Antimicrobial Resistance in Listeria Species

ABSTRACT

For nearly a century the use of antibiotics to treat infectious diseases has benefited human and animal health. In recent years there has been an increase in the emergence of antibiotic-resistant bacteria, in part attributed to the overuse of compounds in clinical and farming settings. The genus Listeria currently comprises 17 recognized species found throughout the environment. Listeria monocytogenes is the etiological agent of listeriosis in humans and many vertebrate species, including birds, whereas Listeria ivanovii causes infections mainly in ruminants. L. monocytogenes is the third-most-common cause of death from food poisoning in humans, and infection occurs in at-risk groups, including pregnant women, newborns, the elderly, and immunocompromised individuals.

Currently, multidrug resistance is not a common feature encountered in Listeria species. However, as has been observed with other pathogens of importance to humans, Listeria species have the ability to rapidly develop resistance to any antimicrobial agent, a feature that represents an emerging and increasing threat to human and animal health. Isolates of Listeria have been reported with varying degrees of resistance to commonly used antibiotics, and the first multidrug-resistant Listeria isolate was identified in France in 1988. These isolates developed resistance through a number of well-known mechanisms, including target gene mutations, such as within genes encoding efflux pumps, together with the acquisition of mobile genetic elements. This article will focus, in particular, on describing the mechanisms that confer resistance in Listeria species to antibiotics, biocides, and heavy metals.

INTRODUCTION TO THE GENUS LISTERIA AND SPECIES

Listeria species are small rod-shaped bacteria, typically 0.5 μm in diameter and 1 to 2 μm in length. They are Gram-positive bacteria, facultative anaerobes, and nonsporulating. They are catalase-positive, oxidase-negative, and motile at low temperatures. They grow at temperatures in the range of 4 to 45°C and pH values of 4.7 to 9.6, and have a low GC content. The genus Listeria comprises 17 recognized species: L. monocytogenes, L. ivanovii, L. grayi, L. innocua, L. seeligeri, L. welshimeri, L. marthii, L. fleischmannii, L. floridensis, L. aquatica, L. newyorkensis, L. cornellensis, L. rocourtiae, L. weihenstephanensis, L. grandensis, L. riparia, and L. booriae (Fig. 1) (1). Listeria species can be classified into Listeria sensu stricto (L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, and L. marthii) and Listeria sensu lato (L. grayi, L. fleischmannii, L. floridensis, L. aquatica, L. newyorkensis, L. cornellensis, L. rocourtiae, L. weihenstephanensis, L. grandensis, L. riparia, and L. booriae) (1).

FIGURE 1.

Listeria species maximum likelihood phylogenetic tree based on concatenated nucleotide sequences of the 16S rRNA genes from all Listeria species. Values on branches represent bootstrap values based on 500 bootstrap replicates; bootstrap values >80% are not displayed. Listeria species are color coded according the new genera classification proposed by Orsi et al. (1).

Phylogenetic studies indicate that the most closely related genus is Brochothrix, and there is a relatedness to other Gram-positive bacteria with low GC content, such as Bacillus, Clostridium, Enterococcus, Staphylococcus, and Streptococcus (2, 3). Orsi and Wiedmann (1) proposed a reclassification of the Listeria genus into four genera, Listeria, Mesolisteria, Paenilisteria, and Murraya (Fig. 1). The species that belong to the Mesolisteria genus are unable to grow at temperatures below 7°C. Listeria sensu stricto species and L. grayi possess motility genes that enable them to move, in contrast to the rest of the Listeria species.

Listeria species can be classified into serotypes based on the serological reactions of listerial somatic antigen (O-antigen) and flagellar antigen (H-antigen) with specific antisera. L. monocytogenes can be differentiated into at least 13 serotypes, and these are divided into 4 lineages, I to IV (Table 1) (4). The majority of L. monocytogenes isolates cluster into lineages I and II, described by Piffaretti et al. (5). Lineage IV is the most recently identified, and currently few isolates belong to this lineage (6, 7).

TABLE 1.

Listeria monocytogenes lineages and serotype distribution

Lineage	Serotypes	Distribution	Genetic characteristics
I	1/2b, 3b, 4b	Commonly isolated from various sources; overrepresented among human isolates	Lowest diversity among the lineages; lowest levels of recombination among the lineages
II	1/2a, 1/2c, 3a, 3c	Commonly isolated from various sources; overrepresented among food and food-related as well as natural environments	Most diverse, highest recombination levels
III	4a, 4b, 4c	Most isolates obtained from ruminants	Very diverse; recombination levels between those for lineage I and lineage II
IV	4a, 4b, 4c	Most isolates obtained from ruminants	Few isolates analyzed to date

L. monocytogenes is an important facultative human foodborne pathogen, and it is the third leading cause of deaths due to foodborne bacteria the United States (8). Listeria outbreaks are distributed globally, causing a significant economic impact on the food industry and public health. This bacterium was first isolated in rabbits and guinea pigs with pronounced monocytosis (9). Listeria species are ubiquitous and widespread in the environment. The pathogenicity of Listeria species is associated with intercellular replication, and its reservoir extends from the environment to humans and animals, providing an ecological niche (10).

LISTERIA SPECIES IN DOMESTIC ANIMALS AND THE FARM ENVIRONMENT

Listeria species have been isolated from a wide range of domestic and wild animals including mammals, birds, fish, and crustaceans (11, 12). The clinical disease is usually observed in domestic ruminants and humans, though occasionally also in poultry, pigs, rabbits, and other species. Clinical listeriosis in birds is rare, but birds tend to be asymptomatic carriers, because they can ingest this bacterium through contaminated food, water, bedding, or soil (13). Younger cohorts of bird populations seem to be more susceptible (14). Like rabbits, guinea pigs are also susceptible, and L. monocytogenes has also been isolated in canine cutaneous infection (15). There have been reports of symptoms such as miscarriages and septicemia associated with L. monocytogenes equine infections (16). The prevalence of Listeria in wildlife species ranges from 1 to 60%, although species were mainly captive or domestic (17–19). Less than 1% prevalence was recorded in wild birds and mammals in their natural habitats in Japan (20), while Listeria species were recorded in 5% of sampled red fox, beech marten, and raccoon populations in Poland (21). Contact with contaminated silage is believed to be an important transmission vector for livestock infection (22).

Listeria can be classified as a saprophytic bacterium, because its reservoirs are the soil and the intestinal tracts of asymptomatic animals, with soil and fecal contamination resulting in the presence of these bacteria on plants and fodder, particularly silage, along with walls, floors, and drains in the agricultural environment (23, 24).

Evidence indicates that farm systems with cattle species have a higher prevalence of L. monocytogenes, including the species that cause human listeriosis, compared to other animals (25). In the addition, it appears that cattle play an important part in the amplification and spread of L. monocytogenes in agricultural environments (Fig. 2), while there is evidence of a difference in epidemiology and transmission between ruminant species (25, 26). L. monocytogenes can be isolated from cattle farms more frequently during the spring, a feature that may be related to variables including housing with silage feeding during the winter and the application of slurry (27). This would indicate that farmyard environments are reservoirs. The latter feature is not surprising since healthy bovine species with fecal carriage of L. monocytogenes as high as 46.3% have been reported (28, 29).

FIGURE 2.

Transmission dynamics of listeriosis involving human and animal hosts. Potential transmission pathways of Listeria species are indicated by arrows, and vehicles are represented by colored boxes.

However, the disease ecology caused by Listeria species is not fully understood, and fundamental questions regarding the interaction between the hosts, the pathogen, and environment remain to be answered. Specifically, the determinants and mechanisms of transmission from the principal reservoirs, i.e., ruminants, within agricultural ecosystems merit further investigations (30).

OVERVIEW OF THE PATHOGENESIS OF LISTERIOSIS

The genus Listeria contains two pathogenic species, L. monocytogenes and L. ivanovii. However, on rare occasions infections caused by L. innocua and L. seeligeri have occurred (31, 32). Listeriosis in humans and many vertebrate species, including birds, is caused by L. monocytogenes, with L. ivanovii infections being specific to ruminants (33). Most infections caused by Listeria result from the ingestion of contaminated food such as raw milk, meat, and fish; ready-to-eat meals; unpasteurized dairy products; and uncooked vegetables in humans and from contaminated silage or other sources of feed in animals. The cycle of infection is completed with the liberation of Listeria into the environment in the feces. Listeria is a saprophyte when it is found in the environment but makes a transition to a physiological state that promotes bacterial survival and replication in host cells (24). Listeria has evolved a number of mechanisms to invade and exploit host processes to multiply, spread from cell-to-cell, and evade the immune system without damaging the host cell. The intracellular infectious cycle consists of distinct stages: (i) invasion of the eukaryotic host cell, (ii) escape from the intracellular vacuole, (iii) intercellular proliferation, (iv) actin filament-based intercellular spread, and (v) dissemination to adjacent cells (Fig. 3) (34).

FIGURE 3.

L. monocytogenes intracellular life cycle. (a) Listeria invades the host cells via a zipper mechanism, by the interaction of surface internalins InlA and InlB with the host cell surface receptors E-cadherin and Met, respectively. (b) Listeria escapes from the phagosome before the fusion with the lysosome occurs, by the action of the secreted proteins, the pore-forming toxin LLO, and phosphatidylinositide phospholipase C (PI-PLC). (c) Listeria may replicate in the cytosol, and (d) it spreads by actin polymerization, which propels the bacteria unidirectionally, (e) promoting cell-to-cell spreading of Listeria. (f) Rupture of the two-membrane vacuole is mainly mediated by the action of LLO and phosphatidylcholine-specific phospholipase C (PC-PLC).

As indicated, Listeria is an intracellular pathogen that spreads from cell to cell, and L. monocytogenes has evolved mechanisms to evade exposure to the host innate immune defenses. One example of immune evasion mechanisms in L. monocytogenes is the O-acetyltransferase (oatA), which encodes for O-acetylation of muramic residues contained in the peptidoglycan. OatA increases the bacterial cell wall resistance to antimicrobial compounds such as lysozyme, thereby favoring bacterial persistence in macrophages and virulence in vivo (34). However, the host has evolved innate and acquired immunity mechanisms, including the induction of apoptosis and the generation of cytotoxic T-cells that recognize and lyse listeria-infected cells, releasing bacteria into the extracellular space and rendering them susceptible to infiltrating phagocytes (35). (For a more complete description of the bacterial mechanisms underpinning the pathogenesis and virulence of Listeria, the following references 33 and 36–39 provide additional information.)

LISTERIOSIS IN FOOD-PRODUCING AND OTHER ANIMALS AND IN HUMANS

Listeria species have been isolated from more than 50 animal species, both domestic and wild (Table 2), with presentation of disease varying according to the host (Table 3). Most infections in animals are subclinical, but invasive listeriosis can occur either sporadically or as part of an outbreak (12). Clinical disease is seen most often in domestic ruminants and humans, though occasional cases have been reported in poultry, pigs, rabbits, and other species.

TABLE 2.

Mammals, birds, and other species from which Listeria species have been isolated

Mammals	Cats, cattle, chinchillas, deer, dogs, ferrets, foxes, goats, guinea pigs, horses, humans, jackals, lemmings, mice, mink, monkeys, moose, pigs, rabbits, raccoons, rats, sheep, skunks, and voles
Birds	Canaries, chaffinches, chickens, cranes, doves, ducks, eagles, geese, hawks, lorikeets, owls, parrots, partridges, pheasants, pigeons, seagulls, turkeys, white grouse, whitethroat grouse, and wood grouse
Other	Ants, crustaceans, lizards, fish, flies, frogs, snails, ticks and tortoises

TABLE 3.

Listeria species, hosts, and forms of disease

Species	Host	Forms of disease
L. monocytogenes	Cattle, sheep, goat Cattle Cats, dogs, horses Pigs Birds	Encephalitis, abortion, septicaemia, ocular form Mastitis (rare) Abortion, encephalitis (rare) Encephalitis, abortion, septicaemia Septicaemia
L. ivanovii	Cattle, sheep	Abortion
L. innocua	Sheep	Encephalitis (rare)

Listeriosis in Food-Producing Domestic Ruminants

In ruminants, infection with Listeria and disease are predominantly due to the consumption of spoiled silage contaminated with L. ivanovii or L. monocytogenes. Listeria species can replicate in poorly fermented silage with pH values above 5.5, and these bacteria can reach numbers of up to 10⁷ colony-forming units/kg. Other contamination sources apart from silage include barn equipment such as bedding, water, and feeding troughs (40), where bacteria can survive within biofilms. Depending on the number of bacteria ingested, the pathogenic properties of the individual bacterial isolate, and the immune status of the host, listeriosis may present as meningoencephalitis, abortion, or septicemia. It is uncommon for different clinical manifestations to be observed during the same outbreak. The following subsections provide a brief synopsis of the clinical features associated with these presentations.

Meningoencephalitis

Meningoencephalitis (rhombencephalitis) is caused by L. monocytogenes and is the most characteristic clinical manifestation of listeriosis in adult ruminants. Indeed, it is among the most common causes of neurological disease in ruminants. A naturally occurring case of ovine meningoencephalitis due to L. innocua has also been reported (41). The incubation period is usually 2 to 3 weeks, and the course of the infection is more acute and frequently fatal in sheep and goats (death may occur within 2 to 3 days after the onset of symptoms) but is subacute to chronic in cattle. Once these bacteria cross the intestinal barrier they can gain access to the central nervous system (CNS) via the blood stream. An alternative route to the CNS is through damaged oral, nasal, or ocular mucosas, where the bacterium can track along the trigeminal nerve terminals with subsequent intraneural direct spread to the brain stem and to further areas of the brain via axonal pathways (42). Clinical presentation depends on the localization and distribution of lesions in the nervous system (30). Early and nonspecific symptoms such as depression and anorexia are followed by neurologic signs, often unilateral, such as facial and tongue paralysis with profuse salivation, nystagmus, absence of palpebral reflex, and dropped ear. The syndrome is known as “circling disease” because it is characterized by a lack of coordination, leaning against objects, head deviation sometimes with head tilt, involuntary torticollis, and walking aimlessly in circles as a result of brainstem lesions. In a further development of the infection, prostration is followed by paddling movements and convulsions before death. The course of the infection is short, extending from 1 to 4 days in sheep and from 1 to 2 weeks in cattle.

Abortion

L. monocytogenes and L. ivanovii can gain access to the developing fetus via hematogenous penetration of the placental barrier. Vaginal transmission can also occur. The depression of cell-mediated immunity during advanced pregnancy is thought to play an important role in the development of listeriosis (33). The incubation period can be as short as 1 day, and the outcome of the fetal infection is mainly abortion, though cases of stillbirths have been reported. Abortion occurs in the last trimester of gestation (after 7 months in cattle [approximately 9 months gestation] and 12 weeks in sheep and goats [approximately 20 weeks gestation]), and abortion storms are more commonly reported in sheep and goats. In both species, the rates of abortion in a group are low but may reach as high as 15%. Mothers have no other clinical signs, with the exception of fever and anorexia, and it is not known yet why these animals never develop CNS infection or overt septicemic disease (33). If the placenta is retained, metritis may result.

Septicemia

The septicemic form of listeriosis is relatively uncommon in adult ruminants and generally, but not invariably, occurs in the neonate as a consequence of in utero infection. It is marked by weakness, depression, inappetence, fever, and death. In this clinical form, multiple foci of necrosis in the liver and, less frequently, the spleen may be noted at necropsy.

Other presentations

Mastitis has rarely been associated with L. monocytogenes infection and is not as frequent as with Brucella species, Mycobacterium bovis, Escherichia coli, Staphylococcus species, or Streptococcus species. However, subclinical L. monocytogenes mastitis is also reported (43). Gastrointestinal infections can occasionally occur in sheep and goats (44), with dullness, inappetence, pyrexia, diarrhea, and death within 24 hours of clinical signs. Unilateral uveitis and keratoconjunctivitis (ocular listeriosis) are also reported in cattle (45) and are attributed to direct ocular contact with contaminated silage.

In addition to the clinical cases of listeriosis, a significant number of animals within a herd may be subclinically infected and excrete Listeria species in their feces, a feature that represents a strong risk factor for contamination of the environment and a means by which the bacterium could gain access to the slaughterhouse (46).

Listeriosis in Other Animals

Birds

Birds are usually subclinical carriers of L. monocytogenes. Secretions and excretions from colonized birds are rich sources of these bacteria, and they can play a major role in transmission and spread of Listeria species to both humans and animals via the ingestion of contaminated food, feed, water, litter, and soil (13). Outbreaks of listeriosis are occasionally observed in the young animals. Predisposing factors include immunosuppression, wet/damp conditions, moist litters, and cold (47). The most frequent form is septicemia occurring with symptoms including depression, listlessness, diarrhea, and emaciation. Peracute deaths occur sometimes without other clinical signs (13). The encephalitic form of listeriosis in birds, meningoencephalitis, is a rare occurrence. However, an outbreak of encephalitic listeriosis in red-legged partridges between 8 and 28 days of age has been recently documented (48). Meningoencephalitis in birds is characterized by incoordination, torticollis, stupor, and paresis or paralysis (49). In young geese, both meningoencephalitis and septicaemia can be seen concurrently. Salpingitis is observed in hens during the acute systemic phase (49).

Rabbits

In rabbits, L. monocytogenes usually causes abortion during late pregnancy or sudden death. Encephalitis is rare. L. monocytogenes was first isolated by Murray et al. as the etiological agent of septicemia in laboratory rabbits and guinea pigs in England in 1926 (9).

Swine

Swine listeriosis is caused by L. monocytogenes, but it is uncommon. The primary manifestation is septicemia in young piglets, with death within 3 to 4 days. Encephalitis in adults and abortions are also seen occasionally.

Others

Septicemia and neurologic signs resembling rabies have been reported in dogs, and L. monocytogenes has also been isolated in canine cutaneous infection (15). Rare cases of encephalitis or septicemia occur in cats, with typical nonspecific symptoms including depression, inappetence, abdominal pain, vomiting, and diarrhea. In horses, there have been reports of meningoencephalitis, abortions, intrauterine infection, septicemia, and kerato-conjunctivitis associated with L. monocytogenes (16). Occasionally, septicemia disease is the usual form of listeriosis in other species, but abortions can also occur.

Listeriosis in Humans

L. monocytogenes is the most commonly reported etiological agent in cases of human listeriosis, though adult meningitis caused by L. seeligeri (31), a fatal bacteremia caused by L. innocua (32), and various cases of listeriosis caused by L. ivanovii (50) have also been reported. The disease mainly affects pregnant women and immunocompromised individuals by either physiological means—due to an immature (in newborns and young children) or suppressed (in the elderly) immune system—by infectious means (arising from immunosuppressive viruses and advanced systemic pathologies), or by iatrogenic means (associated with postchemotherapy or pretransplant immunosuppression). Currently, listeriosis is regarded as a foodborne disease of serious public health concern with a high mortality rate (25 to 30%) and a variable incubation period from 3 to 70 days.

In pregnant women, listeriosis is most commonly noted in the third trimester, but cases have been reported at all stages of pregnancy. The main clinical symptoms documented include mild flu-like symptoms, bloody vaginal discharge, and septicemia, and these may result in premature delivery, abortion, or stillbirth (33). A perinatal listeriosis infection occurs when L. monocytogenes crosses the placenta and elicits an infection in the fetus, but cross-infection during delivery is also possible (51). Perinatal infection is classified on the basis of the clinical symptoms observed: an early onset of listeriosis occurs in the fetus or neonate within the first week after delivery and is characterized by a serious septicemia (granulomatosis infantisepticum). Late onset occurs within the 2nd to 4th week of life and is manifested as meningitis with hydrocephalus as sequela (52). In immunocompromised adults, listeriosis can manifest as a rare but severe form of infection including septicemia or encephalitis (meningoencephalitis or rhombencephalitis), with symptoms appearing after 2 to 10 weeks of infection (33, 53).

Healthy individuals (who are immunocompetent) rarely develop clinical signs after infection, though a milder gastroenteritis with fever, abdominal and back pain, headache, and myalgia can occur. The incubation period is reported to extend from 8 to 48 h, and symptoms are typically self-limiting and resolve within 1 to 3 days. Direct transmission of L. monocytogenes from animals to humans is reported among farmers, veterinarians, and animal handlers who are in frequent direct contact with colonized animals or healthy carriers. A cutaneous form characterized by a nonpainful, nonpruritic pyogranulomatous rash has been reported in individuals who handle infected newborns, fetuses, or aborted cows or who perform necropsies on septicemic animals (54), while cases of conjunctivitis have been reported in workers in poultry processing plants who handled listeria-positive chickens (13).

CHEMOTHERAPEUTIC OPTIONS FOR THE TREATMENT OF ANIMAL AND HUMAN LISTERIOSIS

Cell-mediated immunity is the main host defense strategy against Listeria species and is largely dependent on the action of cytotoxic T-cells—hence the association between listeriosis and conditions involving impairments of cell-mediated immunity (55). The intracellular lifecycle of this bacterium confounds the efficacy of antimicrobial chemotherapy together with the lack of development of anti-Listeria immunity by the host postinfection. However, an anti-listeriolysin O (LLO) neutralizing monoclonal antibody has been reported to increase host resistance to infection in mouse models (56). Anti-LLO antibodies have been shown to help arrest the growth of Listeria species within macrophages, and this approach can be used for the serodiagnosis of these infections in humans and animals (57, 58), though it cannot discriminate between a current or previous infection (59), and furthermore, it provides no indication of the duration of this immunity (46).

Antimicrobial chemotherapy is the only viable option for the treatment of listeriosis. For an antibiotic such as a penicillin-based agent to be effective against this bacterium, it must penetrate into the host cell, maintain a high intracellular concentration, and bind to the penicillin-binding protein 3 (PBP3) expressed by Listeria species (60). High drug doses and early treatment are essential to treat animals with encephalitis, though if signs of encephalitis are severe, death usually occurs despite the intervention. Treatment in sheep and goats usually has little value soon after the appearance of neurological signs or in chronic cases. According to The Merck Veterinary Manual (54), in cattle, chlortetracycline given at a dose of 10 mg/kg body weight per day and administered for 5 days intravenously is effective in the treatment of encephalitis cases. If penicillin G is used, it should be given intramuscularly at 44,000 U/kg body weight per day for 1 to 2 weeks, and the first injection should be accompanied by the same dose given intravenously. Time durations for antimicrobial treatment may vary according to the level of infection. In severe cases, it is often recommended to continue treatment for up to 1 week after the clinical symptoms have disappeared. Supportive care, including the provision of clean housing along with fluids and electrolytes, is an important part of therapy. High-dose dexamethasone (1 mg/kg, given intravenously) at first examination is considered beneficial by some but is controversial and will cause abortion during the last two trimesters in cattle and after day 135 in sheep. Gentamicin has been found to be effective in the treatment of bovine genital listeriosis (61). Other drugs of choice to treat cases of listeriosis in livestock include erythromycin and trimethoprim/sulfamethoxazole. In birds, penicillin, tetracycline, erythromycin, gentamicin, and trimethoprim/sulfamethoxazole may be used successfully to treat the septicemic forms of infection, while treatment of the encephalitic form is usually unsuccessful.

Strategies for vaccine development are currently under study, and these are urgently required to tackle the infection in sheep, though advances in these protocols remain to be validated (12).

In humans, there are specific antimicrobial chemotherapeutic strategies to treat listeriosis depending on the individual and the corresponding diagnosis. Ampicillin or penicillin G combined with an aminoglycoside, classically gentamicin, is the most commonly prescribed treatment for listeriosis (62, 63). However, the combination of trimethoprim/sulfamethoxazole can be used as an alternative treatment. A review by Janakiraman et al. (63) provides information on different treatment options for listeriosis in humans.

RESISTANCE OF LISTERIA SPECIES TO DIFFERENT ANTIMICROBIAL AGENTS

Antimicrobial agents have been used in a wide range of settings to eliminate or inhibit bacterial growth. Most of these compounds target unique bacterial cell features including cell wall synthesis, the bacterial membrane, particular stages of protein synthesis, DNA and RNA synthesis, and folic acid metabolism, and depending on the nature of the drug, these can result in bacterial cell death or the inhibition of growth (64).

Bacteria possess two types of resistance: intrinsic, or naturally occurring, resistance and acquired resistance via mutations in chromosomal genes and by horizontal gene transfer (65). Intrinsic resistance usually arises as a result of inherent structural or functional characteristics of the microorganism. An example of intrinsic resistance arises due to the lack of affinity of the antimicrobial compound for its bacterial target in L. monocytogenes, and this is found in two cases of β-lactam-based compounds, monobactams and broad-spectrum cephalosporins. In these cases, intrinsic resistance is caused by the low affinity of theses drugs for PBP3, the enzyme that catalyzes the final step during cell wall synthesis. Although a few intrinsic resistance mechanisms in Listeria species have been described, most cases of resistance to antimicrobial compounds in this bacterium are due to acquired mechanisms, such as mobile genetic elements including self-transferable plasmids and conjugative transposons (66, 67).

Listeria species are well known for their susceptibility to a wide range of antimicrobial agents. Nonetheless, they possess intrinsic or natural resistance to a select number of antimicrobial compounds. Although natural resistance differs among members of the genus Listeria, all of the Listeria species tested, including L. monocytogenes, L. innocua, L. welshimeri, L. ivanovii, L. grayi, and L. seeligeri, were susceptible or intermediately resistant to aminoglycosides, carbapenems, cefotiam, cefoperazone, first- and second-generation cephalosporins (cefaclor, cefazolin, loracarbef), chloramphenicol, dalfopristin/quinupristin, glycopeptides, lincosamides, macrolides, penicillins (except for oxacillin), and tetracyclines (68). Troxler et al. (68) demonstrated that L. monocytogenes and L. innocua were naturally resistant to fosfomycin and fusidic acid, whereas other Listeria species were found to be naturally resistant only to fusidic acid (Table 4) (68). Listeria species differ in their natural susceptibility to co-trimoxazole, fluoroquinolones, fosfomycin, fusidic acid, rifampicin, and trimethoprim (Table 4), while L. innocua, L. ivanovii, and L. seeligeri elaborated a natural resistance to the fluoroquinolones, enoxacin, and sparfloxacin (Table 4) (68).

TABLE 4.

Intrinsic or natural antibiotic susceptibility and resistance of Listeria species

Listeria spp.	Naturally susceptible	Naturally resistant
L. monocytogenes	Trimethoprim	Fosfomycin
	Co-trimoxazole	Fusidic acid
L. innocua	Trimethoprim	Fosfomycin
	Co-trimoxazole	Fusidic acid
		Enoxacin
		Sparfloxacin
L. welshimeri	Trimethoprim	Fusidic acid
	Co-trimoxazole
L. ivanovii	Fosfomycin	Enoxacin
	Fusidic acida	Fusidic acida
	Trimethoprim	Fleroxacin
	Co-trimoxazole	Pefloxacin
		Sparfloxacin
L. seeligeri	Trimethoprim	Enoxacin
	Co-trimoxazole	Fleroxacin
		Pefloxacin
		Sparfloxacin
		Fusidic acid
L. grayi	All Fluoroquinolones	Fusidic acid
		Trimethoprim
		Co-trimoxazole

^a Both fusidic acid-susceptible and -resistant L. ivanovii isolates occur naturally in the environment.

Listeria species are rarely reported to develop acquired resistance to antimicrobial compounds. Nonetheless, selective pressure imposed in the past following the overuse use of various antimicrobial agents has resulted in members of this genus acquiring target gene mutations and resistance-encoding genes on mobile genetic elements (69). Transfer of resistance genes between Listeria species and other bacteria, such as Enterococcus and Streptococcus, by self-transferable plasmids has also been demonstrated (70).

Multidrug resistance was first documented in L. monocytogenes in 1988 in France (71). Since then, other Listeria species that are resistant to one or more compounds have been isolated, and several antimicrobial-resistance genes have been identified and characterized (62).

The following section provides a summary of the resistance-encoding genes that have been identified and mutations known to be associated with resistance to different classes of antibiotics, biocides, and heavy metals in Listeria species.

Antibiotics

Since the discovery of penicillin in 1929, antibiotics have been commercially used for the treatment of a wide variety of clinical diseases and have also been used for animal production purposes as additives in animal feed (72, 73). Over this period of time, selective pressure arising from the overuse of antimicrobial compounds has led to the emergence of bacteria expressing resistance to one or more of these agents. Similarly, members of the genus Listeria have developed mutations in the chromosome and/or acquired resistance genes carried on mobile genetic elements. In contrast and in comparison to other foodborne pathogens of human health importance, Listeria species do not exhibit these same features. Thus, data describing antimicrobial resistance in Listeria species is limited. Similar to other bacteria, Listeria species can express innate resistance to certain classes of antimicrobial compounds. In addition, several resistance genes have been reported to be located on mobile genetic elements such as conjugative transposons and plasmids. Therefore, members of the genus Listeria may play a role in the dissemination of resistance genes among bacteria.

Multidrug resistance has been documented in various isolates of Listeria, and several antimicrobial-resistance-encoding genes have been identified by means of molecular-based approaches. In the subsections below, a summary of the known mutations and genes associated with resistance to the different classes of antimicrobial agents is provided. The location of resistance genes on mobile genetic elements and the colocation of other markers is discussed.

Resistance to quinolones and fluoroquinolones

Quinolones and fluoroquinolones represent a class of antimicrobial agent which is important for the treatment of severe and invasive infections in animals and humans, with special interest for public and animal health. Quinolones were introduced into clinical use in 1962 in the form of nalidixic acid, and fluoroquinolones were first licensed for veterinary use in several countries in the 1980s (74). Subsequently, their use was followed by the reported emergence of antimicrobial resistance in bacteria. These bacteria were isolated from humans and food-producing animals (69, 75). Resistance to ciprofloxacin is mainly attributed to target gene mutations within the quinolone-resistance-determining regions of genes encoding the bacterial topoisomerase enzymes (76). The presence of plasmid-mediated quinolone-resistance (PMQR)-encoding genes can also contribute to the ciprofloxacin-resistance phenotype (77). Although these PMQR genes confer only low-level resistance to fluoroquinolones, the presence of PMQR (particularly qnr genes) may provide a selective advantage for bacteria exposed to fluoroquinolones and facilitate the subsequent development of high-level chromosomal quinolone resistance. In some Listeria isolates, the quinolone-resistance-determining region of DNA gyrase subunit A was altered; the deduced amino acid sequences revealed substitutions including Ser84 → Thr and Asp/Glu88 → Phe, both representing amino acid changes at hot spots commonly associated with resistance to these agents (78, 79). Another mechanism, involving efflux pumps such as Lde, MdrL, and FepA (see “Concluding Remarks”), has also been associated with resistance to fluoroquinolones in Listeria species (67, 80, 81). Macrolide-based antibiotics and cefotaxime, as well as heavy metals and ethidium bromide, are also exported by MdrL (80).

Resistance to penicillins and cephalosporins

Penicillins and cephalosporins are β-lactam-based antibiotics that inhibit bacterial cell wall synthesis. Listeria species are usually susceptible to penicillins, with the exception of oxacillin, monobactams, and broad-spectrum cephalosporins including cefetamet, cefixime, ceftibuten, ceftazidime, cefdinir, cefpodoxime, cefotaxime, ceftriaxone, and cefuroxime, to which this bacterium is naturally resistant (68). Only, the penA-encoding gene, a known PBP first identified from Neisseria meningitides, has been associated with resistance to penicillin G in L. monocytogenes (82). Some of the previously described mechanisms associated with the innate or natural resistance to cephalosporins most often include cell wall-acting gene products, two-component systems, and efflux pumps. The PBPs are enzymes that are responsible for extending the glycan chains in peptidoglycan and cross-linking the peptides between chains. Several PBPs were identified in Listeria and function to confer a natural resistance phenotype to modern cephalosporins (83). The gene oatA encodes an O-acetyltransferase, which catalyzes the acetylation of muramic acid in peptidoglycan, thus conferring resistance to cefotaxime and gallidermin. OatA is important for pathogenesis in mice, probably via protection from macrophage killing (84). Other mechanisms also confer reduced susceptibility to cephalosporins, including the two-component systems CesR and LiaSR, and efflux pumps (MdrL and AnrAB) (85).

Resistance to aminoglycosides

Aminoglycosides inhibit protein synthesis by binding to the 30S ribosomal subunit. Although more than 170 aminoglycoside-resistance-encoding genes have been described in bacteria, these can be grouped into three major classes: acetyltransferase-acting modifying enzymes, nucleotidyltransferases, and phosphotransferases (86). The streptomycin-resistance gene, aad6, which encodes for 6-N-streptomycin adenylyltransferase, has been identified in L. monocytogenes and L. innocua (69, 70). To date, no other aminoglycoside-resistance genes have been described in Listeria species.

Resistance to tetracyclines

Tetracycline resistance is the most frequent resistance phenotype detected in Listeria species. Tetracyclines inhibit protein synthesis by binding to the 30S ribosomal subunit. More than 50 tetracycline-resistance-encoding genes have been described, and five of these—tet(A), tet(K), tet(L), tet(M), and tet(S)—have been reported in Listeria species. Two known mechanisms of resistance to tetracyclines have been reported in Listeria species. These are mechanisms that involve efflux of the drug mediated by proton antiporters, conferring resistance to tetracycline only [including the genes tet(A), tet(K), and tet(L)] and ribosome protection proteins, conferring resistance to both tetracycline and minocycline [encoded by tet(M) and tet(S)]. Two types of mobile genetic elements, conjugative plasmids and transposons originating from Enterococcus-Streptococcus, are thought to be responsible for the emergence of resistance to tetracycline in Listeria. The tet(M) gene is often associated with the conjugative transposon Tn916, while tet(S) and tet(L) are more often carried on plasmids (87).

Resistance to phenicols

The molecular basis of bacterial resistance to chloramphenicol and its fluorinated derivative florfenicol has been reviewed by van Hoek et al. and Schwarz et al. (88, 89). In Listeria species, enzymatic inactivation of this drug by type A chloramphenicol acetyltransferases (Cat) and the export of chloramphenicol/florfenicol by specific efflux proteins are the dominant resistance mechanisms. A cat gene (type A-8) has been detected in Listeria, located on a plasmid (89, 90). The floR gene is associated with the export of florfenicol in L. monocytogenes, and 50% of floR-positive isolates may also confer resistance to chloramphenicol (82). Florfenicol and chloramphenicol coresistance conferred by floR has also been reported in Gram-negative bacteria such as Pasteurella piscicida and E. coli (82).

Resistance to macrolides (macrolides-lincosamides-streptogramin)

The macrolides (macrolides-lincosamides-streptogramin B [MLS_B]) inhibit protein synthesis through binding to the 50S ribosomal subunit of bacteria. The resistance determinants responsible include rRNA methylases that modify the 23S ribosomal target sites, ATP-binding cassette (ABC) transporters, and efflux proteins of the major facilitator superfamily (MFS), as well as factors such as the ere-encoding genes that function as inactivating enzymes. The most common mechanism of MLS_B resistance is due to the presence of rRNA methylases, encoded by the erm genes. These enzymes methylate an adenine base, which prevents drug binding to the 50S ribosomal subunit, resulting in MLS_B resistance. There are currently 92 MLS_B resistance genes recognized in bacteria (88), but only erm(A), erm(B), and erm(C) have been reported in Listeria species (91, 92). Plasmid pDB2011 from L. innocua, isolated from prepackaged sprouts in Switzerland, contained three antibiotic genes: spc (spectinomycin-adenyltransferase), erm(A) (erythromycin-methylase), and dfrD (trimethoprim-dihydrofolate); spc was located together with erm(A) on the transposon Tn554 (93). In Gram-positive bacteria, resistance to 14- and 15-membered-ring macrolides, such as erythromycin, can also be mediated by efflux pumps belonging to the ABC transporter family, such as msr(A) found in Staphylococcus species, or to the MFS mef(A) found in Streptococcus pneumoniae (69, 92). Although several studies have investigated the possible presence of mef(A) and msr(A) in Listeria species, further studies are necessary to extend our knowledge of the possible role of these efflux pumps in Listeria.

Resistance to trimethoprim

Trimethoprim is a folate pathway inhibitor, and folic acid is essential for the synthesis of adenine and thymine, two of the four bases that are involved as structural components of nucleic acids. Trimethoprim resistance has been described in L. monocytogenes due to the dfrD resistance-encoding gene. High-level trimethoprim resistance in Listeria is mainly due to the replacement of a trimethoprim-sensitive dihydrofolate reductase by a plasmid-, transposon-, or cassette-borne version. To date, more than 40 trimethoprim-resistant dihydrofolate resistance-mediating reductase (dfr) genes have been identified (94). Initially, the dfr genes were classified into two major families according to their amino acid structure, dfrA and dfrB, and currently, six plasmid-mediated families can be distinguished in Gram-positive bacteria, including dfrC, dfrD, dfrG, and dfrK (88).

In Listeria, only two of these—dfrG and dfrD—have been reported. The dfrD gene was found to be encoded on the plasmid pIP823 (95), while the dfrG gene was recently found in Tn6198, a Tn916-like element associated with a trimethoprim-resistance gene (96).

Biocides

Biocides have been widely used to control the microbial ecology of various niches for centuries. European Union regulation no. 528/2102, covering biocidal products (97), divides biocides into four categories: disinfectants, preservatives, pest control agents, and other biocidal products. These compounds are used in a wide variety of settings including the food and cosmetic industries, for personal hygiene, and for applications in veterinary practice and farming (98). Currently, there is a diverse range of chemicals including alcohols, biguanides, bisphenols, chlorine-releasing agents, iodophors, peroxides, and quaternary ammonium compounds (QACs), among others, that constitute biocides. Reduced susceptibility to these agents has been recognized for decades and is increasing (98, 99).

This section provides a summary of the known mechanisms of resistance or tolerance of Listeria species.

Quaternary ammonium compounds

QACs are amphoteric surfactants with a broad spectrum of antimicrobial activity and are widely used as disinfectants in clinical, domestic, and environmental settings (100). The most commonly used QAC is benzalkonium chloride (BC), also known as alkyl dimethyl benzyl ammonium chloride, which targets cytoplasmic membrane permeability functions, causing potassium leakage, osmoregulation disruption, enzyme inactivation, and protein denaturation (101, 102). Tolerance to BC was first described in L. monocytogenes and L. innocua isolates in 1998. BC tolerance may be conferred by transposons or efflux pumps encoded on the chromosome or on plasmids. One such tolerance mechanism previously described in L. monocytogenes was associated with the bcrABC operon, which can be encoded in the chromosome or on a putative IS1216 composite transposon harbored by the large plasmid pLM80 (103, 104). The bcrABC cassette is composed of a TetR-like transcriptional regulator (bcrA) and two small multidrug resistance (SMR) efflux pump-encoding genes (bcrB and bcrC) (103, 104). Another resistance mechanism identified in L. monocytogenes is the chromosomally integrated transposon Tn6188, which is related to other Tn554-like transposons. Transposon Tn6188 is a 5,117-bp structure found integrated within the radC gene, and it consists of three transposase genes (tnpABC), along with genes encoding a putative transcriptional regulator and QacH, an SMR transmembrane protein associated with the export of BC (105, 106). The qacH gene also confers tolerance to a wide range of QACs including BC, benzethonium chloride, cetylpyridinium chloride monohydrate, cetyltrimethylammonium bromide, domiphen bromide, dodecyltrimethylammonium bromide, ethidium bromide, and the sanitizer Weiquat, resulting in higher MIC values being recorded as well as in increased expression of the transporter (106). Although Tn6188 has been widely identified in L. monocytogenes, Tn6188 has not been identified in other Listeria species to date. The putative EmrE SMR-related efflux pump located within Listeria genomic island 1 (LGI1) in L. monocytogenes can also confer tolerance to BC and QAC-based sanitizers (107). There are two MFS efflux pumps, MdrL and Lde, that have also been reported to be partially responsible for BC tolerance in L. monocytogenes (108–110).

Triclosan

Triclosan is a chlorinated aromatic compound with a broad spectrum of antibacterial and antifungal activity, which has been used for over 40 years as an antiseptic, disinfectant, and preservative in clinical and cosmetics settings (111, 112). It is bacteriostatic in nature at low or sublethal concentrations, acting to inhibit a specific enzyme, FabI, an enoyl-acyl carrier protein reductase linked to fatty acid biosynthesis, (113–116). Inhibition of FabI results in reductions in lipid biosynthesis (117). At high or bactericidal concentrations, triclosan has a more general membrane-disrupting action, consistent with potassium leakage from the cell (118, 119). It has been shown that point mutations and overexpression of the fabI-encoding gene conferred tolerance to triclosan in some bacteria, but not in Listeria species (116, 120, 121). Exposure of L. monocytogenes to sublethal concentrations of triclosan can cause cross-resistance to several aminoglycosides and exhibit two types of colony morphology: normal-size and pinpoint colonies. This study demonstrated that adaptation to triclosan may be due to point mutations in several genes including ferrochelatase (hemH) or glutamyl tRNA reductase (hemA) (122).

Heavy Metals

From a saprophytic bacterium (all Listeria species) to intracellular pathogen (L. monocytogenes and L. ivanovii), maintaining heavy metal homeostasis is central to the survival of Listeria species across a broad range of environmental niches. Many heavy metals, such as iron and zinc, are an essential component of cellular function, and bacteria compete to sequester them using a variety of mechanisms such as siderophores and ATP-transporters. As heavy metal concentrations increase, associated toxicity can lead to cell death. Many factors can lead to an increase in heavy metal concentration in niches associated with Listeria species. The use of fertilizers or industrial processing can contaminate soil or water environments with heavy metals to levels which can be toxic to bacteria. Similarly, for pathogenic Listeria species, both heavy metal scavenging and resistance are necessary for survival and infection. It is becoming clear that heavy metals are an integral part of the host immune defense, either through host sequestering to deny availability or by exploiting their toxicity to kill invading pathogens (123–125).

Balancing the intracellular concentration of heavy metals is thus a matter of life or death for bacteria such as Listeria species. Studies investigating heavy metal resistance (HMR) in Listeria largely focus on L. monocytogenes, although these resistance mechanisms are often found in other Listeria species (126, 127). While much progress has been made in identifying mechanisms utilized by Listeria in resistance to certain heavy metals (e.g., cadmium and arsenic), less is known about many others (e.g., mercury and lead).

Many genes for HMR in bacteria are contained on mobile genetic elements such as plasmids and transposons (127–129), and as such, they are often found in association with a variety of other genetic markers, some of which may confer resistance to other antimicrobial agents or environmental stressors. Perhaps the most significant implication of this is the possibility of one resistance phenotype coselecting for another (130). Although mobile genetic element-mediated antibiotic resistance has not been frequently reported in Listeria, the potential coselection of Listeria plasmids containing multiple resistance markers has been reported (127, 131). In particular, heavy metal-mediated coselection of environmental stress resistance markers may contribute to survival and persistence of Listeria species in various environmental niches. Coselection of HMR plasmids may be elicited by a number of factors, including the use of heavy metals in agriculture in fertilizers or supplemented into animal diets as growth promoters or therapeutics (132, 133), heavy metal discharge in industrial wastewater effluent (134), and global mining activities (135). In addition, genetic markers of HMR may be associated with genes coding for disinfectant-resistance determinants (136); as a result, the use of disinfectants in food processing environments, which are frequently colonized by Listeria species (137, 138), may also serve to coselect for HMR (139). A multipronged approach will thus be necessary in any effort designed to limit the maintenance and spread of HMR-mediating mobile genetic elements in bacterial populations including Listeria species and others.

Cadmium resistance

Cadmium is not essential for the growth or survival of Listeria species, and it can be toxic to bacteria at low levels (140). Of all the heavy metals, cadmium-resistance phenotypes and mechanisms are among the most extensively studied in L. monocytogenes (136, 139, 141, 142). Resistance to cadmium was first correlated with plasmid carriage, with a cadAC operon being subsequently identified as the resistance determinant (143, 144). Efflux of Cd²⁺ is driven by the cadA-encoded energy-dependent pump, while cadC serves as a negatively acting regulatory protein of the operon (145, 146). The Listeria cadAC genes were first identified in a plasmid-borne Tn5422 transposon and were initially thought to be more prevalent among serotype 1/2 isolates (147). Recent studies have shown a more uniform distribution across serotype 1/2 and 4 isolates (148). Based on the frequency of plasmid carriage, plasmid profiling along with cadmium and arsenic-resistance phenotyping and serotyping has been suggested as a simpler means of subtyping L. monocytogenes isolates with acceptable discriminatory power (147). Variations of cadA described in Listeria include cadA1, the Tn5422 determinant described above, the cadA2 gene identified on plasmid pLM80 (149), cadA3 harbored by the EGD-e strain (3, 150), and cadA4 located on the chromosome of the outbreak strain ScottA (Fig. 4) (151).

FIGURE 4.

Heavy metal resistance operons in the L. monocytogenes strain ScottA. (A) Arsenic resistance operon. (B) cadAC cadmium resistance operon.

Arsenic resistance

Like cadmium, arsenic is also a nonessential heavy metal for bacterial growth and metabolism but becomes toxic as concentrations increase. Arsenic contamination of soil and water environments can arise from a number of sources including mining, industrial processing, and geothermal activity (152). Organic arsenic is less toxic than the inorganic form, and the use of arsenic has applications in medicine, pest control, and organic arsenic-based drugs. Organoarsenates have also been used as growth enhancers in food production systems (153, 154). Arsenic resistance in Listeria has been well characterized, and an arsenic-resistance cassette initially identified in L. innocua CLIP11262 on plasmid pLI100 has subsequently been identified in a number of L. monocytogenes isolates (Fig. 4) (127, 141, 151). Although harbored on plasmid pLI100, arsenic resistance in L. monocytogenes isolates to date typically show chromosomal association (141, 147). In contrast to cadmium resistance, which does not appear to have serotype-specific association, arsenic resistance has been primarily associated with serotype 4b isolates (139, 147). The use of organoarsenates in intensive poultry food production systems has also been implicated in selecting for arsenic resistance in Listeria species (139).

Zinc resistance

Unlike cadmium and arsenic, zinc is an essential micronutrient for Listeria species and is involved in diverse functions such as metabolism, cell division, and stress resistance (155). Two principle transport mechanisms responsible for sequestering zinc have been characterized in Listeria: the zurLAM and the zinABC operons (155). As with other heavy metals, however, zinc becomes toxic to Listeria species as the concentration increases. Elevated zinc levels are utilized in the host response to pathogen infection, and previous work has shown that this is associated with a bacteriostatic effect on L. monocytogenes (124, 156). Unlike other bacteria, cadA has not been shown to contribute to zinc resistance in Listeria species (128, 144).

Copper resistance

Although copper plays an important role in cellular function, Listeria species must maintain copper homeostasis to combat cytotoxic effects due to elevated concentrations (155). The csoR-copA-copZ operon confers copper resistance in L. monocytogenes (157). Other genetic markers implicated in copper resistance include the copper transporter CptA and a CutC homolog encoded by lmo1018 (155, 157). Copper is used as a growth promoter in food animals and, as such, has implications for selection of copper-resistant Listeria species; future work should address the implications of this selection pressure on coselection of other resistance markers.

MOBILE GENETIC ELEMENTS ASSOCIATED WITH ANTIMICROBIAL RESISTANCE IN LISTERIA SPECIES

DNA elements that can transfer within a genome or to another genome are generally referred to as mobile genetic elements. Transposons and plasmids are among the most commonly described mobile genetic elements in Listeria species and have been identified as one of the most important driving forces underlying the evolution of the species (158). As knowledge of the diversity of these mobile genetic elements increases, it is becoming apparent that they are frequently associated with a plethora of resistance phenotypes. Their dissemination through the Listeria population raises a number of concerns for food safety and public health; mobile genetic elements contributing to resistance of Listeria strains to disinfection regimes utilized in food processing have already been described, and reports are now emerging of resistance to clinically relevant antibiotics (96, 103, 105).

Transposons

Transposons are an important mechanism for the transfer of resistance markers through bacterial populations. A number of transposons and their insert sites (both chromosomal and plasmid) have been identified among Listeria species (129, 158), harboring resistance markers to different classes of antimicrobial agents, some of which include the transposon Tn917, which is implicated in cadmium resistance and is closely related to another plasmid-borne Tn5422 cadmium-resistance transposon (144, 159), the Tn554-like transposon, which carries an arsenic-resistance operon (158), a Tn6188 transposon (also a Tn554-like transposon), which carries the qacH gene and confers increased resistance to QACs, a bcrABC resistance cassette, which harbors an IS1216 composite transposon, a Tn916 to Tn1545 family conjugative transposon associated with tetracycline resistance and which contains tet(M) (160), and a Tn6198 multidrug-resistance transposon that confers resistance to tetracycline and trimethoprim (96).

Previous studies have noted the capacity for the transfer of chromosomal-located resistance determinants between Listeria species as well as other genera (96, 161), highlighting the potential for proliferation of antimicrobial resistance in Listeria species populations. The recent increase in whole-genome sequencing of Listeria genomes will undoubtedly expand our understanding of the repertoire of transposable elements present in Listeria species and provide insights into their putative transfer patterns. Such knowledge will be crucial in directing future control strategies aimed at tackling the issue of horizontal gene transfers among Listeria species.

Plasmids

Maintaining plasmids can impose a fitness cost on the host bacterium, which could lead to that host being outcompeted by other bacteria, in the absence of a selective pressure (162). However, resistance plasmid carriage is widespread in bacterial species, including subgroups of Listeria species (147, 163). Recent advances in understanding the factors underlying their ubiquity among bacterial populations have shown that very low levels of antimicrobial agent, often significantly below the MIC, can select for their carriage (164). This is further exacerbated by coselection, because plasmids often confer resistance to many different antimicrobial compounds, such as antibiotics, heavy metals, or biocides, and thus are maintained in bacterial populations by a multitude of selective pressures (103, 165).

Plasmids sharing replication mechanisms generally cannot exist together in the same host, which is often referred to as plasmid incomparability, and this feature forms the basis of incompatibility (Inc) grouping. Although commonly used for classification of Gram-negative plasmids, its use is less common among Gram-positive bacteria (166, 167). Studies relating to Listeria species plasmids suggest two distinct groupings based on their associated replicon and genetic diversity (127). Few instances of Listeria species harboring more than one plasmid have been reported (168), but more research is needed to understand the dynamics of plasmid carriage and incompatibility in Listeria species.

HMR markers are among the most commonly described with regard to Listeria plasmids, particularly cadmium-resistance genes (129, 144, 169). These plasmids frequently harbor other resistance markers associated with biocide and/or stress resistance, as well as efflux systems (127, 129, 136). These markers may contribute to the survival and persistence of Listeria species in food processing environments and, as such, present a significant concern for food safety and public health (127, 131). Another intriguing observation is the conservation of identical or similar plasmids among isolates from geographically diverse locations, suggesting that strong selective pressures are maintaining plasmid carriage among Listeria species (131).

Although the frequency of antibiotic resistance among Listeria species has been reported to be low (69, 170), plasmid carriage of resistance-encoding mechanisms to antibiotics including chloramphenicol, erythromycin, and tetracycline has been reported (71, 171, 172). In addition, a recently identified multidrug-resistance plasmid in L. innocua conferring resistance to both trimethoprim and spectinomycin exhibited a broad host range (93). The emergence of such resistance mechanisms highlights the need for increased surveillance efforts to attempt to minimize the risk of emergence of clinically relevant antimicrobial resistance in Listeria species and in L. monocytogenes in particular.

EFFLUX PUMPS ASSOCIATED WITH ANTIMICROBIAL RESISTANCE IN LISTERIA SPECIES

Efflux pumps are transmembrane-spanning protein complexes capable of transporting a broad range of chemically and structurally unrelated substrate molecules from inside the bacterial cell to the extracellular matrix (173). Bacterial efflux pumps have been classified into five families: the ABC superfamily, the multidrug and toxic compound extrusion family (MATE), the MFS, the SMR family, and the resistance-nodulation-division superfamily (174). The most commonly identified efflux pumps found in Gram-positive bacteria are those represented by the MFS family, in contrast to those of the resistance-nodulation-division family being found in Gram-negative bacteria. The latter family is rarely reported in Gram-positive bacteria. All of these systems harness the proton motive force as an energy source, with the exception of the ABC transporters, which use direct ATP hydrolysis to drive the transport of substrates (Fig. 5) (175). In the past few years, several efflux pumps have been described in L. monocytogenes.

FIGURE 5.

Diagrammatic representation of the four families of efflux pumps in L. monocytogenes. The ATP-binding cassette (ABC) superfamily, the major facilitator superfamily (MFS), the multidrug and toxic-compound extrusion (MATE) family, and the small multidrug resistance (SMR) family. Common examples of the individual proteins that form each class of efflux pump are shown.

The first efflux pump to be described in L. monocytogenes was cadAC. Shortly thereafter, an MFS efflux pump, denoted MdrL, was described in L. monocytogenes, and it was reported to export cefotaxime, ethidium bromide, heavy metals, and macrolides (Table 5) (80). Several subsequent studies have also reported on the role of MdrL in the adaptation of L. monocytogenes to BC (108, 109). L. monocytogenes encodes two efflux pumps that are highly similar to QacA in Staphylococcus aureus, and they were identified as MdrT and MdrM. Both MFS efflux pumps confer resistance to cholic acid (176, 177). MrdT and MdrM also play a role during bacterial replication within the cytosol of infected cells, by secreting cyclic-di-AMP, which triggers the production of type I interferons, including beta interferon, which promotes L. monocytogenes virulence (178).

TABLE 5.

Multidrug efflux transporters characterzsed in L. monocytogenes

Gene	Efflux pump	Regulator	Substrates
lmo1409	MdrL	LadR	Ethidium bromide Macrolides Cefotaxime Heavy metals Benzalkonium chloride
lmo1617	MdrM	MarR	Rhodamine 6 G Cholic acid
lmo2588	MdrT	BrtAa	Tetraphenylphosphonium Cholic acid Bile
lmo2741	Lde	Unknown	Ciprofloxacin Norfloxacin Ethidium bromide Acridine orange Benzalkonium chloride
lmo1851	EmrE	Unknown	Benzalkonium chloride
lmo2089	FepA	FepRa	Norfloxacin Ciprofloxacin Ethidium bromide
lmo2115	AnrB	VirR	Nisin Bacitracin Gallidermin Cefuroxime Cefotaxime Ampicillin Penicillin
cadAC	CadAC	CadC	Cadmium Benzalkonium chloridea Arsenic
bcrABC	BcrBC	BcrAa	Benzalkonium chloride
qacH	QacH	TetR	Benzalkonium chloride Benzethonium chloride Cetylpyridinium chloride monohydrate Cetyltrimethylammonium bromide Domiphen bromide Dodecyltrimethylammonium bromide Ethidium bromide

^a Belongs to the TetR family.

Two efflux pumps that confer resistance to fluoroquinolones have been characterized in L. monocytogenes, Lde and FepA. The former is encoded by the lde gene, whose protein product is a 12-segment transmembrane-spanning putative MFS efflux pump with 44% amino acid identity with PmrA from S. pneumoniae. Lde was characterized in L. monocytogenes CLIP 21369 by insertional inactivation of the lde gene, and these data demonstrated that this pump conferred resistance to ciprofloxacin and norfloxacin together with a reduced susceptibility to the dyes acridine orange and ethidium bromide (Table 5) (67, 179). Other studies have also suggested that lde may be partly responsible for BC tolerance (110). Lde has other interesting functions, because it may also cooperate with a eukaryotic multidrug resistance-related protein (MRP)-like efflux transporter to reduce the activity of ciprofloxacin, a substrate for both pumps, in J774 macrophages infected with L. monocytogenes (174, 180). Furthermore, the L. monocytogenes in vivo-induced virulence factor Hpt mediates uptake of fosfomycin in L. monocytogenes, thereby conferring a resistant phenotype in vitro and thus constituting an antibacterial in vitro-in vivo paradox, since the bacteria are resistant in vitro but susceptible to the drug in vivo (174, 181).

Another mechanism that may confer resistance to fluoroquinolones in L. monocytogenes is linked with the overexpression of a multidrug and toxic compound extrusion efflux pump, FepA. It has been demonstrated that a single point mutation in the FepA transcriptional regulator fepR, which belongs to the TetR family, produces a frameshift mutation that causes the introduction of a premature stop codon, resulting in an inactive truncated protein. This single point mutation was responsible for the overexpression of fepA, which confers resistance to ciprofloxacin, ethidium bromide, and norfloxacin, thereby confirming the role of FepR as a local repressor of fepA (Table 5) (81).

AnrB, an ABC transporter, confers innate resistance of L. monocytogenes to the lantibiotic nisin. The AnrB function was demonstrated by a nonpolar deletion mutation in the lmo2115 gene, and the loss of this multidrug transporter increased susceptibility to bacitracin, β-lactams, gallidermin, and nisin (Table 5) (182).

In 2015, a novel putative efflux pump, EmrE (324 bp), was described within LGI1 in L. monocytogenes (107). An emrE deletion mutant demonstrated an increase in the susceptibility to BC along with QAC-based sanitizers but exhibited no effect on the MICs for acriflavine, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, tetracycline, and triclosan (Table 5) (107).

CONCLUDING REMARKS

Listeria species are ubiquitous throughout the food chain and the natural environment, and these microorganisms have been isolated from a wide variety of domestic and wild animals. Since 2009, nine new Listeria species have been discovered, and these remain to be further characterized.

Listeriosis may manifest in a mild form, presenting with gastroenteritis-like symptoms, or in a more severe form where Listeria crosses the blood-brain barrier and placenta, invading the CNS and the fetus. Antibiotic treatment strategies for listeriosis vary according to the infection type and the nature of the infected host. In humans, ampicillin or penicillin G in combination with an aminoglycoside is regarded as the front-line treatment (63). The primary antibiotics used to treat listeriosis in animals are chlortetracycline, penicillin, tetracycline, erythromycin, gentamicin, and trimethoprim/sulfamethoxazole (61). Although the incidence of antibiotic resistance in Listeria species remains low, multidrug-resistant Listeria species strains have been isolated from numerous sources including clinical isolates, retail foods, and the environment (69, 92, 183). Furthermore, the range of antibiotics to which resistance has been acquired is broad and includes compounds currently used to treat listeriosis, such as ampicillin, penicillin, trimethoprim, tetracycline, and erythromycin, among others (88, 183). Resistance to erythromycin, rifampicin, trimethoprim, and especially, to tetracycline has been commonly reported in Listeria species from Europe and North America and is consistent with the identification of tet-, erm-, aad-, and dfr-encoding genes (79). This development may be problematic for patients who are allergic to penicillin because the combination of trimethoprim and sulfamethoxazole is used for the treatment of listeriosis (63).

Multidrug resistance in Listeria species has also been linked to the presence of efflux pumps, which confer resistance to a wide variety of antimicrobials, including antibiotics, biocides, heavy metals, and other antimicrobial compounds. Lde and AnrB confer resistance to fluoroquinolones, whereas EmrE, BcrABC, and QacH confer tolerance to BC. Tolerance to biocides is of special concern in food-producing plants and farms, because these agents are used as sanitizers and their decreasing efficacy hinders the elimination of Listeria from these environments, increasing the risk of cross-contamination to the final product or animals. Efflux pumps and antimicrobial-resistance genes can be encoded chromosomally or on mobile genetic elements, e.g., plasmid pDB2011 from L. innocua, which contains three antibiotic resistant genes, spc, erm(A), and dfrD, and confers resistance to spectinomycin, erythromycin, and trimethoprim, and Tn6188, which encodes for the qacH gene and confers resistance to QACs. Furthermore, Listeria can acquire antimicrobial genes from foreign sources by self-transferable plasmids or conjugative transposons from other bacterial species such as Enterococcus and Streptococcus (62, 170, 183).

Despite the ever-increasing threat of antimicrobial resistance to human health and the fact that Listeria is widespread in the environment and agriculture, relatively little research has been focused on this topic to date.