Genetic loci that are regulated/putatively regulated by SigB and that may contribute to the tolerance of antimicrobial agents

SigB contributes to bacteriocin tolerance.

The growth rate of a sigB mutant (a nonpolar internal deletion mutant) (24) was comparable to that of the wild type when grown in brain heart infusion (BHI) broth at 37°C, indicating that SigB is not required for growth under normal physiological conditions (data not shown). Addition of sublethal levels of nisin (a bacteriocin currently used as a biopreservative) (5) or lacticin 3147 (a bacteriocin that shows potential as a biopreservative) (11) had noticeable effects on growth (Fig. 1A and B). Survival assays performed with lethal levels of each bacteriocin revealed more-dramatic differences at every concentration tested. Although there was an initial kill of the parent strain at the levels shown in Fig. Fig.1,1, it was detected at high numbers over the 6-h experiment, whereas the mutant was not detected after 1 h in the broth supplemented with nisin or after 4 h in the broth supplemented with lacticin. At bacteriocin levels higher than these, the parent was rapidly and completely inactivated. Interestingly, agar well diffusion assays (not shown), overlay assays (Fig. 1E and F), and MIC determinations by the broth dilution method (using twofold serial dilutions of the bacteriocins in BHI broth) (data not shown) did not reveal any differences between the strains. However, these experiments are analyzed at a single time point after 24-h incubation periods, after which the numbers of wild-type and mutant bacteria were identical in our experiments (data not shown). Overall, our data strongly suggest an important role for SigB in bacteriocin tolerance, which may be important for the survival of L. monocytogenes in foods. The results also highlight the importance of assay selection in the assessment of the contribution of a specific locus to bacteriocin tolerance. For example, Moorehead and Dykes (17) concluded that SigB did not play a role in L. monocytogenes tolerance of the bacteriocins nisin and sakacin A as determined by overlay assays.

FIG. 1.
(A to D) Growth and survival of wild-type L. monocytogenes 10403S ([filled square]) and the sigB mutant (○) in broth supplemented with bacteriocins. Overnight cultures were inoculated (3%) into BHI broth supplemented with either ~45 AU/ml lacticin ...

SigB contributes to antibiotic tolerance.

The abilities of the wild type and the sigB mutant to withstand exposure to penicillin G and ampicillin were compared, because these are the antibiotics of choice in the treatment of listeriosis (12). Initial agar diffusion experiments with antibiotic disks (Oxoid) revealed that the diameters of the zones of bacterial growth inhibition surrounding the filter disks were similar for the two strains (Fig. 2E and F). In addition, there was no difference in MICs as determined by the broth dilution method (using twofold serial dilution of the antibiotics in BHI broth) (data not shown). However, detailed growth curves (Fig. 2A and B) and survival assays (Fig. 2C and D) revealed that the sigB mutant was significantly impaired in growth in sublethal levels of each antibiotic and was killed more rapidly at lethal levels. It is therefore possible that SigB contributes significantly to the survival of L. monocytogenes in clinical settings. Designing future therapies to target SigB may improve the treatment of listeriosis, which is presently inefficient.

FIG. 2.
(A to D) Growth and survival of wild-type L. monocytogenes 10403S ([filled square]) and the sigB mutant (○) in broth supplemented with antibiotics. Overnight cultures were inoculated (3%) into BHI broth supplemented with either 0.02 μg/ml penicillin ...

Role of SigB in the tolerance of antimicrobial agents.

The exact role of SigB in L. monocytogenes tolerance of antimicrobial agents has yet to be determined. It is likely that SigB plays a role in controlling membrane characteristics (e.g., charge or lipid composition) and that altering these properties significantly affects the cell's ability to tolerate antimicrobial compounds. It is noteworthy that bacteriocins and antibiotics act on bacterial cell walls and that L. monocytogenes sigB mutants have been shown to be significantly more sensitive than parent cells to stresses that exert their effects on the cell wall, such as bile (3). In addition, cell surface alterations have previously been shown to be important in the tolerance of several bacteriocins and antibiotics (4, 9, 10, 18, 23). SigB may also regulate general stress proteins or proteins involved in extrusion of antimicrobials out of the cell. Indeed, htrA (lmo0292), which encodes a putative molecular chaperone that has been shown to be involved in tolerance of penicillin G (21), and mdrL (lmo1409), which encodes an antibiotic efflux pump (15), both possess consensus SigB binding sites.

In conclusion, we report our novel observation that SigB contributes positively to L. monocytogenes tolerance of the bacteriocins nisin and lacticin 3147 and of the antibiotics ampicillin and penicillin G. Because SigB may contribute to the survival of L. monocytogenes both in the food-processing environment and in vivo during infection, it may represent an attractive target for the development of new control and treatment strategies for this important pathogen.

Acknowledgments

We acknowledge the funding received from the Irish Government under the National Development Plan 2000—2006 and through funding of the Alimentary Pharmabiotic Centre by the Science Foundation of Ireland Centres for Science Engineering and Technology (CSET) scheme.

We also thank Martin Wiedmann (Cornell University) and Paul Cotter (University College Cork) for supplying L. monocytogenes and Lactococcus lactis strains, respectively.

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