Listeria monocytogenes was identified as an animal pathogen more than 75 years ago but only gained prominence as a human pathogen in the last twenty-five years. Since its first appearance, this organism has been implicated as the causative agent in several outbreaks of food-borne listeriosis in North America and Europe (Jacquet et al., 1995; Dalton et al., 1997; Miettinen et al., 1999; Aureli et al., 2000; Anonymous, 2002; 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 immuno-compromised, 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 US population is aging; in 2000 this population was 35 million but is projected to be 72 million by 2030, when the elderly will constitute 20% of the US population (US Census Bureau, 2005). Cancer is also one of the leading causes of death in the United States (ACCT, 2001; Anonymous, 2006a; Anonymous, 2006b), and new AIDS cases are reported each year. Due to a rise in the number of cases of immuno-compromised 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.
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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 eliminates other competitors 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 to the changes in food production practices and the increase in number of susceptible individuals, the ubiquitous nature of this organism allows it to encounter various physical, chemical and biological 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. Since the discovery of penicillin in 1929, the use of antibiotics in food husbandry has been on the rise (WHO, 2002). 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).
While 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.
Ecology of Listeria monocytogenes
Listeria monocytogenes is a non-spore forming Gram-positive rod shaped bacterium that does not form capsules, is motile at 20 to 25oC but non motile at 37oC. L. monocytogenes grows well on most commonly used bacteriological media such as brain heart infusion (BHI) and nutrient agar, and exhibits aerobic, facultative anaerobic and microaerophilic characteristics (Lungu et al., 2009). L. monocytogenes is widely distributed in nature and can be found on decaying vegetation, in soil, human and animal feces, sewage, silage, food processing environments, milk of normal and mastitic cows and water (Welshimer and Donker-Voet, 1971; Botzler et al., 1974; Weis and Seeliger, 1975; Sanaa et al., 1993; Lawrence and Gilmour, 1994; Yoshida et al., 2000; Thimothe et al., 2002; Garrec et al., 2003). L. monocytogenes has been isolated from cattle, sheep, goats, poultry and fish, and wild animals (Weis and Seeliger, 1975; Grønstøl, 1979; Dijkstra, 1981; Løken et al., 1982; Hofer, 1983; Fenlon, 1985; Fenlon et al., 1996; Yoshida et al., 2000; Thimothe et al., 2002; Nightingale et al., 2004).
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L. monocytogenes is able to survive a wide variety of environmental stresses including low temperature, nutrient deprivation, oxidative stress, osmotic stress, preservative stress, temperature stress, acid stress and antibiotic stress and still maintain virulence (Lou and Yousef, 1996, 1997; Ferreira et al., 2001; Herbert and Foster, 2001; Ferreira et al., 2003; Christiansen et al., 2004; Mendonca et al., 2004; Begley et al., 2005). These stressors can be encountered by the organisms in the processing, preparation and storage environments in food processing plants as well as in cafeterias/kitchens of hospitals, schools, nursing homes, and other institutions. L. monocytogenes has been frequently isolated from drains, pooled water, floors and residues or runoffs within processing plant environments (Lawrence and Gilmour, 1994; Smoot and Pierson, 1998a, b).
Pathogenesis and epidemiology of L. monocytogenes
L. monocytogenes causes a disease known as listeriosis and accounts for an estimated 2500 illnesses and 500 deaths per year (Mead et al., 1999). Unlike infection with other common food borne pathogens such as Salmonella, which rarely result in deaths, listeriosis is associated with a mortality rate of approximately 30%. The fatality rate in untreated patients or those treated late may rise to 70% (Mead et al., 1999). Symptoms of listeriosis vary depending on the individual but may include fever, diarrhea, septicemia and meningitis. L. monocytogenes can also cause spontaneous abortion, meningioencephalitis, endocarditis, endophthalmitis, osteomylitis, brain abscesses and peritonitis (Muriana and Kushwaha, 2006). Food borne L. monocytogenes is the main source of infections in humans (Swaminathan and Gerner-Smidt, 2007). L. monocytogenes has been associated with raw milk, soft cheeses, fresh and frozen meat, poultry and seafood products, and fruits and vegetables, particularly ready-to-eat (RTE) products such as milk, soft cheeses, luncheon meats and raw vegetables (Sanaa et al., 1993; Lawrence and Gilmour, 1994; Uyttendaele et al., 1999; Samelis et al., 2002; Thimothe et al., 2002; Vitas et al., 2004,).
Approximately 2 to 6% of healthy people shed L. monocytogenes without having symptoms (Jensen, 1993; Schuchat et al., 1993) and some of these may work in food service establishments or in hospitals and thus may be able to transmit the bacteria to other susceptible individuals. Results of an investigation of a California outbreak in 1985 revealed that community acquired outbreaks could be amplified through secondary transmission by fecal carriers (Mascola et al., 1992). The minimum infective dose is not known and the severity of the disease is dependent on the individual, thus the USDA and FDA hold a zero tolerance level for this organism on RTE foods (i.e. 0 CFU/25 g). The severity and high fatality rates of the disease make it imperative that this human pathogen is controlled and its mechanisms of survival are understood. All of these traits make L. monocytogenes a major concern for public health as well as the food production and processing industry. Because of the increasing elderly population as well as increases in immuno-compromised individuals in the United States a high proportion of individuals are potential victims of listeriosis (Kaiser Foundation, 2006).
Antibiotic therapy for listeriosis
Invasive listeriosis is treated mainly by supportive therapy along with IV 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). L. monocytogenes often causes meningitis in infected persons, and is associated with significant morbidity and mortality (Hof et al., 1997). 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. For patients that are allergic to Î²-lactams, co-trimoxazole has been recommended for treatment (Hof et al., 1997). 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 on 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 old quinolones (such as ciprofloxacin) while being generally susceptible to fluoroquinolones (new 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.
Antibiotic resistance overview
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In common usage the term antibiotic refers to a substance produced by a mold or bacterium that inhibits or kills bacteria. Antibiotics are a subset of the broader group, antimicrobials, a general term for the drugs, chemicals, or other substances that either kill or slow the growth of microorganisms, including bacteria, viruses, fungi and parasites (CDC, 2006). Antibiotics first became commercially available in the 1940s, and they have been used universally for clinical diseases of bacterial origin. Many of these antibiotics originate from natural sources such as bacteria that produce toxins against other bacteria. In response, bacteria develop defenses, including resistance to the antibiotics produced by other organisms. Alexander Fleming, who won a Nobel Prize for his discovery of penicillin, warned in 1945 that misuse of the drug would lead to resistance in bacteria (Rosenblatt-Farrell, 2009). Antibiotics are used in food-producing animals for short periods of time not only to treat sick animals but also to prevent infections. Antibiotics can also be used at low doses for long periods of time as growth promotants (CDC/NARMS, 2005). Scientists from academia, industry and the government have concluded that any use of antimicrobials creates the potential for the development of antimicrobial resistant bacteria (Isaacson and Torrence, 2002).
Mechanisms of antibacterial activity
Antibiotics exhibit a wide range of bactericidal mechanisms, as can be seen in Table 1. They also represent a wide range of therapeutic indices; the therapeutic index is a ratio of the amount of antibiotic that causes the therapeutic effect to the amount that causes death (Schneiderman et al., 1964). Penicillins, cephalosporins, carbapenems, and vancomycin damage or inhibit the production of cell walls (Lancini et al., 1993), and are the most selective antibiotics and have a high therapeutic index. The high therapeutic index of these cell wall active antibiotics is due to the unique structure of bacterial cell walls not found in eukaryotes. Aminoglycosides, chloramphenicol, tetracyclines, and macrolides have effects on protein synthesis (Lancini et al., 1993). These drugs discriminate between prokaryotic and eukaryotic ribosomes and so also have a fairly high therapeutic index. Some antibiotics have effects on bacterial DNA or RNA biosynthesis and/or function, such as the quinolones and rifampin. These have a lower therapeutic index due to there being minimal difference in nucleic acid synthesis in eukaryotes and prokaryotes. Some antibiotics have effects on metabolism (trimethoprim or sulfonamides) by competitive inhibition (Lancini et al., 1993). Sulfonamides have a high therapeutic index because they block the production of folic acid, but humans cannot synthesize folic acid and thus must derive it from dietary sources. Just as there are a variety of mechanism by which antibiotics work, bacteria also have a variety of mechanisms for being or becoming resistant to antibiotics.
Genetics of antibiotic resistance
Some of the known mechanisms of antibiotic resistance used by bacteria are listed in Table 2. Bacteria are said to have "intrinsic" resistance when the normal characteristics of the genus make them immune to the effect of the antibiotic; for instance the cell wall of Gram-negative bacteria makes them inherently resistant to penicillin (Levy, 1998). Conversely, bacteria may acquire resistance to an antibiotic through mutation or transfer of genetic material between bacteria, or they may use "efflux pumps" to remove the antibiotic, or use enzymes to break down the antibiotic (Levy, 1998). When bacteria are able to perform more than one of these functions they may be resistant to more than one type of antibiotic (Bennett, 2008). Multidrug resistant bacteria may acquire multiple genes, each one coding for a resistance to one drug, on resistance plasmids (Nikaido, 2009). There may also be increased expression of genes that code for multidrug efflux pumps that force out a wide range of antibiotics (Nikaido, 2009).
Spontaneous mutations are relatively rare, occurring only once per 107 to 1010 bacteria (Mulvey and Simor, 2009) and this type of resistance is not transferrable to other organisms. Mutations of this type are highly unlikely to lead to multiple antibiotic resistance. Of much more consequence are the mobile genetic elements. Mobile genetic elements fall into two general types, elements that can move from one genetic location to another in the same cell, and elements that can move from one bacterial cell to another. Mobile genetic elements that move from one cell to another include resistance plasmids and conjugative resistance transposons (Bennett, 2008).
Plasmids replicate separately from the bacterial chromosome. They carry only those genes that are useful for bacteria to adapt to particular environmental conditions, including survival and growth in the presence of a potentially lethal antibiotic (Stanisich, 1988). Many resistance plasmids are conjugative, that is they encode the functions necessary to promote cell-to-cell DNA transfer, particularly their own transfer; conjugation is a replicative process that leaves both donor and recipient cells with a copy of the plasmid (Wilkins, 1995). Most, if not all, of the antibiotics currently in use are encoded on resistance plasmids (Bennett, 2008). 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 (Perez-Diaz et al., 1982). Another broad-host-range plasmid, pAM1 of Enterococcus faecalis, conferring resistance to erythromycin, was transferred successfully by conjugation from E.Â faecalis to L.Â monocytogenes, was found to replicate in the new host and be transferred by conjugation between strains of L.Â monocytogenes and from L.Â monocytogenes back to E.Â faecalis (Flamm et al., 1984). In another study, a plasmid carrying the vanA gene cluster, conferring glycopeptide resistance, was transferred from E.Â faecium to L.Â monocytogenes, L.Â ivanovii, and L.Â welshimeri (Biavasco et al., 1996).
Conjugative transposons are DNA elements that can either reintegrate into another site on the DNA of the same host cell or transfer by conjugation to a recipient cell and become integrated into the genome of the recipient. Conjugative transposons have a very broad host range, and may contribute as much as plasmids to the spread of antibiotic resistance genes among bacterial genera (Salyers et al., 1995). The conjugative transposon designated as Tn916 has a broad host range, carries the tetM tetracycline resistance gene and was originally found in E. faecalis (Rice, 1998). Vicente et al. (1988) demonstrated the transfer by conjugation of Tn916 from E. faecalis to L. 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).
Efflux pumps are proteins found in both Gram-positive and -negative bacteria that are involved in the removal of toxic substrates, including antibiotics, from within cells into 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). Bacteria have five major classes of efflux pumps: major facilitator (MF), multidrug and toxic efflux (MATE), resistance-nodulation-division (RND), small multidrug resistance (SMR) and ATP binding cassette (ABC) (Lomovskaya et al., 2001). The ABC pumps are primary active transport systems that directly use ATP hydrolysis to drive export, while the others are secondary active transport systems that use proton motive force as an energy source (Paulsen et al., 1996). All of the bacterial genomes studied contain several different efflux pumps, indicating an origin that predates antibiotics rather than an evolution in response to antibiotic pressure (Saier and Paulson, 2001). There have been two efflux pumps described in L. monocytogenes, MdrL and Lde. The genes which 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). Fluorquinolone resistance is associated with Lde as are resistance to the DNA intercalating dyes acridine orange and ethidium bromide (Godreuil et al., 2003).
Antibiotic modification is another method of antibiotic resistance in which the resistant bacteria modify the antibiotic in some way so that it is no longer active against the target cell. For example Î² lactamases, widespread among both Gram-positive and Gram-negative bacterial species, enzymatically cleave the four membered Î² lactam ring rendering the penicillin family of antibiotics inactive. Most Î² lactamases act to some degree against both penicillins and cephalosporins (Livermore, 1995). Modification is mainly based on either the hydrolysis of the antibiotics or a modification by transfer of acetyl-, adenyl- or phosphorus groups to specific sites of the antibiotics (Quintilliani et al., 1999; Kayser, 1996). These enzymes usually have a narrow substrate spectrum which is limited to the respective antibiotics and are not involved in physiological cell metabolism. The genes coding for these enzymes are most often located on mobile genetic elements.
Antibiotic resistant Listeria monocytogenes in food and the food processing environment
A comprehensive list of L. monocytogenes antibiotic resistant strains isolated from food and food producing and processing areas is presented in Table 3. Rota et al. (1996) found that Listeria strains isolated from cheese and pork were resistant to multiple antimicrobial agents, although more than 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 4 of the 6 antibiotics tested, while organically grown poultry (which were presumably not exposed to any antibiotics) were resistant to 2 of the 6. 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.
In 1988, Espaze and Reynaud found very little antibiotic resistance in Listeria spp. The studies discussed here as well as the additional ones listed in Table 3 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 processing environments increased. Studies have also demonstrated that antibiotic resistance transfers and exchanges take place in L. monocytogenes (Perez-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 Listeria monocytogenes
Antibiotic resistance genes identified in L. monocytogenes are listed in Table 4. 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 while 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. Walsh et al. (2001) established 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. 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). In addition L. monocytogenes isolates exhibited resistance to more than one antibiotic in different combinations. All isolates were resistant to cephalosporin C, streptomycin and trimethoprim. 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.
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 Enterococcus faecalis, Streptococcus 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 (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 while all Listeria isolates were sensitive to penicillins and aminoglycosides, but exhibited resistance to cephalosporins.
While L. monocytogenes strains with resistance to one or more antibiotics have been isolated, it is important to again note that 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. There also can occur transfer by conjugation of plasmids and transposons carrying antibiotic resistance genes from Enterococcus-Streptococcus to Listeria (Doucet-Populaire et al., 1991).
Antibiotic resistance and environmental stress exposure
L. monocytogenes commonly encounters 'safe' 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. 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. Acid adapted L. monocytogenes may also be less susceptible to antibiotic stress. Short chain fatty acids (SCFA) have been widely used as preservatives, and can also be found in the gastrointestinal tract. Kwon and Ricke (1998) reported that Salmonella Typhimurium exhibited increased acid resistance and virulence after adaptation to various SCFA. They also demonstrated that the percentage of survival of this pathogen was higher under anaerobic conditions after adaptation. Overall they concluded that SCFA-induced acid resistance was enhanced by acid pH, anaerobiosis and prolonged exposure to SCFA. Like S. Typhimurium, L. monocytogenes will also encounter SCFA as a food preservative and in the gastrointestinal tract; therefore it would be of interest to determine the role short chain fatty acids play in the growth and survival of L. monocytogenes. It would also be interesting to determine whether adaptation of L. monocytogenes to SCFA would make them more resistant to other environmental stresses including antibiotic stress.
Subtypes of L. monocytogenes previously thought susceptible have become more resistant to one or more antimicrobial drugs (Srinivasan et al. 2005; Walsh et al. 2001) 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 while 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.
As previously described, starvation allowed L. monocytogenes cells to become more resistant to other quality control 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 to 45oC. Lou and Yousef (1996) showed that heat shocking L. monocytogenes cells at 45oC 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, 2005; 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 normal flora and pathogens found in animals that are fed low levels of antibiotics all become resistant (Phillips et al., 2004). In the US, 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 Enterococcus faecium (Welton et al., 1998). However, avoparcin is not commonly used in the US 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). It has been demonstrated that 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 less than 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, some researchers have suggested that the use of substances such as copper as feed supplements might lead to co-selection of antibiotic resistance when the two resistance determinants are linked on the same plasmid or transposon (Hasman and Arestrup, 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 Î² lactamases has allowed researchers to develop new semi-synthetic 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 multi-drug resistance, and a number of efflux pump inhibiters (EPI) have been studied. Griffith et al. (2000) 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 in order 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. Interestingly, 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 have been conducted.
With the considerable current knowledge available on S. aureus and E. faecalis antibiotic resistance patterns and mechanisms, it would seem that interest in L. monocytogenes would also be on the rise. Perhaps the difficulties that go along with Listeria uptake of DNA or plasmid elements as well as the misconception that antibiotic resistance is not a problem with L. monocytogenes plays a role in this lack of information. More work still needs to be conducted on combination therapies as well as use of antibiotics in multiple hurdle systems as methods to reduce the likelihood of resistance selection. Single antibiotic use for long durations simply allows for selection of antibiotic resistant strains. The limited information on antibiotic usage, antibiotic quantities and types currently in use by the industry as well as antibiotic resistance patterns in L. monocytogenes makes it difficult to make meaningful control strategies to curb antibiotic resistance so this is information that is sorely needed. It is imperative that we glean knowledge on the transmission dynamics of L. monocytogenes in production agriculture systems as this may give us an idea of how best to control of strains of this pathogen whether they have developed antibiotic resistance or not. Looking at how this pathogen grows and survives through the gastrointestinal tract of humans and animals may give us an idea of how this organism is able to acquire resistance during exposure to this environment. Stress factors and original environmental growth conditions as well as genetic history of L. monocytogenes may play a role in antibiotic resistance and rate of resistance acquisition. Since the first antibiotic resistant strain of L. monocytogenes was isolated, little scientific research has been targeted towards determining the mode of action and resistance mechanisms of antibiotics against L. monocytogenes. There is a need to do more genetic studies involving transcriptional analysis and mutational analysis to determine the mode of action and mechanism.
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 processing areas. Reducing the use of antibiotics both in agriculture and for human treatment 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 strains of Listeria in nature. In addition, more consumer education on the bacterial risks from consumption of certain foods and proper food handling practices should be pursued.