Introduction

Background on Listeria monocytogenes

Listeria monocytogenes is a gram-positive bacteria that causes an infection known as listeriosis. Listeriosis is a serious foodborne disease which affects 0.1 and 11.3/1,000,000 of the population depending on the country.^{1 2} 

L. monocytogenes is a common bacteria and everyone is routinely exposed to it. Despite this, listeriosis is a relatively rare disease¹ and, in most people, it has no symptoms or only causes mild symptoms for a few days, such as:^{3 4}

• Fever
• Aches and pains
• Chills
• Diarrhea 
• Feeling or being sick

Common sources and transmission routes

L. monocytogenes is an ubiquitous bacteria and it has been found in groundwater, soil and animal and human faeces. ⁵  One study examined surface water and point source pollution samples from an urban and a rural municipal water supply watershed in Nova Scotia, Canada, over 18 months. The researchers found that 30.3% and 34.5% of samples were positive in rural and urban watersheds respectively with higher prevalences during the fall and winter seasons.⁶

L. monocytogenes is also commonly found and has been isolated from market or restaurant products like dairy, processed and ready-to-eat food, cold-stored meat and fresh vegetables.⁷

Cross-contamination can be common when handling contaminated products in the kitchen. Washing raw vegetables reduces the risk of contamination.⁸

Importance of studying antibiotic resistance

Antibiotic resistance is a public world threat, WHO considers it one of the top 10 global public health threats confronting humanity.⁹ It is estimated that antibiotic resistance caused 1.27 million deaths in the world in 2019 and contributed to 4.95 million deaths.¹⁰ The reported causes of the rise in antibiotic resistance include the overuse of antimicrobials in humans, animals and plants.⁹ The resistance to antibiotics in bacteria jeopardises numerous advancements in modern medicine. It complicates the treatment of infections and increases the risks associated with various medical procedures and treatments, including surgeries, caesarean sections, and cancer chemotherapy.⁹ It is estimated that between 38.7% and 50.9% of pathogens causing surgical site infections and 26.8% of pathogens causing infections after chemotherapy are resistant to standard prophylactic antibiotics in the USA.¹² Also, it has significant economic costs. The World Bank estimates that  antibiotic resistance could increase in 1 trillion  dollars of additional healthcare costs by 2050.¹¹

Overview of listeria monocytogenes

Biology and behaviour

L.monocytogenes is a gram-positive bacteria which can survive in the absence of oxygen (it is a facultative anaerobe). Unlike most pathogens, it is resistant to acid and high salt concentrations. It is also capable of growing at low temperatures, which means it can multiply in refrigerated foods. In addition, L.monocytogenes produces biofilm which increases its survival (it has been described to survive more than 10 years).¹³

Epidemiology and risk factors

There are two clinical forms of listeriosis: invasive and non-invasive. Non-invasive listeriosis is a self-limiting clinical disease with non-specific symptoms and therefore, it is probably underdiagnosed. It can be present as febrile gastroenteritis (inflammation of the digestive tract) and as cutaneous listeriosis. Cutaneous listeriosis is very rare and usually related to occupational exposure in people who work with animals.¹

Invasive listeriosis occurs when bacteria spread from the intestines to the bloodstream and can produce sepsis, bacteremia and meningoencephalitis among others in vulnerable people. Despite adequate treatment, invasive listeriosis has an average mortality rate of 20–30%.^{1 2}

L.monocytogenes is particularly dangerous in pregnant women because the pathogen is transmitted to the foetus from the mother through the placenta and while the mother usually has flu-like symptoms, the foetus develops a systemic infection because its immune system is not developed. That can cause death or premature birth of a severely ill baby.¹

Other risk factors for suffering invasive listeriosis are old age, malignancies, diabetes mellitus, alcoholism, liver, renal and autoimmune diseases and other immunosuppressive conditions or treatments. Due to its long incubation period between a few days and up to 90 days and often exceeding 30 days, it is difficult to determine the source of the outbreak.^{1 2}

Antibiotic resistance in listeria monocytogenes

L. monocytogenes is susceptible to most commonly used antibiotics: penicillins, tetracycline, aminoglycosides, trimethoprim, macrolides, and vancomycin. It is naturally resistant to cephalosporins, sulfamethoxazole,¹ and current fluoroquinolones.¹³ Acquired antibiotic resistance is rare in clinical strains¹⁴ but has been found with a significant frequency in animal food and environmental samples.¹⁵ However, these studies are very variable, finding higher frequencies in medium-low income countries, indicating that maybe this data should be reviewed.

The most frequently acquired resistance in human and food isolates of L. monocytogenes is tetracycline resistance. It was observed in the first multiresistant isolates, in 1988.^{15 16}

However, a review of some studies found that the highest acquired resistance in various ready-to-eat food and chicken samples came from penicillins (80%).¹⁷ The current treatment of invasive listeriosis is supportive therapy accompanied by high doses of intravenous penicillin or ampicillin,  often in combination with an aminoglycoside (gentamicin).¹⁷ This finding is concerning and indicates that resistance in clinical human isolates may emerge. Fortunately, gentamicin resistance is still very uncommon or nonexistent in L.monocytogenes. It has only  been documented in one clinical case of meningitis in Greece and in a single isolate from market food in England.¹⁶

Despite all these findings, and unlike other pathogenic bacteria, L.monocytogenes, at least in clinical strains, remains largely resistant to antibiotics.^{14 16} Listeria monocytogenes has a lot of variability between some strains which do not all cause listeriosis (or at least not invasive listeriosis).¹⁶

Mechanisms of antibiotic resistance

Two main mechanisms of antibiotic resistance have been described in L.monocytogenes. The first one is the horizontal gene transfer or acquisition of resistance genes from a donor cell in a process called conjugation. The other one are the efflux pumps, a protein complex which expels toxic substances out of the cell. This is a natural resistance mechanism but it can also give rise to acquired resistance when mutations occur in the genes that encode or regulate efflux pumps, changing the bacterial susceptibility to antibiotics.

Antibiotic resistance mediated by conjugation 

Bacterial conjugation is the transfer of genetic materials between two bacterial cells by direct cell-to-cell contact. The transfer is done by movable genetic elements like plasmids and transposons that go from the donor cell to the receptor through a pilus. It is the main mechanism to acquire resistance to antibiotics used by L.monocytogenes and the main donors are Enterococci and Streptococci.¹⁵ Some studies showed that they were responsible for the emergence of antibiotic resistance in L.monocytogenes.¹⁵ Apparently, this bacterial conjugation between Enterococcus or Streptococcus and L.monocytogenes most likely takes place in the gastrointestinal tract of humans.¹⁵ 

Also, it has been found that L.monocytogenes could acquire resistance genes from Staphylococcus aureus (common bacteria which causes problems due to resistant strains) and other members of the phylum firmicutes in food processing environments.^{14 15}  

The emergence of tetracycline resistance in L.monocytogenes is mainly due to the conjugative plasmids and transposons originating from Enterococcus or Streptococcus.¹⁵ 

The conjugative transfer of plasmids and transposons has also carried other antibiotic resistance (i.e. chloramphenicol) to L.monocytogenes from Enterococcus, Streptococcus, and other Listeria species.^{14 15}

Antibiotic resistance mediated by efflux pumps 

Efflux pumps are protein structures located in the cell membrane that remove toxic substances out of the cell. They are involved in the protection from heavy metals and disinfectants but also have a main role in bacterial antibiotic resistance.¹⁸

Mutations in the genes that encode for efflux pumps can change their functionality, increasing the bacteria resistance to antibiotics, although they can make it more sensitive to heavy metals and other toxic substances like ethidium bromide.¹⁵

L.monocytogenes has three efflux pumps, which operate to extrude antibiotics, heavy metals and ethidium bromide. Efflux pumps give L.monocytogenes its natural resistance to fluoroquinolones.¹⁵

Sublethal exposure to antibiotics can induce overexpression of genes that code or regulate the efflux pumps and as a result, increase antibiotic resistance. That has been observed in one study in China with food isolates resistant to ciprofloxacin.¹⁹

Also, sublethal exposure to disinfectants can activate some genes and increase resistance to antibiotics. That has been observed in a study where several strains of L.monocytogenes were gradually exposed to quaternary ammonium compound and developed more resistance to fluoroquinolones ²⁰

Other mechanism of resistance

Since β-lactam antibiotics like penicillin inhibit proteins in the cell membrane called penicillin binding proteins (PBPs), mutations in these proteins could grant L.monocytogenes resistance to penicillins.¹⁶

Summary

Antibiotic resistance is an emerging world problem, even clinical relevant strains of Listeria monocytogenes remain susceptible to the main antibiotics, many resistant isolates have been found in several studies in food, environmental, animal and human samples. Even though many of these isolates do not cause the invasive form of listeriosis in most people, the increasing of antibiotic resistance in L.monocytogenes is a concern in terms of public health due to the common exposure of all humans to the bacteria and the susceptibility of some vulnerable people to a systemic infection. An increase of surveillance is necessary to detect emerging resistant isolates. Furthermore, controlling the  overuse and misuse of antibiotics and the research of new drugs and treatments is mandatory to prevent a public health crisis due to antibiotic resistance.