Listeria monocytogenes : Antibiotic Resistance in Food Production

Abstract

Listeria monocytogenes is an opportunistic human pathogen that causes listeriosis, a disease that mainly affects the immunocompromised, the elderly, infants, and pregnant women. Listeriosis has become increasingly common in the last 25 years since the first foodborne outbreak was noted. Treatment for listeriosis currently consists primarily of supportive therapy in conjunction with the use of intravenous antibiotics. Antibiotics have been commercially available for over 60 years for treatment of a myriad of clinical diseases. Bacteria resistant to antibiotics have been developing over this same period. This review seeks to elucidate the extent of antibiotic resistance in L. monocytogenes, the possible transmission mechanisms, and contributing factors to distribution of antibiotic resistance among Listeria species, and possible control strategies.

Introduction

L isteria monocytogenes was identified as an animal pathogen >75 years ago but only gained prominence as a human pathogen in the last 25 years. Since its first appearance, this organism has been implicated as the causative agent in several outbreaks of foodborne listeriosis in North America and Europe (Jacquet et al., 1995; Dalton et al., 1997; Miettinen et al., 1999; Aureli et al., 2000; Graves et al., 2005). Currently, L. monocytogenes is considered an opportunistic human pathogen of high public concern that causes listeriosis, a disease that mainly affects the immunocompromised, the elderly, infants, and pregnant women.

The emergence of listeriosis as an important disease could be the result of changes in social and economic patterns. During the past 50 years, improvements in medicine, public health, sanitation, and nutrition have resulted in increased life expectancy particularly in developed countries. The U.S. population is aging; in 2000 the elderly population was 35 million but is projected to be 72 million by 2030, when the elderly will constitute 20% of the U.S. population (U.S. Census Bureau, 2005). Cancer is also one of the leading causes of death in the United States (ACCT, 2001; Anonymous, 2006a, 2006b), and new AIDS cases are reported each year. Due to a rise in the number of immunocompromised individuals as a result of the emergence of diseases such as AIDS, the use of intensive cancer therapies, immunosuppressive drug therapies, organ transplants, and the rise in number of elderly people from the aging baby boomer population, L. monocytogenes has become a pathogen of serious concern.

Changes in food production practices, particularly centralization and consolidation, make ideal hygiene practices very challenging in the food-processing and food preparation environments. The increased use of refrigeration temperatures for food preservation also allows for prolonged L. monocytogenes proliferation in foods. Low temperature storage prevents the growth of other microorganisms in food allowing L. monocytogenes, which is generally a poor competitor, to grow (Cole et al., 1990; Walker et al., 1990; Okamoto et al., 1994). Finally, the demand for fresh minimally processed or natural foods among consumers has also increased. Most of these foods, including fruits and vegetables, require little cooking or preparation and do not contain preservatives that would prevent L. monocytogenes growth and survival.

In addition, the ubiquitous nature of this organism allows it to encounter various factors that are aimed at food preservation, improving animal growth performance, efficiency of feed conversion in animal husbandry, and reducing or killing pathogenic bacteria. One such measure is the use of antibiotics as growth promoters. Since the discovery of penicillin in 1929, the use of antibiotics in food husbandry has been on the rise (WHO, 2002). The U.S. Food and Drug Administration approved the use of antibiotics for animal feed additives without a veterinary prescription in 1951 (Jones and Ricke, 2003). At about this same time most European countries also approved use of antibiotics as animal feed additives (Castanon, 2007). In recent years, the food industry has seen an emergence of antibiotic-resistant bacteria strains, including pathogens of public health importance such as Salmonella, Staphylococcus aureus, Escherichia coli, and L. monocytogenes (Van den Bogaard et al., 2001; White et al., 2001; Dar et al., 2006; Li et al., 2007). The first antibiotic-resistant strain of L. monocytogenes was described in France in 1988 and since then many more resistant strains have been isolated from food (Poyart-Salmeron et al., 1990; Rota et al., 1996; Walsh et al., 2001; Antunes et al., 2002) and human sporadic Listeria cases (Tsakris et al., 1997; Safdar and Armstrong, 2003). Partially in response to this increase in antibiotic resistance in foodborne pathogens, the European Union has banned the use of antibiotics as animal feed additives (with the exception of coccidiostats) as of January 2006 (Castanon, 2007).

Although our understanding of the culture characteristics, environmental ecology, and virulence of L. monocytogenes has improved, it is clear that very limited information exists on the antibiotic resistance patterns of L. monocytogenes. Antibiotic usage in food animals is prevalent and in most cases these antibiotics have historically been used in large quantities for prolonged periods (National Research Council, 1999). This would allow for selection of resistant bacteria, which may infect humans, and treatment of these antibiotic-resistant strains would become difficult especially if strains arise that are resistant to current antibiotic regimens used to treat listeriosis. Therefore, there is a need to understand the extent of antibiotic resistance, the antibiotic resistance profile, and the transmission dynamics of antibiotic resistance as well as the antibiotic resistance acquisition of L. monocytogenes.

Antibiotic Therapy for Listeriosis

Invasive listeriosis is treated mainly by supportive therapy along with intravenous penicillin or ampicillin in combination with an aminoglycoside such as gentamicin (Swaminathan and Gerner-Smidt, 2007). For patients who are allergic to penicillin, vancomycin/teicoplanin or trimethoprim/sulfamethoxazole can be used (Swaminathan and Gerner-Smidt, 2007). Scheld et al. (1979) compared penicillin, ampicillin, gentamicin, rifampicin, penicillin plus rifampicin, penicillin plus gentamicin, and ampicillin plus gentamicin for treating listerial meningitis in a rabbit model and found that the combination of ampicillin plus gentamicin was the most effective treatment. Since even with ampicillin plus gentamicin treatment significant mortality still occurs (Levidiotou et al., 2004), Sipahi et al. (2008) examined the effects of a potential alternative antibiotic moxifloxacin for treating listerial meningitis. Sipahi et al. (2008) found that moxifloxacin was as effective as ampicillin plus gentamicin, but not more effective. L. monocytogenes has been found to be naturally susceptible to penicillins, aminoglycosides, trimethoprim, tetracycline, macrolides, and vancomycin, but has either reduced susceptibility or resistance to sulfomethoxazole, cephalosporins, and first-generation quinolones while being generally susceptible to fluoroquinolones (second-generation quinolones) (Troxler et al., 2000). It is rare to find acquired antimicrobial resistance in human clinical strains (Hansen et al., 2005), but resistant strains have been found with increasing frequency in animals (Srinivasan et al., 2005). This finding is a cause for concern and suggests that resistance in clinical human isolates may emerge in the near future.

Mechanisms of Antibiotic Resistance in L. monocytogenes

Antibiotic resistance in L. monocytogenes is chiefly caused by three mobile genetic elements: self-transferable plasmids, mobilizable plasmids, and conjugative transposons (Charpentier et al., 1999). Efflux pumps have also been reported to be present in Listeria (Godreuil et al., 2003).

Resistance mediated by conjugation

Plasmid pIP501, which has a broad host range and confers resistance to chloramphenicol, macrolides, lincosamides, and streptogramins, was first identified in Streptococcus agalactiae (Evans and Macrina, 1983). This plasmid was the first reported to be transferable by conjugation to L. monocytogenes, replicate within Listeria, be transferable between species of Listeria as well as transfer back to Streprococcus (Pérez-Diaz et al., 1982). Another broad-host-range plasmid, pAMβ1 of Enterococcus faecalis, conferring resistance to erythromycin, was transferred successfully by conjugation from E. faecalis to L. monocytogenes, was found to replicate in the new host and be transferred by conjugation between strains of L. monocytogenes and from L. monocytogenes back to E. faecalis (Flamm et al., 1984). In another study, a plasmid carrying the vanA gene cluster, conferring glycopeptide resistance, was transferred from Enterococcus faecium to L. monocytogenes, Listeria ivanovii, and Listeria welshimeri (Biavasco et al., 1996). Plasmid pIP823 has a broad host range, including L. monocytogenes, E. faecalis, and E. coli. Charpentier et al. (1999) were able to transfer pIP823 between L. monocytogenes and E. faecalis, and between L. monocytogenes and E. coli. Transfer of antibiotic resistance from lactic acid bacteria to Listeria by conjugation in a food matrix was studied by Toomey et al. (2009). They were able to transfer erythromycin resistance in vitro from the lactic acid bacteria to Listeria, but in a food matrix transfer of resistance was seen only between lactic acid bacteria (Toomey et al., 2009).

The conjugative transposon designated as Tn916 has a broad host range, carries the tetM tetracycline resistance gene, and was originally found in E. faecalis (Franke and Clewell, 1981). Vicente et al. (1988) demonstrated the transfer by conjugation of Tn916 from E. faecalis to Listeria innocua. It could also mediate its own transfer from L. innocua to other Listeria species, and from there back to E. faecalis (Celli and Trieu-Cuot, 1998). A Tn916-related transposon, Tn1545, was transferred from E. faecalis to L. monocytogenes both in vitro and in vivo (Doucet-Populaire et al., 1991; Poyart-Salmeron et al., 1992).

Resistance mediated by efflux pumps

Efflux pumps are proteins found in both Gram-positive and Gram-negative bacteria that are involved in the removal of toxic substrates, including antibiotics, from within cells to the external environment (Bambeke et al., 2000). These pumps may be specific to a substrate or may transport compounds with dissimilar structures, allowing them to transport antibiotics of many classes and thus may be associated with multiple drug resistance (Webber and Piddock, 2003). There have been two efflux pumps described in L. monocytogenes, MdrL and Lde. The genes that encode for these efflux pumps, mdrL and lde, seem to be universally present in L. monocytogenes (Mereghetti et al. 2000). Macrolide antibiotics and cefotaxme as well as heavy metals and ethidium bromide are extruded by MdrL (Mata et al., 2000). Fluoroquinolone resistance is associated with Lde as are resistance to the DNA intercalating dyes acridine orange and ethidium bromide (Godreuil et al., 2003).

Antibiotic-Resistant L. monocytogenes in Food and the Food-Processing Environment

A comprehensive list of L. monocytogenes antibiotic-resistant strains isolated from food and food-producing and food-processing areas is presented in Table 1. Rota et al. (1996) found that Listeria strains isolated from cheese and pork were resistant to multiple antimicrobial agents, although >80% of the strains of both food origins were found to be susceptible to penicillin G and ampicillin, whereas the proportion of isolates resistant to the cephalosporins cefotaxime and cefoxitin was nearly 100%. Walsh et al. (2001) reported that 0.6% of L. monocytogenes isolates from retail foods were resistant to one or more antibiotics compared to 19.5% of L. innocua isolates, suggesting that the ability to acquire antibiotic resistance may be species related. Antunes et al. (2002) also observed that L. monocytogenes isolates from poultry carcasses exhibited resistance to one or more antibiotics, implicating poultry as a potential vehicle for antibiotic-resistant foodborne illness. Srinivasan et al. (2005) isolated L. monocytogenes resistant to one or more antimicrobials from dairy farm environments, with all isolates exhibiting resistance to cephalosporin C, streptomycin, and trimethoprim. Miranda et al. (2008) isolated L. monocytogenes from conventionally and organically grown poultry and examined the antibiotic resistance patterns; conventionally grown poultry were resistant to four of the six antibiotics tested, whereas organically grown poultry (which were presumably not exposed to any antibiotics) were resistant to two of the six. Schwaiger et al. (2010) compared antibiotic resistance of Listeria spp. isolated from eggs from hens raised either conventionally or organically in Germany. They reported that all isolates were sensitive to the tested antibiotics, whether the birds were grown organically or in cages. Lyon et al. (2008) isolated L. monocytogenes from a turkey further processing plant and found that 59% of the isolates were resistant to ceftriaxone, 3% were resistant to ciprofloxacin, and 90% were resistant to oxacillin. Harakeh et al. (2009) found that all L. monocytogenes isolates from dairy based food products were resistant to at least one antibiotic. Pesavento et al. (2010) found that L. monocytogenes isolated from ready-to-eat foods had high levels of resistance to ampicillin, gentamycin, and methicillin.

Table 1.

Antibiotic-Resistant Listeria monocytogenes Isolated from Food and Food-Producing and Food-Processing Environments

Antibiotic	Reference	Isolated from
Ampicillin	Njagi et al. (2004)	Listeria monocytogenes type strains
	Srinivasan et al. (2005)	Dairy farm environment
	Yucel et al. (2005)	Meat products
	Walsh et al. (2001)	Retail meats
	Harakeh et al. (2009)	Dairy-based foods
Amikacin	Rota et al. (1996)	Pork sausage
Augmentin	Njagi et al. (2004)	L. monocytogenes type strains
Bacitracin	Li et al. (2007)	Bison
Cefotaxime	Li et al. (2007)	Bison
	Rota et al. (1996)	Cheese, pork sausage
Cefoxitin	Rota et al. (1996)	Cheese, pork sausage
Ceftriaxone	Lyon et al. (2008)	Processing plant
Cefuroxime	Njagi et al. (2004)	L. monocytogenes type strains
Cephalosporin C	Srinivasan et al. (2005)	Dairy farm environment
Cephalothin	Prazak et al. (2002)	Cabbage, environment
	Yucel et al. (2005)	Meat products
Chloramphenicol	Srinivasan et al. (2005)	Dairy farm environment
	Rota et al. (1996)	Cheese, pork sausage
	Harakeh et al. (2009)	Dairy-based food
	Miranda et al. (2008)	Poultry meat
Ciproflaxin	Prazak et al. (2002)	Cabbage
Clindamycin	Prazak et al. (2002)	Cabbage, water, environment
	Antunes et al. (2002)	Poultry carcasses
	Safdar and Armstrong (2003)	Clinical isolates
	Harakeh et al. (2009)	Dairy-based food
Doxycycline	Miranda et al. (2008)	Poultry meat
Enrofloxacin	Antunes et al. (2002)	Poultry carcasses
Erythromycin	Prazak et al. (2002)	Cabbage, water
	Rota et al. (1996)	Pork sausage
	Harakeh et al. (2009)	Dairy-based food
	Miranda et al. (2008)	Poultry meat
Florfenicol	Srinivasan et al. (2005)	Dairy farm environment
Fosfomycin	Li et al. (2007)	Bison
Gentamicin	Prazak et al. (2002)	Environment
	Harakeh et al. (2009)	Dairy-based foods
Kanamycin	Yucel et al. (2005)	Meat products
Linomycin	Li et al. (2007)	Bison
Nalidixic acid	Yucel et al. (2005)	Meat products
Nitrofurantoin	Prazak et al. (2002)	Cabbage, water,
Oxacillin	Prazak et al. (2002)	Cabbage, water, environment,
	Li et al. (2007)	Bison
	Harakeh et al. (2009)	Dairy-based food
Penicillin	Prazak et al. (2002)	Cabbage, water, environment
	Srinivasan et al. (2005)	Dairy farm environment
	Harakeh et al. (2009)	Dairy-based food
Rifampin	Prazak et al. (2002)	Cabbage, water
Rifampicin	Srinivasan et al. (2005)	Dairy farm environment
Rifamycin	Srinivasan et al. (2005)	Dairy farm environment
Streptomycin	Prazak et al. (2002)	Cabbage, water
	Srinivasan et al. (2005)	Dairy farm environment
	Antunes et al. (2002)	Poultry carcasses
Tetracycline	Prazak et al. (2002)	Cabbage, environment
	Srinivasan et al. (2005)	Dairy farm environment
	Rota et al. (1996)	Pork sausage
	Bertrand et al. (2005)	Human and food origins
	Walsh et al. (2001)	Retail foods
	Harakeh et al. (2009)	Dairy-based food
Tobramycin	Prazak et al. (2002)	Cabbage, environment
	Rota et al. (1996)	Pork sausage
Trimethoprim/sulfamethoxazole	Prazak et al. (2002)	Cabbage, water
	Srinivasan et al. (2005)	Dairy farm environment
	Yucel et al. (2005)	Meat products
	Harakeh et al. (2009)	Dairy-based foods
	Miranda et al. (2009)	Poultry meat
Vancomycin	Harakeh et al. (2009)	Dairy-based foods

Espaze and Reynaud (1988) found very little antibiotic resistance in Listeria spp. The studies discussed here as well as the additional ones listed in Table 1 indicate that over time, changes are occurring in the nature and incidence of antibiotic resistance in the genus Listeria and in L. monocytogenes in particular. Espaze and Reynaud (1988) predicted that resistant strains of Listeria spp. would emerge as the prevalence of Listeria spp. in food-producing and food-processing environments increased. Studies have also demonstrated that antibiotic resistance transfers and exchanges take place in L. monocytogenes (Pérez-Diaz et al., 1982; Flamm et al., 1984; Vicente et al., 1988; Biavasco et al., 1996; Celli and Trieu-Cuot, 1998) and could possibly occur in foods and food environments.

Multiple Resistance and Antibiotic Resistance Genes in L. monocytogenes and Transmission of Antibiotic Resistance in L. monocytogenes

Antibiotic resistance genes identified in L. monocytogenes are listed in Table 2. The appearance of resistance to antibiotics as well as antibiotic resistance genes in foodborne Listeria isolates suggests the need for more prudent use of antibiotics by farmers, veterinarians, and physicians. Roberts et al. (1996) demonstrated that erythromycin resistance in Listeria species was associated with ermC genes, which encode for rRNA methylases and this was found to be transferable to L. monocytogenes, L. innocua, and E. faecalis. In a study conducted by Li et al. (2007), the possibility of the potential for transfer of resistance to L. monocytogenes from L. innocua was raised. Li et al. (2007) demonstrated that L. monocytogenes strains from bison were susceptible to the antibiotics commonly used to treat human listeriosis; however, they also detected the presence of antibiotic-resistant L. innocua in the bison. The gene tetM was detected in the antimicrobial-resistant L. innocua species giving rise to the notion that L. innocua could potentially transfer resistance to L. monocytogenes. In an earlier study conducted by Bertrand et al. (2005) a collection of 241 Listeria isolates yielded three L. monocytogenes strains that were resistant to tetracycline due to the presence of the tetM gene. The tetM genes found in one of the isolates were similar to tetM genes previously found in S. aureus, whereas the sequences of tetM found in the other two isolates were associated with a member of the Tn916-Tn1545 family of conjugative transposons and were closely related to SHG lll, which harbors Enterococcal tetM genes associated with Tn916. Nineteen of the 38 L. monocytogenes strains isolated from four dairy farms contained more than one antimicrobial resistance gene sequence, and a high frequency of floR was detected followed by penA, strA, tetA, and sulI (Srinivasan et al., 2005). Antunes et al. (2002) also found that L. monocytogenes isolates from poultry carcasses exhibited resistance to one or more antibiotics. Likewise, Rota et al. (1996) reported that Listeria strains isolated from cheese and pork were resistant to multiple antimicrobial agents. Resistance testing by Mayrhofer et al. (2004) found no L. monocytogenes isolates from pork, beef, and poultry that were resistant to antibiotics commonly used to treat listeriosis, including tetracycline, vancomycin, cotrimazole, erythromycin, chloramphenicol, and streptomycin.

Table 2.

Antibiotic Resistance Genes Found in Listeria Spp.

Gene	Antibiotic	Species	Reference
tetA	Tetracycline	Monocytogenes	Srinivasan et al. (2005)
tetK	Tetracycline	Innocua	Facinelli et al. (1993)
tetL	Tetracycline	Monocytogenes	Poyart-Salmeron et al. (1992)
tetM	Tetracycline	Innocua	Chen et al. (2010)
		Monocytogenes	Poyart-Salmeron et al. (1992)
		Innocua	Facinelli et al. (1993)
tetS	Tetracycline	Monocytogenes	Hadorn et al. (1993)
			Poyart-Salmeron et al. (1990)
			Quentin et al. (1990)
aad6	Streptomycin	Innocua	Charpentier et al. (1995)
ermC	Erythromycin	Monocytogenes Innocua	Roberts et al. (1996)

L. monocytogenes strains isolated from Italian meat products were resistant to antibiotics such as tetracycline, co-trimoxazole, and erythromycin, but the resistance was not plasmid mediated (Barbuti et al., 1992). However, Poyart-Salmeron et al. (1990) demonstrated that antibiotic resistance to chloramphenicol, erythromycin, streptomycin, and tetracycline in L. monocytogenes was found to be mediated by a 37 kb plasmid, which was self-transferable as well as transferable to other organisms such as E. faecalis, S. agalactiae, and S. aureus. This suggests that emergence of antibiotic resistance in L. monocytogenes could be due to acquisition of a plasmid originating in the enterococci–streptococci. Slade and Collins-Thompson (1991) reported that most Listeria species isolated from raw milk were resistant to sulfisoxazole. Abrahim et al. (1998) noted that some Salmonella strains isolated from sausage samples in Greece were resistant to ampicillin, chloramphenicol, and tetracycline, whereas all Listeria isolates were sensitive to penicillins and aminoglycosides, but exhibited resistance to cephalosporins.

Although L. monocytogenes strains with resistance to one or more antibiotics have been isolated, overall resistance to antibiotics commonly used to treat listeriosis has rarely been observed. However, the presence of such resistance in other Listeria species raises the possibility of future acquisition of resistance by L. monocytogenes. Transfer by conjugation of plasmids and transposons carrying antibiotic resistance genes from Enterococcus–Streptococcus to Listeria also can occur (Doucet-Populaire et al., 1991).

Antibiotic Resistance and Environmental Stress Exposure

L. monocytogenes commonly encounters low levels of antibiotics and other antimicrobials in the agricultural and food sector. This may serve as pre-exposure adaptation, which subsequently allows L. monocytogenes to resist higher levels of antibiotics or antimicrobial drugs. Short chain fatty acids have been widely used as preservatives, and can also be found in the gastrointestinal tract (Kwon and Ricke, 1998). Van Schaik et al. (1999) demonstrated that acid-adapted L. monocytogenes displayed enhanced tolerance against the lantabiotics nisin and lacticin 3147. This increased resistance after acid adaptation could allow increased survival in food products as well as in the host or in the environment. Mild acidic pH induces an adaptive acid tolerance response in L. monocytogenes, which gives protection against later exposure to severe low pH challenges (Davis et al., 1996). Acid adaptation at pH 5.5 also confers protection against subsequent challenges with acetate (Phan-Than and Montagne, 1998). Alonso-Hernando et al. (2009) also found that L. monocytogenes exposed in vitro to acidified sodium chlorite was more resistant to some antibiotics, although additional studies are needed under field conditions to confirm these results.

Subtypes of L. monocytogenes previously thought susceptible have become more resistant to one or more antimicrobial drugs (Walsh et al., 2001; Srinivasan et al., 2005) perhaps as a result of extensive usage in the agricultural and food sector. It has also been proven that transmission of resistance among species occurs. For example, Lemaitre et al. (1998) demonstrated transmission of antibiotic resistance from L. monocytogenes to S. aureus, whereas Trieu-Cuot et al. (1993) demonstrated transmission from E. coli to S. aureus and L. monocytogenes. Enterococci and Streptococci represent a reservoir of antibiotic resistance genes for L. monocytogenes. The gastrointestinal tracts of humans and animals are considered essentially anaerobic environments; however, L. monocytogenes is a facultative pathogen and persists in the gut (Sleator et al., 2009). The gastrointestinal tract of humans and animals has been reported to serve as a site for transmission of resistance genes from enterococci and streptococci to L. monocytogenes (Doucet-Populaire et al., 1991). However, little or no work has been conducted to further clarify the role of anaerobiosis on the response of this pathogen to various conditions, including antibiotic stress or antibiotic resistance transfer and acquisition.

Starvation allows L. monocytogenes cells to become more resistant to other pathogen reduction methods such as heat and irradiation (Lou and Yousef, 1996, 1997; Mendonca et al., 2004); therefore, the added benefits of lack of nutrients in areas of processing plants may confer protection against antibiotics as well. L. monocytogenes can grow in a wide range of temperatures ranging from 0°C to 45°C. Lou and Yousef (1996) showed that heat shocking L. monocytogenes cells at 45°C for 1 h increased the resistance of this pathogen to ethanol and sodium chloride. However, the response of heat-adapted L. monocytogenes to antibiotic stresses has not been documented. The molecular and physiological response of L. monocytogenes to low temperature has been studied (Borezee et al., 2000; Wemekamp-Kamphuis et al., 2002, 2004; Zhu et al., 2005), but more work needs to be done on the response of low-temperature-adapted cells to other environmental stresses, including antibiotics.

Resistance to metal ions is often related to antibiotic resistance and common plasmids seem to be involved. For example, an E. coli strain carrying the robA plasmid from a cyclohexane tolerant mutant exhibited increased tolerance to solvents and resistance to antibiotics and heavy metals such as silver, mercury, and cadmium (Nakajima et al., 1995). Silver and Misra (1988) found that S. aureus strains that were resistant to mercury also carried penicillinase plasmids. Hayashi et al. (1993) also reported that Vibrio species that were resistant to lead acetate, cobalt chloride, sodium arsenate, and nickel sulfate were also resistant to aminobenzylpenicillin. Mullapudi et al. (2008) characterized L. monocytogenes isolates from a turkey-processing plant as to resistance to benzalconium chloride (BC), arsenic, and cadmium. All BC-resistant strains were also resistant to cadmium, although no correlation was found between BC resistance and resistance to arsenic, which overall was low (6%). These findings indicate that the processing plant environment may constitute a reservoir for L. monocytogenes with resistance to metals or disinfectants and raises the possibility of common genetic elements or mechanisms mediating resistance antibiotics. There is a need for more work to be conducted in the area of metal ion resistance and antibiotic resistance in L. monocytogenes. This is especially important in the seafood industry where many of the organisms found on seafood come into contact with heavy metals. Overall, it is important to understand how specific preservation factors as well as other environmental stress factors affect the sensitivity of target cells to antibiotics.

Control/Containment of Antibiotic-Resistant Strains

Three basic strategies exist to overcome the problem of antibiotic resistance in bacteria: (1) reduce the level of current antibiotic use, (2) strengthen the action of existing antibiotics by modifying them or providing a decoy molecule to tie up enzymes associated with resistance, or (3) interfere with the mechanisms of bacterial resistance (Tan et al., 2000).

Reducing current levels of antibiotic use

The movement to reduce the levels of antibiotics used each year has been aimed at both the human and agricultural uses of antibiotics. However, agricultural use of antibiotics is the focus of many of the studies under the theory that use of antibiotics as growth promoters is unwise and is an important source of antibiotic resistance in human pathogens (Witte, 1998). The use of low levels of antibiotics in animal husbandry selects for bacteria resistant to antibiotics (Phillips et al., 2004). In the United States, virginiamycin has been widely used as a growth promoter in animal husbandry and it is common to find resistance to the streptogramin class of antibiotics in E. faecium (Welton et al., 1998). However, avoparcin is not commonly used in the United States and thus glycopeptides resistance (vancomycin) is very low in animal enterococci (Harwood et al., 2001). In contrast, before the ban of growth promoters in Denmark up to 75% of E. faecium isolates from broilers were found to be resistant to vancomycin (Schouten et al., 1999). Resistance to some antibiotics decreases when the use of the antibiotic decreases or ceases, such as in Denmark after the growth promoter ban when the resistance levels for vancomycin were <5% (Bager et al., 2002). However, there is also data that indicate that some resistance persists after the use of antibiotics is discontinued. Langlois et al. (1986) observed that tetracycline resistance of fecal coliforms in pigs fluctuated between 20% and 44% even after 13 years without antibiotics being used on the farm. In addition, the use of substances such as copper in feed supplements might lead to coselection of antibiotic resistance when the two resistance determinants are linked on the same plasmid or transposon (Hasman and Aarestrup, 2002). Although reduction of antibiotic use clearly has some beneficial effects on the level of antibiotic resistance, other avenues should be explored to further reduce this problem.

Strengthening the action of existing antibiotics

One potential avenue for combating antibiotic resistance is modifying current antibiotics so that they are effective in microorganisms that have resistance to the parent antibiotic. Understanding the mechanism of β lactamases has allowed researchers to develop new semisynthetic penicillins such as ertapnem and faropenem that are not subject to inactivation by these enzymes (Jones et al., 2003; Hammond, 2004). Nelson and Levy (1999) developed a tetracycline analog, 13-cyclopentylthio-5-OH-TC (13-CPTC), that was able to competitively inhibit tetracycline translocation by the Tet(B) protein, blocking the uptake of tetracycline into vesicles and therefore preventing the efflux of tetracycline.

Interference with resistance mechanisms

Efflux pumps are one of the chief mechanisms of antibiotic resistance and are likely responsible for multidrug resistance, and a number of efflux pump inhibiters (EPI) have been studied. Griffith et al. (2001) demonstrated that the use of an EPI (MC-02,595) potentiated the use of levofloxacin in a mouse sepsis model against Pseudomonas aeruginosa possessing the MexAB-OprM efflux pump. These compounds apparently function by competing with antibiotic substrates for binding to the pumps. The EPI have no antibacterial activity alone, but they can potentiate the activity of antibiotics, and reverse acquired resistance attributable to efflux mutations (Lomovskaya et al., 2001; Renau et al., 2003). Another approach is to develop inhibitors of resistance enzymes that can be administered with the antibiotics, thereby blocking resistance and rescuing the antimicrobial activity of the drugs. This approach has been used with great success to overcome resistance to the penicillinases by the use of clavulinic acid, sulbactam, and tazobactam (Lee et al., 2003). These latter two approaches both require an in-depth knowledge of the molecular mechanisms of antibiotic resistance to develop the means to prevail over antibiotic resistance.

Conclusion and Implications

Given the increasing number of antibiotic-resistant L. monocytogenes strains being isolated around the world, it is imperative that we gain a better understanding of the extent of antibiotic resistance in L. monocytogenes, the antibiotic resistance gene patterns of this pathogen, and the ability of this pathogen to acquire resistance from other bacterial species. Most of the commensal organisms found in the gastrointestinal tract are obligate anaerobes, and these organisms may be antibiotic resistant. The pH conditions of the gut that range from acidic to slightly alkaline may provide environments that cause variations in the antimicrobial activity of the antibiotics, thus allowing more of these commensals and other pathogens to become antibiotic resistant. These would in turn provide a gene pool of antibiotic-resistant genes and plasmids for L. monocytogenes. Some microorganisms require a carbon dioxide-enriched atmosphere to grow. Carbon dioxide has been known to lower the pH of culture media, which may cause variations in the antibacterial activity of the antibiotics; for example, fluoroquinolones are less active at acidic pH. L. monocytogenes is a facultative anaerobe but grows better at oxygen tensions lower than that of atmospheric air conditions. Therefore, it would be interesting to determine the role anaerobiosis plays in the acquisition of resistance or tolerance to antibiotics by this organism and the commensals it comes in contact with in the gastrointestinal tract.

It has become increasingly obvious that external stresses and growth phase play major roles in bacterial physiology, resistance, cell morphology, and gene expression, including virulence gene expression. Therefore, looking at the response of L. monocytogenes to antibiotics under various conditions is a necessity. With the exception of bacteriocin studies, namely, nisin-resistant L. monocytogenes, little to no molecular work characterizing the mechanisms by which L. monocytogenes acquires antibiotic/antimicrobial resistance or tolerance has been conducted.

Although the occurrence of antibiotic resistance in L. monocytogenes is still low, there is an emerging pattern of resistance in strains isolated from food, the environment, and clinical cases. In addition, the range of antibiotics to which L. monocytogenes has acquired resistance is broad. Within this range of antibiotics are several that are traditionally used to treat listeriosis, such as penicillin and gentamicin. Multiresistant strains are not common, but evidence for emergence is available (Srinivasan et al., 2005). With the exception of penicillin, ampicillin, and trimethoprim-sulfamethoxazole, the Clinical and Laboratory Standards Institute (CLSI) has not determined resistance breakpoints for L. monocytogenes (CLSI, 2006). For other antibiotics, researchers often use the criteria for other Gram-positive organisms, such as the staphylococci (Conter et al., 2009).

Antibiotic-resistant bacteria in foods have been found with increasing frequency (Soto et al., 2001; Sherley et al., 2003; Wang et al., 2006). It appears that L. monocytogenes is rapidly acquiring a wide variety of antibiotic resistance genes, many of which may come from the commensal organisms found in foods and food-growing and food-processing areas. Reducing the use of antibiotics both in agriculture and for treatment of human diseases would reduce the emergence of more antibiotic-resistant bacteria, but those bacteria that already possess resistance disappear at a much slower rate than new antibiotic-resistant strains emerge in nature (Butler et al., 2007). Clearly, more research is needed in the areas of plasmid curing and inhibition of efflux pumps to control emergence of antibiotic-resistant strains of Listeria. In addition, more consumer education on the bacterial risks from consumption of certain foods and proper food-handling practices should be pursued.

Footnotes

Acknowledgments

This review was supported by U.S. Department of Agriculture Food Safety Consortium to S.C.R. and M.G.J., and American Meat Institute Foundation grant to P.G.C. and S.C.R.

Disclosure Statement

No competing financial interests exist.

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