Universidade de Aveiro Ano 2012/2013

Departamento de Biologia

Sara Maria Cunha Oliveira Silva

RESPOSTA AO STRESS DE Listeria monocytogenes

Universidade de Aveiro Ano 2012/2013

Departamento de Biologia

Sara Maria Cunha Oliveira Silva

STRESS RESPONSE OF Listeria monocytogenes

Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Biologia Molecular e Celular, realizada sob a orientação científica da Professora Doutora Paula Cristina Maia Teixeira, Professora Auxiliar da Escola Superior de Biotecnologia da Universidade Católica Portuguesa e co-orientação do Professor Doutor António Carlos Matias Correia, Professor Catedrático do Departamento de Biologia da Universidade de Aveiro e Doutora Joana Gabriela Laranjeira Silva, investigadora da Escola Superior de Biotecnologia da Universidade Católica Portuguesa.

DECLARAÇÃO Declaro que este relatório é integralmente da minha autoria, estando devidamente referenciadas as fontes e obras consultadas, bem como identificadas de modo claro as citações dessas obras. Não contém, por isso, qualquer tipo de plágio quer de textos publicados, qualquer que seja o meio dessa publicação, incluindo meios eletrónicos, quer de trabalhos académicos.

Dedico este trabalho à minha família e amigos.

o júri

presidente Prof. Doutora Maria Adelaide de Pinho Almeida professora auxiliar do Departamento de Biologia da Universidade de Aveiro

Doutor Artur Jorge da Costa Peixoto Alves investigador auxiliar, Centro de Estudos do Ambiente e do Mar, Universidade de Aveiro

Prof. Doutora Paula Cristina Maia Teixeira professora auxiliar da Universidade Católica Portuguesa, Escola Superior de Biotecnologia

agradecimentos

Gostaria de deixar aqui o meu agradecimento à Escola Superior de Biotecnologia – Universidade Católica Portuguesa, pela oportunidade e condições proporcionadas para a realização deste Projeto de Dissertação.

À minha orientadora, Prof. Doutora Paula Teixeira, por me ter aceite como sua aluna de mestrado, pela sua paciência e constante disponibilidade, pelo conhecimento que me transmitiu e acima de tudo pela sábia contribuição para a minha formação profissional. Ao Prof. Doutor António Correia, meu co-orientador por me ter aceite igualmente como sua aluna de mestrado, pela sua disponibilidade, pelos conselhos prestados e valiosas contribuições. À Prof. Doutora Joana Silva, minha co-orientadora, pelo disponibilidade, acompanhamento e amizade ao longo deste período. A todos os colegas e amigos que fiz durante este projeto no Laboratório das Lácticas e do Pescado do Centro de Biotecnologia e Química Fina (CBQF), bem como nos Serviços de Microbiologia do Centro de Inovação e Apoio Empresarial (CINATE) pelo fantástico ambiente de trabalho, ajuda e paciência. À Prof. Doutora Teresa Brandão pela sua incansável disponibilidade para me ajudar e por tornar a análise estatística deste trabalho mais fácil. Ao Prof. Paul Gibbs por simpaticamente ter corrigido o inglês desta dissertação e pela partilha do seu conhecimento e sabedoria. À minha família e aos meus amigos, pelo incentivo, motivação e apoio dados durante este período.

Por fim, a todos aqueles que direta ou indiretamente contribuíram para que os objectivos deste trabalho pudessem ser alcançados.

palavras-chave

Listeria monocytogenes, stresse ácido, stresse alcalino, tracto gastro-intestinal, resistência a antibióticos.

resumo

Trinta e cinco isolados de Listeria monocytogenes provenientes de alimentos (n=20) e pacientes humanos com listeriose (n=15) e com diferentes perfis de resistência a antibióticos foram caracterizados e comparados com base na: (i) sua capacidade de sobrevivência à passagem pelo trato gastrointestinal simulado, (ii) sua capacidade de sobrivência a condições extremas de pH, (iii) potencial relação entre a resistência a antibióticos e a resistência às condições de stresse investigadas. A resposta às várias condições de stresse demonstrou ser estirpe- e stresse-dependente e não foi observada nenhuma relação entre isolados alimentares e clínicos (p > 0.05). Os resultados mostraram que L. monocytogenes sobrevive em condições ácidas e alcalinas extremas e não sobrevive quando submetida à passagem pelo trato gastrointestinal simulado, ou seja, à atividade dos sais biliares após ação conjunta do ácido clorídrico e pepsina. Não foi observada qualquer correlação entre a resistência a antibióticos e a resposta às condições de stresse aplicadas para os isolados estudados.

keywords

Listeria monocytogenes, acid stress, alkali stress, gastro-intestinal tract, antibiotic resistance.

abstract

Thirty-five Listeria monocytogenes isolates previously collected from food (n=20) and human patients suffering from listeriosis (n=15), with different antibiotic resistance profiles were characterized and compared based on: (i) their ability to survive through sequential conditions that parallel the digestive tract; (ii) their capacity to survive extreme pH values; (iii) the potential relationship, between antibiotic resistance and the resistance of L. monocytogenes isolates to the stress conditions investigated. The response was shown to be strain- and stress-dependent and no relation between food and clinical isolates was observed (p > 0.05). The results showed that L. monocytogenes is able to survive under extreme acid and alkaline conditions and did not survive when submitted to simulated sequential gastro-intestinal transit, i.e. the activity of bile salts after combined action of hydrochloric acid and pepsin. No correlation was observed between antibiotic resistance and response to the stress conditions applied to the isolates investigated.

i

Table of contents

List of abbreviations iii

List of figures v

List of tables vi

1. Listeria monocytogenes, a foodborne pathogen 1 1.1. History 1 1.2. Characteristics of the organism 1 1.3. Listeriosis 3 1.4. Background and occurrence 4 1.5. Stress resistance 6 1.6. Acid Stress 7

1.6.1. Induction of proteins 8 1.6.2. pH homeostasis 9 1.6.3. Glutamate decarboxylase system 9 1.6.4. Sigma factor (σB) 10 1.6.5. Two-component regulatory system 10

1.7. Alkali Stress 11 1.8. Antimicrobial resistance and stress resistance 12 1.9. Aim of the thesis 13

2. Material and Methods 15 2.1. Origin of isolates 15 2.2. Growth and storage conditions 16 2.3. Inoculum 17 2.4. Simulation of gastrointestinal conditions 17 2.5. Acid and alkaline treatments 18 2.6. Enumeration 18 2.7. Statistical analysis 19

3. Results and discussion 20 3.1. Effect of simulated digestion conditions 20 3.2. Effect of the pH in the survival of Listeria monocytogenes 26

ii

3.2.1. Acidic stress 26 3.2.2. Alkaline stress 30

3.3. Antimicrobial resistance and stress resistance 34

4. Conclusions 35

5. Proposals for future work 36

6. References 37

iii

List of abbreviations

AITR Alkali-Tolerance Response

ANOVA One-way analysis of variance

AR Antibiotic Resistant

AS Antibiotic Sensitive

ASP Alkali-Shock Proteins

ATP Adenosine triphosphate

ATR Acid Tolerance Response

BPW Buffered Peptone Water

CFU Colony Forming Units

DNA Deoxyribonucleic Acid

ECDC European Centre for Disease Prevention and Control

EFSA European Food Safety Authority

EU European Union

GABA Glutamate-γ-aminobutyrate

GAD Glutamate decarboxylase

GI Gastrointestinal

GSI Gastric and small intestinal

GSP General Stress Proteins

iv

HCl Hydrochloric acid

HPP High Pressure Processing

MA Massachusetts

MIC Minimum inhibitory concentration

NaHCO3 Sodium Bicarbonate

NaOH Sodium Hydroxide

PCR Polymerase Chain Reaction

PEF Pulsed Electric Field

RTE Ready-to-eat

TSAYE Tryptone Soy Agar supplemented with 0.6% Yeast Extract

TSBYE Tryptone Soy Broth supplemented with 0.6% Yeast Extract

UK United Kingdom

USA United States of America

UV Ultraviolet

VTEC Verocytotoxigenic Escherichia coli

WT Wild type

v

List of figures

Page Figure Title

4 1 Successive steps of human listeriosis (in Lecuit et al., 2007)

25 2 Survival (%) of food (A) and clinical (B) isolates of Listeria

monocytogenes resistant (C) or susceptible (D) to antibiotics during

stomach conditions simulation. Data are the mean of three independent

experiments; error bars represent the standard error of the mean.

29 3 Survival (%) of food (A) and clinical (B) isolates of Listeria

monocytogenes resistant (C) or susceptible (D) to antibiotics after

treatment with 10 mM HCl. Data are the mean of three independent

experiments; error bars represent the standard error of the mean.

33 4 Survival (%) of food (A) and clinical (B) isolates of Listeria

monocytogenes resistant (C) or susceptible (D) to antibiotics after

treatment with 100 mM NaOH. Data are the mean of three independent

experiments; error bars represent the standard error of the mean.

vi

List of tables

Page Table Title

15 1 Listeria monocytogenes isolates selected for the study and characterization

according to the serogroup and resistance to erythromycin, ciprofloxacin

and nitrofurantoin

18 2 Processing conditions used in each step of simulated digestion

23 3 Survival of thirty-five Listeria monocytogenes isolates when exposed to

simulated gastrointestinal conditions

28 4 Survival of thirty-five Listeria monocytogenes isolates when exposed to

acidic treatment with 10 mM HCl

32 5 Survival of thirty-five Listeria monocytogenes isolates when exposed to

alkaline treatment with 100 mM NaOH

1

1. Listeria monocytogenes, a foodborne pathogen

1.1. History Murray et al. first described Listeria monocytogenes in 1926. These researchers

isolated L. monocytogenes as the etiological agent of sudden death of young rabbits in the

animal-breeding establishment of the Department of Pathology of Cambridge, England

(Murray et al., 1926). In 1927, Pirie (Pirie, 1927), during investigations of unusual deaths

verified in gerbils in South Africa, isolated an identical bacterium.

The first documented case of human listeriosis was reported in 1929 in Denmark

and involved a soldier at the end of World War I (Nyfeldt, 1929; Allerberger, 2002). After

an increase in the number of reports on Listeria isolations in the late 70’s and early 80’s,

the first human listeriosis outbreak directly linked to the consumption of Listeria-

contaminated food was reported in 1983 (Schlech et al., 1983). Since then, several

epidemic outbreaks of listeriosis in humans occurred in North America and Europe

implicating L. monocytogenes as a causative agent (Farber and Peterkin, 1991) and

recognizing it as an important foodborne infection (Vazquez-Boland et al. 2001).

1.2. Characteristics of the organism Listeria spp. are Gram-positive; non spore-forming; non-capsulated, facultative

anaerobic; catalase positive; oxidase negative bacteria, with low G+C content (36 to 42%)

closely related to members of the genus Bacillus (Sallen et al., 1996), Clostridium,

Enterococcus, Streptococcus and Staphylococcus (Vazquez-Boland et al. 2001;

Swaminathan and Gerner-Smidt, 2007). At the time of writing, this genus is composed of

ten species: L. monocytogenes, L. ivanovii, L. welshimeri, L. innocua, L. seeligeri, L. grayi

(Vazquez-Boland et al. 2001), L. marthii (Graves et al., 2010), L. rocourtii (Leclercq et al.,

2010), L. fleischmannii (Bertsch et al., 2012) and L. weihenstephanensis (Halter et al.,

2012). Although species of the genus Listeria are environmental bacteria (Leclercq et al.,

2010), it also includes mammalian pathogenic species (i.e., L. monocytogenes, a human

and animal pathogen and L. ivanovii, an animal pathogen) (Orsi et al., 2008).

Listeria monocytogenes exhibits a wide range of temperature for growth, -1.5 to 45

2

ºC, with optimal growth between 30 ºC and 37 ºC (Gray and Killinger, 1966; Bell and

Kyriakides, 2005). It also grows in a wide range of pH values, i.e. 4.1 to 9.0 (Giotis et al.,

2007) and at high salt concentrations, e.g. 10% NaCl solutions (Duché et al., 2002).

Strains of Listeria monocytogenes species are divided into serotypes on the basis of

group-specific surface proteins, such as somatic (O) and flagellar (H) antigens

(Jeyaletchumi et al., 2010). Through examination of these particular proteins in slide

agglutination, at least 13 serotypes have been recognized (i.e. 1/2a, 1/2b, 1/2c, 3a, 3b, 3c,

4a, 4ab, 4b, 4c, 4d, 4e and 7) (Jeyaletchumi et al., 2010).

Molecular subtyping studies have established that L. monocytogenes can be divided

into four major evolutionary lineages, including lineages I and II, which are common, and

lineages III A/C and IIIB, which are rare (Roberts et al., 2006; Orsi et al., 2008). Isolates

from all four lineages have been associated with human listeriosis (Orsi et al., 2008).

Lineage I encompasses serotypes 1/2b, 3b, 3c, and 4b strains, whereas lineage II includes

serotypes 1/2a, 1/2c, and 3a (Nightingale et al., 2005; Liu et al., 2006). Most human

listeriosis cases and outbreaks have been associated with lineage I isolates, in particular

those of serotype 4b. On the other hand, lineage II isolates seem to be overrepresented

among isolates from ready-to-eat (RTE) and other food products, environmental sources

and animal listeriosis cases; and under-represented among human clinical cases

(Nightingale et al., 2005; Orsi et al., 2008; Ward et al., 2008).

Within lineage III, three different lineages have been identified (lineage IIIA, IIIB

and IIIC) (Liu et al., 2006) and represent serotypes 4a, 4b and 4c (Roberts et al., 2009).

These strains are mainly associated with isolates from animals with clinical listeriosis

(Roberts et al., 2009).

Serological and genetic typing schemes are clinically relevant since they

demonstrate that serotypes 1/2a, 1/2c, 1/2b, and 4b (lineages I and II) are involved with

around 98% of the documented human listeriosis cases and that serotypes 4a and 4c

(lineage III) are rarely associated with outbreaks of the infection (Liu et al., 2006).

3

1.3. Listeriosis

On one hand, L. monocytogenes is well-adjusted as a saprophyte for peaceful

survival in soil, water and decaying vegetation (Schuchat et al., 1991), but on the other

hand, it is a facultative intracellular pathogen responsible for causing listeriosis (Gray et

al., 2006), a severe foodborne, opportunistic infection, both in humans and animals (Ripio

et al., 2007).

Although L. monocytogenes is considered as a significant human and animal

pathogen responsible for listeriosis, L. innocua, L. ivanovii, L. welshimeri and L. seeligeri

have also been associated with occasional human infections (Rocourt et al., 1986; Andre

and Genicot, 1987; Cummins et al., 1994; Perrin et al., 2003; Guillet et al., 2010). Though

some cases of cutaneous listeriosis have been reported in veterinarians and farmers, who

have had direct contact with infected animals (Cain et al., 1986; Schuchat et al.,

1991; McLauchlin et al., 1994; Swaminathan and Gerner-Smidt, 2007), the major source

of human infections is due to ingestion of contaminated food (Farber and Peterkin, 1991;

Vázquez-Boland et al., 2001). Human invasive listeriosis can occur in healthy individuals,

but manifests primarily in pregnant women and neonates, the elderly (55 to 60 years and

older) and immunocompromised or debilitated individuals with underlying diseases

(Vázquez-Boland et al., 2001). The virulence of the infecting organism, the susceptibility

of the host and the size of the inoculum can influence the severity of the disease (Schuchat

et al., 1991).

The different steps of human listeriosis are shown schematically in Figure 1. After

the ingestion of Listeria-contaminated food, the bacteria are able to survive the stomach’s

acid conditions and reach the gastrointestinal tract. There, they have the capacity to cross

the intestinal barrier and are thought to disseminate from the mesenteric lymph nodes to

the spleen and liver (Lecuit et al., 2007). If not controlled properly by the immune system,

L. monocytogenes infection may reach the brain or placenta, resulting in infections of the

central nervous system mostly in immunocompromised patients and intrauterine or

cervical infections in pregnant women, which may lead to spontaneous abortion (2nd/3rd

trimester), stillbirth and neonatal infection with high mortality (Vázquez-Boland et al.,

2001).

4

Figure 1. Successive steps of human listeriosis (in Lecuit et al., 2007)

In healthy individuals, the ingestion of highly contaminated food products may lead

to a self-limited gastroenteritis (Lecuit et al., 2007). In groups at risk for invasive

listeriosis, general signs of infection often occur after an incubation period that can vary

between 10 and 70 days, making it difficult to identify the contaminated source in sporadic

cases (Lecuit et al., 2007).

1.4. Background and occurrence Listeria monocytogenes has been isolated from a wide range of raw foods and RTE

meats, poultry, dairy products, seafood and vegetables and from processed food that

becomes contaminated after processing such as soft cheeses (Lou and Yousef, 2007). From 2010 until 2012 a survey was carried out on L. monocytogenes with the aim

to estimate the prevalence of the pathogen in certain RTE foods at retail in the European

Union (EU) (European Food Safety Authority, 2013). 13,088 food samples including fish,

meat products and soft or semi-soft cheeses were collected and examined for the presence

of L. monocytogenes (European Food Safety Authority, 2013). The prevalence across the

entire European Union in fish samples was of 10.4% at the time of sampling, and of 10.3%

at the end of shelf-life. For meat and cheese samples, at the end of shelf-life, the

prevalence was of 2.07% and 0.47% respectively (European Food Safety Authority, 2013).

The data provided by this survey, gathered in all EU, will be useful in assessing the

exposure of EU consumers to L. monocytogenes via the specific three RTE food categories

5

tested that are thought to pose a particular risk for L. monocytogenes (European Food

Safety Authority, 2013).

In 2006, listeriosis was reported in 23 EU Member States as well as in Bulgaria (in

EU since 2007) and Norway and was the fifth most common zoonotic infection in Europe

after Campylobacter, Salmonella, Yersinia and verocytotoxigenic Escherichia coli (VTEC)

infections (Denny and McLauchlin, 2008). Listeriosis occurs infrequently (0.3 cases per

year per 100,000 of the population in the whole EU) (Denny and McLauchlin, 2008). On

one hand, from the 1,583 cases reported from the Member States in 2006, 64% were

accounted for in Germany, France and United Kingdom (Denny and McLauchlin, 2008).

On the other hand, Denmark, Finland and Luxembourg reported the highest incidence rates

of t 0.9 cases per 100,000 population (Denny and McLauchlin, 2008).

Listeriosis is not a reportable disease in Portugal nor has an active surveillance

system been established, thus available data are scarce (Almeida et al., 2006).

Nevertheless, some studies have been helping to understand the disease in Portugal

(Almeida et al., 2006; Almeida et al., 2009; Almeida et al., 2010). Almeida et al. (2006)

performed a retrospective study on listeriosis in Portugal and gathered all the data available

from 1994 until 2003. During that period, at least 35 cases of listeriosis were registered,

with a death rate of 37.5% for the 16 cases for which full case histories were available

(Almeida et al., 2006). According to the data collected in the study, the year with most

cases was 2003 with an estimated incidence of at least 1.4 cases per million inhabitants

(Almeida et al., 2006). These results must be assessed carefully since they are only an

initial estimate: not all the country's healthcare units were contacted, clinical symptoms

were sometimes not evident, thus making diagnosis difficult, and due to the fact that in

Portugal the notification of listeriosis is not legally mandatory (Almeida et al., 2006).

Despite the fact that there are no official reports of listeriosis cases in Portugal, this study

showed that L. monocytogenes is a threat to public health in a similar dimension to what

happens in other countries (Almeida et al., 2006). The only official data are from 2004,

recorded in the report of the European Food Safety Authority (EFSA)/European Centre for

Disease Prevention and Control (ECDC), reporting 38 human cases that year (EFSA,

2007). This authority has not reported data before or after 2004 for Portugal (EFSA, 2007;

EFSA, 2011; EFSA 2013). Between 2004 and 2007, Almeida et al. (2009) gathered cases

of listeriosis diagnosed in Portugal: 67 cases were identified with a death rate above 40%

6

in 24 cases of which full cases histories were available (Almeida et al., 2009). The number

of cases has increased over the years with an annual rate of 2.3 cases per million

inhabitants, roughly double the value reported in 2003 (Almeida et al., 2009; Almeida et

al., 2010).

For the presence of L. monocytogenes in foods, Mena et al. (2004) investigated its

presence on products sold in Portugal (collected from Portuguese producers and retailers).

From the 1035 samples, 7.0% were positive for L. monocytogenes (Mena et al., 2004). The

occurrence of L. monocytogenes in raw meat, raw chicken, raw milk, raw fish, flour and

frozen vegetables reflects the ubiquity of this organism (Mena et al., 2004). These products

showed the highest incidence of the pathogen with 17.7%, 60%, 16.7%, 12%, 18.5% and

12.9% of positive samples respectively (Mena et al., 2004). Although values are high, the

presence of L. monocytogenes in these products is not as problematic as its presence in

RTE products as these raw foods are typically cooked or pasteurized before consumption

(Mena et al., 2004). In RTE products, an occurrence of 25% in ham, 8.3% in dried fruits,

4.1% in pastry and 4% in fresh cheese was recorded; foods that are part of the eating habits

of Portuguese people in general (Mena et al., 2004).

1.5. Stress resistance Bacteria are adapted for optimal functioning in their normal physiological

environments (Beales, 2004). Although changing environments inflict stress on an

organism (Beales, 2004), bacteria have developed adaptive networks to face the challenges

and survive (Abee and Wouters, 1999).

Yousef and Courtney (2003) defined microbial stress as the imposition of

detrimental nutritional conditions, toxic chemicals and suboptimal physical conditions that

not necessarily cause physical damage but alter organism behaviour (Wesche et al., 2009).

Biological stressors could include competitive microorganisms (e.g., lactic acid bacteria),

microbial metabolites and antagonism (Yousef and Courtney, 2003). Hence, traditional or

novel food preservation techniques such as use of brine, high pressure processing (HPP),

pulsed electric field (PEF), ionizing radiation and ultraviolet (UV) irradiation can be

considered as microbial stressors (Lado and Yousef, 2002). These “hurdles” can cause

different degrees of stress in different bacteria that will influence the physiology, function

7

and activity of the microorganisms (Donnelly, 2002).

Once microorganisms sense stress, the cells respond in various ways in order to

survive: promote changes in membrane fluidity (e.g. cold shock), alter cell protein

structure or disrupt ribosomes (e.g. heat) or harm nucleic acids (e.g. γ radiation) (Yousef

and Courtney, 2003). At a molecular level, stress response includes transcription leading to

the synthesis of regulatory proteins that cope with the imposed stress (Yousef and

Courtney, 2003). Depending on the magnitude of the stress involved, stress can be

distinguished as either “sub-lethal” or “lethal or severe” (Donnelly, 2002). Sub-lethal stress

implies damage to structures within cells, the expression of which involves some loss of

cell function that may be transient or permanent (Wesche et al., 2009). It can result in

microbial “injury” and can be manifested as either retarded growth or complete prevention

of growth (Donnelly, 2002). When microorganisms are exposed to sub-lethal stress, it is

generally thought that this exposure can induce a transient adaptation (adaptive response)

accompanied by a temporary physiological change that often results in increased tolerance

to subsequent applications of the same type of stress or at times to a different stress (Hill et

al., 2002; Wesche et al., 2009). However, lethal or severe stress causes irreversible damage

to the microbial cells (Lou and Yousef, 1997) killing some, but not necessarily all,

bacterial cells (Wesche et al., 2009). When only a fraction of the population experiences

lethality, additional gene responses and adaptive mutations may actually improve survival

of the overall population (Wesche et al., 2009).

1.6. Acid Stress A common method of food preservation is its acidification and it is used worldwide

(Gahan et al., 1996). It is achieved either via fermentation or by addition of specific acid

food preservatives such as acetic, lactic and propionic acids (Hazan et al., 2004). The weak

acids in non-dissociated form permeate the cell membrane and, once inside the cytoplasm,

the acid dissociates (Phan-Thanh et al., 2000; Hazan et al., 2004). In this way, the acid

cannot diffuse out, decreasing the intracellular pH to dramatic values, which disrupts the

metabolic activities of the cell (Phan-Thanh et al., 2000; Hazan et al., 2004). Strong acids

completely dissociate into H+ and anions (Phan-Thanh et al., 2000). Because cell

membranes have a very low permeability to H+, protons enter and exit cells by interacting

8

with systems that control H+ transport, such as F0F1-ATPase, Na+/H+ antiporters, electron

transport systems (respiratory chains) (Phan-Thanh et al., 2000).

The acid tolerance response (ATR) is an induced protective response in

microorganisms against acid stress (Foster and Hall, 1990). The microbial response to acid

stress includes changes in membrane composition; increase in proton efflux; increase in

amino acid catabolism and induction of DNA repair enzymes (Yousef and Courtney,

2003). ATR is dependent on growth phase (exponential and stationary phase cells) and

differs among different bacterial species (Jordan et al., 1999; Yousef and Courtney, 2003).

The variations in intracellular or extracellular pH can be a signal for induction of acid

shock or adaptation proteins (Yousef and Courtney, 2003). Membrane bound proteins may

sense external or periplasmic pH (Yousef and Courtney, 2003). Internal pH may affect

gene expression or modulate a regulatory element involved in gene expression (Yousef and

Courtney, 2003).

Listeria monocytogenes is a neutrophile that is not able to grow at a pH below 4.5

(Walecka and Bania, 2012). During its life cycle, the pathogen faces a low-pH

environment at a number of stages, including in acidic foods, during gastric passage,

following exposure to fatty acids in the intestine and in the phagosome of the macrophages

(Cotter and Hill, 2003). In order to survive, Listeria possesses some stress adaptation

mechanisms (Cotter and Hill, 2003) such as,

x Induction of proteins;

x pH homeostasis;

x Glutamate decarboxylase system;

x Sigma factor (σB);

x Two-component regulatory systems.

1.6.1. Induction of proteins

In order to study the expression of proteins, Phan-Thanh and Mahouin (1999)

examined cells exposed to lethal acid pH (acid stress) and non-lethal acid pH (acid

adaptation). Although they observed that more proteins were induced in cells exposed to

lethal pH, the majority of induced proteins were common to both pH conditions used

(Phan-Thanh and Mahouin, 1999). Along with protein GroEL, which is also induced

9

during low temperatures (cross-protection), various transcriptional regulators and ATP

synthase were induced (Phan-Thanh and Mahouin, 1999). These results suggest that acid

adaptation also provides cross-protection against other stress factors (Phan-Thanh and

Mahouin, 1999; Gandhi and Chikindas, 2006). This phenomenon has great implications for

the food industry (Gandhi and Chikindas, 2006).

1.6.2. pH homeostasis

pH homeostasis allows microorganisms to maintain their intracytoplasmic pH via

proton transport across the cell membrane (Gandhi and Chikindas, 2006).

Listeria monocytogenes is a facultative anaerobic bacterium that can use the active

transport of H+ coupled with electron transport in respiratory chains (aerobic organisms) or

carry out H+ transport via H+-ATPase molecules using energy from ATP hydrolysis

(Shabala et al., 2002). Cotter et al. (2000) decided to study a multi-subunit enzyme called

F0F1-ATPase, highly conserved that had been extensively studied in other bacteria, namely

Escherichia coli. This enzyme serves as a channel for proton translocation across the cell

membrane by using ATP (Cotter et al., 2000). F0 rotation is caused by proton gradient and

followed by rotation of portion F1 that leads to ATP synthesis. Cotter et al. (2000)

observed a 3 log reduction under acid stress in the presence of proton inhibitor (DCCD-

N,N’-dicyclohexylcarbodiimide) concluding that F0F1-ATPase has a role in acid adaptation

of Listeria (Cotter et al., 2000).

1.6.3. Glutamate decarboxylase system

Glutamate decarboxylase (GAD) system is another mechanism that L.

monocytogenes also uses to survive acid stress (Gandhi and Chikindas, 2006). This system

is composed of three genes gadA, gadB and gadC (Gandhi and Chikindas, 2006). The first

two genes encode for two glutamate decarboxylases and gadC codes for a glutamate-γ-

aminobutyrate (GABA) antiporter (Gandhi and Chikindas, 2006). It is thought that

glutamate is taken up by the cell via a specific transporter followed by its decarboxylation,

which happens in the cytoplasm (Gandhi and Chikindas, 2006). In the process, γ-

10

aminobutyrate is produced resulting in the use of an intracellular proton (Gandhi and

Chikindas, 2006). Afterwards, an antiporter located in the cell membrane exports γ-

aminobutyrate from the cell (Gandhi and Chikindas, 2006). Due to proton loss from the

cell, the pH in the cytoplasm increases (Gandhi and Chikindas, 2006). Cotter et al. (2001)

studied the role of the GAD system in the acid resistance of L. monocytogenes during

gastric passage and found that the addition of glutamate increased the survival of the wild

strain in gastric fluid (Cotter et al., 2001). Deletion of the genes gadA, gadB and gadC

resulted in an increased sensitivity to low pH (Cotter et al., 2001). These results

demonstrated the importance of a functional GAD system in acid resistance in Listeria

(Cotter et al., 2001).

1.6.4. Sigma factor (σB)

Wiedmann et al. (1998) studied the role of general stress transcription factor (σB)

on the acid resistance of L. monocytogenes. The acid resistance of a sigB mutant, in

stationary phase, was lower than in the wild type (WT) (Wiedmann et al., 1998). This

study suggested that the rate of survival of L. monocytogenes is dependent on the

expression of σB-dependent proteins upon exposure to an acidic environment (Wiedmann

et al., 1998). Kazmierczak et al. (2003) observed that the alternative σB factor regulates the

expression of gadB gene involved in acid stress survival and regulates virulence gene

expression in L. monocytogenes.

1.6.5. Two-component regulatory system

Cotter et al. (1999) identified the role of a two-component regulatory system

consisting of lisR and lisK, which encode the response regulator and histidine kinase,

respectively in L. monocytogenes. This study by Cotter et al. (1999) showed that these

two-component signal transduction systems can sense alterations in the environment such

as low pH by a membrane-associated histidine kinase and the response regulator enables

the cell to respond by altering gene expression. Thus, the signal LisRK transduction system

is involved in response to acidic stress and regulates the virulence gene expression in L.

11

monocytogenes (Cotter et al., 1999).

Because acidic pH of foods helps to prevent the growth of foodborne pathogens,

the ATR observed in L. monocytogenes is noteworthy, since the exposure of the pathogen

to mild acidic conditions could confer resistance to more severe acidic conditions (Gandhi

and Chikindas, 2006).

1.7. Alkali Stress The resistance of L. monocytogenes to moderate and severe alkaline pH conditions

is of particular concern especially when alkali cleaners and sanitizers are used in cleaning

and decontamination within the food industry (Sharma et al., 2003; Giotis et al., 2007;

Giotis et al., 2008a). Such exposure can induce alkaline adaptation and it may account for

this bacterium’s persistence in food-processing environments (Giotis et al., 2008b). These

adaptations enable the pathogen to resist high pH conditions related to human defence

mechanisms, e.g. alkali conditions in phagolysosomes and pancreatic secretions in the

small intestine (Sharma et al., 2003; Giotis et al., 2007; Giotis et al., 2008a). Strongly

alkaline cleaners, like sodium hydroxide (NaOH), are used, for example, to remove heavy

soils (proteins and fats) in meat processing plants (Sharma et al., 2003).

The ability of Listeria to induce an alkali-tolerance response (AlTR) could be a

significant factor in the persistence and clinical impact of this pathogen in alkaline food-

processing systems and pathogen’s virulence within human gastrointestinal tract (Giotis et

al., 2008a).

Little is known about the factors or mechanisms by which AlTR initiates (Giotis et

al., 2008a). In fact, knowledge about the mechanisms used by Gram-positive bacteria to

adapt and grow at alkali pHs comes mainly from studies of alkaliphilic strains of Bacillus

species (Giotis et al., 2008b). The alkali stress promotes a specific response in these

organisms resulting in a transient enhanced expression of a subset of genes that increase

metabolic acid production, changes in cell surface properties and an increase of expression

and activity of monovalent- cation/proton antiporters (Padan et al., 2005). These response

mechanisms allow the maintenance of neutral cytoplasmic pH and therefore enable the

growth of Bacillus in alkali conditions (Padan et al., 2005). On the other hand, other

bacteria, such as Enterococcus faecalis, use other response mechanisms different from the

12

maintenance of neutral cytoplasmic pH (Mugikura et al., 1990; Flahaut et al., 1997). Their

adaptation to alkali environments is mainly accomplished due to de novo protein synthesis

by induction of proteins/enzymes capable of remaining active at high pH values (Giotis et

al., 2008b). These proteins provide either a specific protective function to alkali pH

(Alkali-Shock Proteins, ASP) (Giotis et al., 2008b) or provide a rather non-specific

protective function (General Stress Proteins, GSP), regardless of the type of stress (i.e. heat

shock proteins and proteins involved in the SOS response) (Giotis et al., 2008b).

Giotis et al. (2008b), in order to enlighten the response of L. monocytogenes

towards alkali stress, attempted to identify the contribution of protein synthesis in the

induction of AlTR. They concluded that alkali pH provides L. monocytogenes with non-

specific multiple-stress resistance and AlTR functions to minimize excess alkalization and

energy expenditures while mobilizing available carbon sources (Giotis et al., 2008b).

1.8. Antimicrobial resistance and stress resistance

Antibiotics were, originally, developed to treat human infectious diseases (Barbosa

and Levy, 2000). However, soon after, several were applied into animal husbandry where

they have proven to be highly effective in treating bacterial infections of significant animal

health consequence in possibly every food animal species, including poultry and

aquaculture (Doyle et al., 2013). Broad use and misuse of antibiotics created a strong

selective pressure, which consistently has resulted in the survival and spread of resistant

bacteria (Barbosa and Levy, 2000), which constitutes a major public health threat (Zhang

et al., 2013). Nevertheless, the occurrence of resistance among targeted pathogens in food

animals is not what raises most public-health and food safety concerns (Doyle et al., 2013).

Instead, it is the unintentional consequences of the effects of the antibiotics on the bacteria

that often reside in the gastrointestinal tracts of the food animals (Doyle et al., 2013).

Other implication of antibiotic resistant pathogens is that the acquisition of

antibiotic resistance may influence the behaviour of such organisms during typical food

processing conditions (Duffy et al., 2006). There are reports in the literature that suggest

that bacteria resistant to antibiotics may display different resistance to stresses, such as

acid or heat (McGee, 2003; Walsh et al., 2005). McGee (2003) suggested a possible link

between the acquisition of antibiotic resistance and survival of Escherichia coli O157:H7

at a low pH. This study is however not in accordance with other published study by Duffy

13

et al. (2006) that showed that there were no differences in acid resistance between

antibiotic-resistant and –susceptible E. coli strains. Other reports in the literature also

propose a possible link between the acquisition of antibiotic resistance and pathogen

thermal resistance (Walsh et al., 2001; Walsh et al., 2005). Walsh et al. (2001) reported

that the presence or absence of antibiotic resistance genes did not modulate the heat

resistance of the L. monocytogenes strains studied. Later, in 2005, Walsh et al. concluded

that no significant differences were observed in the heat resistance of sensitive strains of

Salmonella Enteritidis and Salmonella Typhimurium, and their laboratory-acquired

antibiotic-resistant mutant strains. However, a potential link between multiantibiotic

resistance and heat resistance in S. Typhimurium DT104 was suggested (Walsh et al.,

2005).

Considering the continuing increase, albeit slow, of the antimicrobial resistance of

L. monocytogenes particularly of strains isolated from food and production environments

(Barbosa et al., 2013; Conter et al., 2009), further investigation on the stress responses of

such strains, in particular for multiple antibiotic resistant strains, is justified (Walsh et al.,

2001).

1.9. Aim of the thesis

Since L. monocytogenes was recognized as a foodborne pathogen in the 1980s,

extensive research on the pathogen’s cell biology, virulence mechanisms and epidemiology

has been done (Walecka and Bania, 2012). Before causing infection, L. monocytogenes has

to overcome numerous stresses encountered both in the environment as well as in the host

(Walecka and Bania, 2012). Thus, understanding microbial stress resistance and response

to sub-lethal stresses may be of great value for food safety (Walecka and Bania, 2012). It

is possible that knowledge about the role of the factors affecting stress responses could

influence the design of modern food preservation methods (Walecka and Bania, 2012). In

this study, 35 L. monocytogenes isolates from different origins (clinical and food) were

characterized with the following objectives:

x To assess the survival of L. monocytogenes isolates based on their capacity to

survive through conditions that are typically encountered in the gastrointestinal

tract;

14

x To evaluate the survival of L. monocytogenes isolates when submitted to different

stress conditions (acidic and alkaline);

x To investigate the potential relationship, if any, between antibiotic resistance and

the resistance of L. monocytogenes isolates to the stress conditions investigated.

15

2. Material and Methods

2.1. Origin of isolates The thirty-five isolates of L. monocytogenes used during this study are listed in

Table 1. All isolates belong to L. monocytogenes culture collection of Escola Superior de

Biotecnologia-Universidade Católica Portuguesa and were selected based on their origin

(clinical and food) and resistance profile to erythromycin, ciprofloxacin and nitrofurantoin.

Some isolates had previously been characterized by multiplex-PCR for determination of

their serotype accordingly to Doumith et al. (2004). Listeria monocytogenes_2542 isolate

was selected as it was isolated in the course of the Portuguese outbreak of listeriosis that

occurred in Região de Lisboa e Vale do Tejo, in 2010.

Table 1. Listeria monocytogenes isolates selected for the study: characterization according

to the serogroup and resistance to erythromycin, ciprofloxacin and nitrofurantoin

Resistance tob

L. monocytogenes

isolatea Source Serogroup Erythromycinc Ciprofloxacind Nitrofurantoine

462 Food Frozen bread 4b R R R

654 Food RTE 4b S S S

949 Food RTE 1/2b S R S

971 Food RTE 1/2b S R S

1043 Food RTE 1/2b S S R

1162 Food Cheese 4b S S R

1216 Food Raw chicken 1/2a S S S

1305 Food Fresh

sausage 1/2c S S S

1535 Food Seafood 1/2b S S R

1728 Food Cheese 4b S R R

1382/1 Food Seafood 1/2b S S R

1486/1 Food Cheese 1/2b S S R

16

1604/2 Food Cheese 4b S S S

1852/3 Food Alheira 1/2b R R R

1940/1 Food Fermented

sausage 4b S R S

800/2 Food Bread 1/2b S S R

841/2 Food Garlic bread 4b R S R

863/1 Food Garlic bread 1/2c S S S

925/1 Food Cheese 1/2b S S S

969/3 Food Garlic bread 1/2b S S R

1062 Clinical Blood 1/2b S S R

1543 Clinical Blood 4b S S S

1547 Clinical Blood 1/2b S S S

2065 Clinical Blood 1/2b S S S

2086 Clinical Blood 1/2a S S S

2103 Clinical Blood 1/2a S R S

2104 Clinical Blood 4b S S R

2264 Clinical Blood 4b S R S

2265 Clinical Blood 4b S R S

2388 Clinical Peritoneal

fluid 1/2a S R S

2542 Clinical Placenta 4b S S S

2571 Clinical CSF 4b S R S

2658 Clinical Blood 1/2b S R R

3390 Clinical CSF 4b S R R

3391 Clinical Blood 1/2a S R R aCodes of L. monocytogenes isolates of the Listeria culture collection of Escola Superior de

Biotecnologia-Universidade Católica Portuguesa. bGrowth (R, resistant) or no growth (S,

susceptible) on Mueller-Hinton agar with 3% (v/v) lysed horse blood. cMinimum inhibitory

concentration (MIC, μg/mL) ≥ 8; dMIC (μg/mL) ≥ 4; eMIC (μg/mL) ≥ 128

2.2. Growth and storage conditions Stock cultures were grown on Tryptic Soy Agar (Pronadisa, Madrid, Spain)

17

supplemented with 0.6% (w/v) of Yeast Extract (Lab M, Lancashire, United Kingdom)

(TSAYE) at 37ºC for 24h and preserved at -20ºC in Tryptic Soy Broth supplemented with

0.6% (w/v) of Yeast Extract (TSBYE) containing 30% (v/v) of glycerol (Sigma,

Steinheim, Germany).

2.3. Inoculum The preparation of the inoculum was done according to Ramalheira et al. (2010).

One pure colony of each isolate was transferred from TSAYE incubated at 37 ºC for 24 h

to 10 mL of TSBYE and incubated in the same conditions. This culture was then

subsequently diluted 1:100 in TSBYE and incubated at 37 ºC for 24h to yield cells in the

stationary phase of growth. Cells were harvested by centrifugation (7000 g, 10 min, 4 ºC;

Rotina 35R, Hettich, Germany), re-suspended in 10 mL of sterile Ringer’s solution (Lab

M, Lancashire, United Kingdom) and mixed to obtain an inoculum of approximately 107

CFU/mL.

2.4. Simulation of gastrointestinal conditions The simulated gastrointestinal pathway was performed according to Madureira et

al. (2011) in buffered peptone water (BPW; Lab M) and is described in Table 2 – including

the compounds and concentrations used, the period of time and the intensities of stirring in

all steps (stirring was used to somehow simulate the peristaltic movements).

0.5 mL aliquots of inoculum (prepared as described in section 2.3) were added to

glass flasks with 49.5 mL of BPW adjusted to pH 2.5 with Hydrochloric Acid (1 M HCl,

Pronalab, Lisbon, Portugal) and 25 mg/mL of pepsin (Sigma), prepared in 0.1 N HCl. The

glass flasks were kept at 37 ºC and samples were taken just before inoculation, and after 15

min, 45 min, 70 min and 90 min (esophagus-stomach step). Following exposure to

stomach conditions, the duodenum step simulation used 12 g/L bile salts (Pronadisa)

diluted in 0.1 M Sodium Bicarbonate (NaHCO3). This solution was added at the beginning

of this step at a rate of 0.25 mL/mL and a sample was taken at time 110 min (intestinal

phase). Finally, the ileum step was brought about by an increase of pH to 6.5 using 0.1 M

18

NaHCO3 and a sample was taken at the end of this step.

All enzyme solutions were prepared just before use and filter-sterilized using a 0.22

μm - membrane filter (Milipore, Billerica MA, USA) prior to use. After sterilization, all

solutions were maintained in an ice bath during the entire period of the simulation prior to

gradual addition (when appropriate). For each bacterial isolate, all assays were performed

in triplicate. For each experiment an aliquot of 0.5 mL of inoculum was placed into glass

flasks with 49.5 mL of BPW at pH 7.0 and used as control.

Table 2. Processing conditions used in each step of simulated digestion

Compartment Added compounds Stirring (rpm) Final pH Time (min)

Oesophagus-stomach

Pepsin (25 mg/mL)

130 2.5 90

Duodenum Bile salts (12

mg/mL) 45 5.0 20

Ileum - 45 6.5 90 – none

2.5. Acid and alkaline treatments The acid and alkaline treatments were performed according to Liu et al. (2005). 0.5

mL aliquots of inoculum (prepared as described in section 2.3) were added to glass flasks

with 49.5 mL of BPW acidified to a final concentration of 10 mM HCl or alkalized to a

final concentration of 100 mM NaOH. 1 mL of each treated sample was taken just before

inoculation, and aftern 30 and 60 min. For each bacterial isolate, all assays were performed

in triplicate. For each experiment an aliquot of 0.5 mL of inoculum was placed into glass

flasks with 49.5 mL of BPW at pH 7.0 and used as control.

2.6. Enumeration

Viable cell counts were determined by preparing serial decimal dilutions with

sterile Ringer solution that were subsequently plated on TSAYE using the Miles and Misra

method (1938) and spread-plate method. After incubation at 37ºC for 48h the colonies

were counted and the CFU/mL calculated.

19

2.7. Statistical analysis Microbial counts were transformed to logarithmic reduction using the equation: log

(N/N0), where N is the microbial cell density at a particular sampling time and N0 is the

initial cell density. One-way analysis of variance (ANOVA) was carried out to assess

statistically any significant differences among all experimental data on the survival of L.

monocytogenes in simulated gastrointestinal (GI) tract and acid and alkaline treatments.

All tests were performed to a 5% significance level, using Kaleidagraph software (version

4.4, Synergy Software, Reading, USA).

20

3. Results and discussion

3.1. Effect of simulated digestion conditions

The decreases in viability of the thirty-five isolates of L. monocytogenes tested in

the simulated gastrointestinal model is plotted in figure 2; a minimum threshold of 3 log

CFU/mL was take into account, owing to the intrinsic sensitivity limitation of Miles and

Misra’s plating method of enumeration. The survival in each step of digestion is tabulated

in Table 3.

Examination of Figure 2, for stomach conditions, indicates that the majority of the

isolates were susceptible to the treatment showing a survival of 15% or lower. The isolate,

L. monocytogenes_1043, from a RTE food, showed the highest resistance to the decrease

in pH in the presence of pepsin, with approximately 53% survivors (Figure 2). Upon

addition of bile salts (110 min, Table 1), all the isolates that resisted until that moment

(stomach conditions), were reduced to values below the detection limit of the enumeration

technique. This step was dramatically detrimental: the viable cell counts dropped more

than 4 log cycles from their initial value, even for isolate L. monocytogenes_2542, clinical

4b, associated with the deadly Portuguese outbreak of listeriosis in 2010. No correlation

was found between origin (clinical or food), antibiotic resistance profile and survival of L.

monocytogenes (p>0.05) in simulated gastric conditions. During exposure to oesophagus-stomach simulated conditions, five of the thirty-

five L. monocytogenes isolates (L. monocytogenes_1604/2, L. monocytogenes_1852/3, L.

monocytogenes_863/1, L. monocytogenes_1543, L. monocytogenes_2065) were incapable

to survive to the simulated gastric juice, which corresponds to pH 2.5. Upon exposure to

duodenum conditions (i.e. bile salts), viable cell numbers of all isolates decreased abruptly.

Apparently, L. monocytogenes cells were in a fragile state when they reached this stage of

digestion with several injuries caused by acid before exposure to bile salts. It is important

to recall that bile salts are natural detergents that facilitate the digestion and absorption of

hydrophobic components of the diet (Sánchez et al., 2007). However, their antimicrobial

nature makes them strongly inhibitory for bacteria (their detergent property dissolves

bacterial membranes), thus, restricting their survival throughout the GI tract (Sánchez et

al., 2007). In contrast to this deleterious action of acid and pepsin conjugated with bile

21

salts, Melo et al. (2013) and Olier et al. (2004) reported that all L. monocytogenes strains

were able to survive the exposure to the gastric fluid as well to the intestinal fluid.

However, it is important to point out that Melo et al. (2013) exposed their L.

monocytogenes strains to a cheese-simulated medium previously to the GI stress. In fact, a

simulated or a real food matrix confer resistance to GI passage due to the protection

conferred by food components as other studies have demonstrated (Barmpalia-Davis et al.,

2009; Peterson et al., 2007). Nevertheless, our results are in accordance with those

reported from Barbosa et al. (2012), Jiang et al. (2010), King et al. (2003) and Ramalheira

et al. (2010) (studies also carried out in broth) who proved that an increase in bile salts

susceptibility was observed when cells had first been exposed to the low pH environment

of the gastric system.

It is important to note that in the current study two of the three L. monocytogenes

isolates with the highest percentages of resistance to GI passage were recovered from RTE

foods. Note also that RTE foods do not undergo any treatment to ensure their safety before

consumption and therefore the risk of foodborne disease must be considered if L.

monocytogenes cells are present in the food.

Comparison of survival patterns to different stress conditions of clinical versus

food isolates is well documented in the literature (Avery and Buncic, 1997; Barmpalia-

Davis et al., 2008; Dykes and Moorhead, 2000; Lianou et al., 2006; Olier et al., 2004;

Ramalheira et al., 2010; Viallete et al., 2003; Werbrouck et al., 2008). Avery and Buncic

(1997) investigated the effect on pathogenicity and growth characteristics of thirty isolates

of L. monocytogenes (fifteen clinical and fifteen from meat) after long-term storage at 4 ºC.

Clinical isolates demonstrated higher resistance to the effects of unfavourable storage

conditions, concerning pathogenicity and lag-phase duration at 37 ºC (Avery and Buncic,

1997). Barmpalia-Davis et al. (2008a) compared the ability to survive in a dynamic GI

model of thirteen strains of L. monocytogenes (outbreak origin/food associated and

human). Survival of GI tract conditions was demonstrated to be growth phase and strain-

dependent (Barmpalia-Davis et al. (2008a). The highest acid susceptibility was observed

for two strains of clinical origin (Barmpalia-Davis et al. (2008a). In the current study, the

survival of the acidic phase also proved to be strain-dependent as had been demonstrated in

other studies (Faleiro et al., 2003;; Francis and O’Beirne, 2005;; Lianou et al., 2006). Dykes

and Moorhead (2000) tested thirty L. monocytogenes strains of different origins (clinical

22

and meat) for their capacity to resist acid stress. Although they reported an increased acid

resistance in all clinical L. monocytogenes strains, no significant differences were apparent

based on source (Dykes and Moorhead, 2000). Lianou et al. (2006) characterized and

compared twenty-five L. monocytogenes strains (clinical and food) representing several

serotypes, based on growth rates and heat and acid resistance. These authors reported that

though no clear origin-related trends were observed under the test conditions (heating at 55

ºC or pH 3), outbreak-related isolates of serotype 4b demonstrated the highest acid

resistance (Lianou et al., 2006). In the current study and probably due to the unequal

distribution of each serotype investigated, no relationship between the serotype and

simulated GI passage was observed (p> 0.05). Olier et al. (2004) reported that there was no

relationship between the origin (human, food or food processing environment) of fifty

strains of L. monocytogenes and capacity to degrade bile salts. Ramalheira et al. (2010)

showed that clinical isolates were more resistant than food isolates to GI passage

performed in BPW. Vialette et al. (2003) investigated the growth rates of eight L.

monocytogenes strains (four clinical associated to fish products and four from other

seafoods) with respect to stress conditions that can occur during food processing. Clinical

strains displayed better adaptation to pH and osmotic stress than seafood strains in

stationary phase Vialette et al. (2003). Werbrouck et al. (2008) did not find any clear

correlation between the origin (clinical and meat) and survival of L. monocytogenes

serotype 4b in synthetic gastric fluid.

Although static gastric and small intestinal (GSI) models do not reproduce the

dynamic processes occurring during human digestion such as gastric emptying or

continuous changes in pH and secretion flow rates, the generated information would be a

valuable contribution for further studies in the context of microbiological risk assessments.

23

Table 3. Survival of thirty-five Listeria monocytogenes isolates when exposed to simulated gastrointestinal conditions

log CFU/mL

Oesophagus-stomach

L. monocytogenes isolatea

Source

0 min

15 min

45 min

70 min

90 min

462 Food 7.0±0.02 6.5±0.01 6.1±0.03 4.6±0.06 4.6±0.06 654 Food 7.1±0.02 6.4±0.01 6.0±0.03 5.9±0.01 5.9±0.01 949 Food 7.2±0.03 7.0±0.01 6.9±0.02 6.6±0.16 6.6±0.06 971 Food 7.2±0.00 7.1±0.03 7.0±0.03 6.3±0.32 6.0±0.05

1043 Food 7.2±0.01 7.0±0.03 7.0±0.03 6.9±0.01 6.9±0.01 1162 Food 7.0±0.01 7.0±0.01 6.9±0.00 6.3±0.17 6.2±0.13 1216 Food 7.7±0.24 7.1±0.01 6.4±0.03 6.3±0.01 6.1±0.04 1305 Food 7.0±0.01 6.8±0.17 6.4±0.25 6.2±0.42 5.7±0.44 1535 Food 7.3±0.02 7.2±0.02 7.0±0.03 7.0±0.00 6.3±0.02 1728 Food 7.4±0.08 6.3±0.06 6.0±0.03 6.0±0.03 6.0±0.00

1382/1 Food 7.0±0.01 5.9±0.01 5.4±0.13 4.4±0.31 4.2±0.11 1486/1 Food 7.4±0.05 7.2±0.04 6.8±0.00 6.6±0.03 6.5±0.10 1604/2 Food 7.0±0.03 5.4±0.08 4.3±0.09 <2.8±0.00 <2.8±0.00 1852/3 Food 7.0±0.00 6.1±0.08 <2.8±0.00 <2.8±0.00 <2.8±0.00 1940/1 Food 7.1±0.08 6.2±0.02 6.1±0.03 5.5±0.04 5.4±0.01 800/2 Food 7.0±0.00 5.9±0.28 5.5±0.25 5.3±0.02 5.2±0.08 841/2 Food 6.9±0.01 6.3±0.07 6.0±0.00 5.4±0.13 4.5±0.10 863/1 Food 7.4±0.04 6.1±0.09 4.4±0.02 <2.8±0.00 <2.8±0.00 925/1 Food 7.0±0.00 6.5±0.08 6.1±0.07 5.9±0.01 5.3±0.01 969/3 Food 7.0±0.00 6.1±0.05 6.0±0.03 5.9±0.01 5.3±0.32 1062 Clinical 7.0±0.03 7.0±0.03 5.5±0.41 4.4±0.25 4.0±0.01 1543 Clinical 7.2±0.03 7.1±0.02 5.4±0.25 <2.8±0.00 <2.8±0.00

24

1547 Clinical 7.0±0.01 6.5±0.07 6.1±0.01 5.3±0.31 4.4±0.36 2065 Clinical 6.9±0.01 6.2±0.00 5.3±0.14 4.0±0.00 <2.8±0.00 2086 Clinical 7.1±0.01 7.0±0.01 6.9±0.01 6.3±0.33 5.9±0.01 2103 Clinical 7.0±0.00 6.9±0.16 6.4±0.00 6.1±0.11 6.0±0.00 2104 Clinical 7.1±0.01 7.0±0.00 6.6±0.04 6.3±0.00 6.0±0.02 2264 Clinical 7.5±0.01 7.3±0.04 6.5±0.02 6.4±0.01 6.0±0.01 2265 Clinical 7.4±0.01 7.3±0.02 6.5±0.04 6.3±0.01 6.0±0.01 2388 Clinical 7.1±0.05 7.0±0.01 6.5±0.19 6.3±0.00 6.3±0.00 2542 Clinical 7.2±0.03 7.1±0.01 7.0±0.01 6.4±0.10 6.2±0.06 2571 Clinical 7.4±0.01 7.3±0.03 6.7±0.20 6.5±0.05 6.2±0.10 2658 Clinical 7.2±0.03 7.2±0.03 7.0±0.00 6.1±0.02 6.0±0.03 3390 Clinical 6.9±0.01 6.6±0.03 6.4±0.02 6.3±0.03 6.1±0.08 3391 Clinical 7.0±0.00 6.6±0.02 6.4±0.03 6.4±0.02 6.3±0.03

aCodes in L. monocytogenes collection of Escola Superior de Biotecnologia-Universidade Católica Portuguesa All the isolates were reduced to values

below the detection limit of the enumeration technique in duodenum and ileum steps.

25

Figure 2. Survival (%) of food (A) and clinical (B) isolates of Listeria monocytogenes resistant (C) or susceptible (D) to antibiotics during stomach

conditions simulation. Data are the mean of three independent experiments; error bars represent the standard error of the mean.

0%

20%

40%

60%

80%

100%

462

949

971

1043

1162

1305

1535

1728

1382

/1

1486

/1

1852

/3

1940

/1

800/

2

841/

2

969/

3

% S

urvi

val

Listeria monocytogenes

AC

0%

20%

40%

60%

80%

100%

1547

2065

2086

2103

2104

2264

2265

2388

2542

2571

3391

% S

urvi

val

Listeria monocytogenes

BC

0%

20%

40%

60%

80%

100%

654 1216 1604/2 863/1 925/1

% S

urvi

val

Listeria monocytogenes

AD

0%

20%

40%

60%

80%

100%

3390 2658 1062 1543

% S

urvi

val

Listeria monocytogenes

BD

26

3.2. Effect of the pH in the survival of Listeria monocytogenes Concerning exposure to HCl and NaOH, Figures 3 and 4 demonstrate that survival

in the conditions studied, was strain dependent.

The ability of L. monocytogenes strains to tolerate and survive under harsh

environmental conditions such as extreme pH values (<4.0 or >9.0) is well documented in

literature (Cotter and Hill, 2003; Dykes and Moorhead, 2000; Giotis et al., 2008b; Giotis et

al., 2008a, Lianou et al., 2006; Liu et al., 2005; Metselaar et al., 2013). External stresses

often result in inhibition of growth and once the stress factors are removed, L.

monocytogenes is capable of multiplying (Giotis et al., 2007; Liu et al., 2005). The

mechanisms underlying L. monocytogenes response to environmental stress conditions,

such as acid, alkali, oxidative and carbon-starvation stresses have become clearer,

following the identification of stress response genes (Gandhi and Chikindas, 2007). In

particular, an alternative sigma factor gene σB that plays a vital role in the general stress

response of L. monocytogenes to acid and alkali hurdles (Giotis et al., 2008a; Sue et al.,

2004).

3.2.1. Acidic stress

Regarding the 10 mM HCl challenge, after 1 h treatment, pH 1.5, all L.

monocytogenes isolates displayed reductions in their mean viable cell numbers (Table 4).

Of thirty-five L. monocytogenes isolates, only three, two food (L. monocytogenes_1852/3

and L. monocytogenes_800/2) and one clinical (L. monocytogenes_2065), all belonging to

serogrup 1/2b, were not detected after 1 h treatment (Table 4). In fact, viable cell numbers

decreased to values below the detection limit of the enumeration technique following

inoculation for the isolates L. monocytogenes_1852/3 and L. monocytogenes_2065, while

for the isolate L. monocytogenes_800/2 this was observed after 30 min of treatment. In

contrast, L. monocytogenes_462, isolated from frozen bread, 4b, showed, alongside garlic

bread isolate L. monocytogenes_863/1, 1/2c, and L. monocytogenes_1547, isolated from

blood, 1/2b, the highest resistance to acid treatment with 68% and approximately 30%

survivors respectively (Figure 4). Regarding this challenge, isolate L.

27

monocytogenes_2542, clinical 4b, associated with the Portuguese outbreak, showed a

survival of 16% (Figure 4). As in the previous challenge, no significant differences in the

resistance to both treatments were detected between food and clinical isolates or between

antibiotic sensitive and antibiotic resistant isolates (p > 0.05).

Although, a number of researchers have reported the inhibitory effects of low pH

on L. monocytogenes (Conner et al., 1990; Ita and Hutkins, 1991; Phan-Thanh et al., 2000;

Vasseur et al., 1999), the results of the current study demonstrated that L. monocytogenes

isolates possibly evolved adaptive networks that allow them to tolerate low pH as

previously reported in other studies (Barmpalia-Davis et al., 2008; Dykes and Moorhead,

2000; Francis and O’Beirne, 2005;; Liu et al., 2005). Indeed, of thirty-five L.

monocytogenes isolates, only two (L. monocytogenes_1852/3; L. monocytogenes_2065)

were not detected after 30 minutes exposure and one (L. monocytogenes_800/2) after one

hour. Nonetheless the pH tested in this treatment was very low, pH 1.5, Ita and Hutkins

(1991) observed that low intracellular pH was not the major factor in the inhibition of L.

monocytogenes at acid pH. In fact, cells treated with organic acids or HCl at pH values as

low as 3.5 were able to maintain their cytoplasmic pH at a value near 5 (Ita and Hutkins,

1991). Budde and Jakobsen (2000), Shabala et al. (2002) and Siegumfeldt et al. (1999)

also demonstrated that L. monocytogenes maintains its intracellular pH within a narrow

range of 7.6 to 8.0 at extracellular pH values of 5.0 to 8.0 and at pH 4.0 in the presence of

glucose. Confronted with acidic conditions, bacterial cells attempted to resist by

maintaining its intracellular homeostasis whose failure leads to loss of cell viability

(Chitarra et al., 2000). Listeria, a facultative bacterium, may use aerobic or anaerobic

processes to maintain its intracellular pH homeostasis (Phan-Thanh et al., 2000); indeed, in

aerobic bacteria, the active transport of H+ is coupled to the process of electron transport in

respiratory chains; in anaerobic bacteria, H+ transport is carried out through a specific H+

channel in the F0F1-ATPase molecule (proton pump) using energy from ATP hydrolysis

(Cotter et al., 2000).

28

Table 4. Survival of thirty-five Listeria monocytogenes isolates when exposed to acidic

treatment with 10 mM HCl

log cfu/mL

L. monocytogenes isolatea Source 0 min 30 min 60 min 462 Food 7.2±0.06 7.1±0.06 7.1±0.02 654 Food 6.7±0.43 5.8±0.36 5.0±0.01 949 Food 6.9±0.19 6.3±0.37 5.6±0.01 971 Food 6.4±0.39 5.6±0.24 5.2±0.02

1043 Food 6.1±0.40 5.2±0.15 4.9±0.02 1162 Food 6.1±0.26 5.4±0.05 5.2±0.02 1216 Food 7.0±0.05 6.6±0.01 5.9±0.02 1305 Food 6.9±0.16 6.4±0.03 6.2±0.01 1535 Food 6.7±0.20 5.6±0.02 5.4±0.01 1728 Food 6.9±0.36 6.3±0.34 5.4±0.01

1382/1 Food 6.7±0.52 4.7±0.21 4.3±0.01 1486/1 Food 6.9±0.02 6.4±0.11 5.6±0.02 1604/2 Food 7.1±0.03 5.9±0.02 5.3±0.02 1852/3 Food 6.3±0.13 <2.8±0.00 <2.8±0.00 1940/1 Food 7.0±0.25 5.3±0.01 5.2±0.03 800/2 Food 5.9±0.41 4.0±0.01 <2.8±0.00 841/2 Food 7.1±0.23 6.5±0.32 6.1±0.04 863/1 Food 5.9±0.27 5.4±0.09 5.2±0.07 925/1 Food 5.9±0.32 4.5±0.05 4.4±0.01 969/3 Food 5.7±0.23 4.3±0.04 4.1±0.03 1062 Clinical 6.8±0.14 4.7±0.10 4.4±0.01 1543 Clinical 6.8±0.15 6.1±0.09 5.5±0.02 1547 Clinical 6.1±0.28 5.6±0.33 5.5±0.33 2065 Clinical 7.2±0.09 <2.8±0.00 <2.8±0.00 2086 Clinical 6.9±0.12 5.6±0.15 5.3±0.01 2103 Clinical 7.1±0.02 6.5±0.14 5.9±0.00 2104 Clinical 6.9±0.13 6.3±0.01 6.1±0.01 2264 Clinical 6.9±0.13 6.4±0.07 6.2±0.05 2265 Clinical 6.8±0.18 5.8±0.38 5.2±0.33 2388 Clinical 6.7±0.24 5.8±0.33 5.2±0.33 2542 Clinical 7.0±0.03 6.2±0.15 6.2±0.02 2571 Clinical 6.8±0.13 5.8±0.26 5.3±0.02 2658 Clinical 6.9±0.04 6.3±0.16 6.2±0.02 3390 Clinical 7.0±0.02 6.4±0.12 6.1±0.09 3391 Clinical 7.3±0.19 6.6±0.04 6.2±0.18

aCodes in L. monocytogenes collection of Escola Superior de Biotecnologia-Universidade Católica

Portuguesa.

29

Figure 3. Survival (%) of food (A) and clinical (B) isolates of Listeria monocytogenes resistant (C) or susceptible (D) to antibiotics after the treatment with

10 mM HCl. Data are the mean of three independent experiments; error bars represent the standard error of the mean.

0%

20%

40%

60%

80%

100%

462

949

971

1043

1162

1305

1535

1728

1382

/1

1486

/1

1852

/3

1940

/1

800/

2

841/

2

969/

3

% S

urvi

val

Listeria monocytogenes

AC

0%

20%

40%

60%

80%

100%

1547

2065

2086

2103

2104

2264

2265

2388

2542

2571

3391

% S

urvi

val

Listeria monocytogenes

BC

0%

20%

40%

60%

80%

100%

654 1216 1604/2 863/1 925/1

% S

urvi

val

Listeria monocytogenes

AD

0%

20%

40%

60%

80%

100%

1062 1543 2658 3390

% S

urvi

val

Listeria monocytogenes

BD

30

3.2.2. Alkaline stress

With reference to 100 mM NaOH treatment, after 1 h at pH 12.0, L. monocytogenes

isolates displayed reductions in their mean viable cell numbers (Table 5). Although this

noteworthy capacity to tolerate this challenging condition, two isolates, L.

monocytogenes_863/1, garlic bread 1/2c, and L. monocytogenes_1547, blood 1/2b, failed

to recover after 30 min of such treatment (Table 5). On the other hand, the alheira isolate

L. monocytogenes_1852/3, 1/2b, and L. monocytogenes_2542, clinical 4b, associated with

the Portuguese outbreak, demonstrated the highest resistance to alkaline treatment with

approximately 38% and 31% survivors respectively (Figure 5). One hour of exposure to

pH 12.0 resulted in 0.8 log cycle reduction for isolate L. monocytogenes_2542 (Table 5).

No significant differences or clear trends in overall reduction were apparent based on

source (clinical or food) or antibiotic resistance profile (p > 0.05).

The use of alkaline cleaners and sanitizers is widespread in food processing

environments, thus L. monocytogenes may be exposed to alkaline conditions in a variety of

pre- and post- processing environments (Sharma et al., 2002). Alkaline solutions, such as

NaOH are generally used in detergents to remove heavy soils (proteins and fats), eliminate

carbonized sediment, oil or grease since they facilitate protein denaturation, fats

saponification and have a bactericidal activity (Sharma et al., 2002; Vasseur et al., 1999).

Our results have shown a great tolerance to high pH values since only two (L.

monocytogenes_863/1, L. monocytogenes_1547) of thirty-five L. monocytogenes isolates

were not detected after one hour of exposure to such conditions. Growth of L.

monocytogenes at pH > 9.5 is unusual (Gray and Killinger, 1966), but a number of recent

studies demonstrated that the expression of factor σB-dependent genes plays a fundamental

role in cellular survival of L. monocytogenes under adverse conditions, such as alkaline

environments (Becker et al., 2000; Ferreira et al., 2001; Giotis et al., 2008b; Giotis et al.,

2008a; Sharma et al., 2002; Wemekamp-Kamphuis et al., 2004; Wiedmann et al., 1998).

Cheroutre-Vialette et al. (1998) investigated the effects of water activity and pH shifts on

the growth of three L. monocytogenes strains using different osmotic solutes among them

NaOH. They concluded that cells were not affected by the presence of NaOH (Cheroutre-

Vialette et al., 1998). Vasseur et al. (1999) performed a similar study and evaluated the

responses of five L. monocytogenes strains to an alkaline treatment by the addition of

31

NaOH in order to achieve different pH values. They saw that increased alkalinity induced

an increase in lag phases for all strains (Vasseur et al., 1999). However, at pH 11 (highest

pH value tested) three of five strains were more tolerant to alkaline conditions (Vasseur et

al., 1999).

From a food safety point of view, an obvious implication from this study is that use

of acid or alkali in a food processing environment may not be sufficient to eliminate all

cells of L. monocytogenes.

The absence of a clear association of acid or alkaline resistance with origin of the

isolate (clinical or food) and antibiotic resistance profile could be due to the limited

statistical power of the analysis resulting from the small number of clinical and AS

isolates.

32

Table 5. Survival of thirty-five Listeria monocytogenes isolates when exposed to alkaline

treatment with 100 mM NaOH

log cfu/mL L. monocytogenes

isolate Source 0 min 30 min 60 min

462 Food 7.4±0.02 7.2±0.06 6.4±0.01 654 Food 7.1±0.05 6.5±0.28 5.9±0.01 949 Food 7.3±0.06 6.5±0.02 5.3±0.00 971 Food 7.3±0.19 5.5±0.23 4.5±0.00

1043 Food 7.3±0.08 6.9±0.36 6.1±0.04 1162 Food 7.1±0.24 6.4±0.31 5.0±0.03 1216 Food 7.1±0.03 6.4±0.02 5.2±0.00 1305 Food 7.0±0.23 6.5±0.04 5.3±0.00 1535 Food 7.0±0.22 6.5±0.65 5.9±0.61 1728 Food 7.5±0.03 7.0±0.02 6.3±0.00

1382/1 Food 7.2±0.38 6.7±0.33 5.5±0.00 1486/1 Food 6.9±0.19 6.2±0.02 5.5±0.00 1604/2 Food 7.3±0.03 6.7±0.29 6.1±0.04 1852/3 Food 6.9±0.15 5.6±0.16 5.4±0.00 1940/1 Food 7.0±0.25 6.6±0.15 6.4±0.00 800/2 Food 6.8±0.27 5.9±0.29 5.2±0.33 841/2 Food 7.2±0.08 6.6±0.20 4.9±0.00 863/1 Food 5.7±0.37 5.3±0.12 <2.8±0.00 925/1 Food 6.6±0.31 5.6±0.29 4.6±0.00 969/3 Food 6.6±0.14 6.0±0.34 5.4±0.32 1062 Clinical 6.8±0.24 5.7±0.19 5.4±0.00 1543 Clinical 7.1±0.08 6.1±0.24 5.4±0.35 1547 Clinical 6.3±0.21 5.0±0.05 <2.8±0.00 2065 Clinical 7.4±0.05 6.6±0.19 6.2±0.00 2086 Clinical 6.9±0.20 5.6±0.00 4.7±0.00 2103 Clinical 7.0±0.06 6.1±0.09 5.5±0.00 2104 Clinical 7.2±0.09 6.9±0.25 6.2±0.03 2264 Clinical 7.0±0.15 5.6±0.28 5.3±0.33 2265 Clinical 6.9±0.20 5.4±0.49 4.5±0.24 2388 Clinical 7.0±0.27 6.2±0.48 5.9±0.57 2542 Clinical 7.2±0.14 6.5±0.34 6.4±0.35 2571 Clinical 7.0±0.22 5.7±0.35 5.4±0.05 2658 Clinical 7.1±0.14 6.8±0.42 6.3±0.37 3390 Clinical 6.9±0.33 6.4±0.55 5.9±0.60 3391 Clinical 6.9±0.07 5.8±0.32 5.7±0.34

aCodes in L. monocytogenes collection of Escola Superior de Biotecnologia-Universidade Católica

Portuguesa

33

Figure 4. Survival (%) of food (A) and clinical (B) isolates of Listeria monocytogenes resistant (C) or susceptible (D) to antibiotics after the treatment with

100 mM NaOH. Data are the mean of three independent experiments; error bars represent the standard error of the mean.

0%

20%

40%

60%

80%

100%

462

949

971

1043

1162

1305

1535

1728

1382

/1

1486

/1

1852

/3

1940

/1

800/

2

841/

2

969/

3

% S

urvi

val

Listeria monocytogenes

AC

0%

20%

40%

60%

80%

100%

1547

2065

2086

2103

2104

2264

2265

2388

2542

2571

3391

% S

urvi

val

Listeria monocytogenes

BC

0%

20%

40%

60%

80%

100%

654 1216 1604/2 863/1 925/1

% S

urvi

val

Listeria monocytogenes

AD

0%

20%

40%

60%

80%

100%

1062 1543 2658 3390

% S

urvi

val

Listeria monocytogenes

BD

34

3.3. Antimicrobial resistance and stress resistance

The antibiotic-resistant profiles of the thirty-five L. monocytogenes isolates used

are shown in Table 1. In this study, the results obtained for antibiotic resistant (AR)

isolates did not differ from those obtained for antibiotic sensitive (AS) isolates under any

of the conditions examined (Figures 2-4), suggesting that antibiotic resistance mechanisms

did not confer any cross-resistance to gastrointestinal passage, acid or alkaline stresses.

35

4. Conclusions

Once L. monocytogenes is ingested by a mammalian host through contaminated

foods, its survival within that host depends upon the bacterium’s ability to survive the acid

environment of the stomach, the secretion of proteolytic enzymes and the inhibitory action

of bile salts in the small intestine. In this study the survival of different isolates of L.

monocytogenes through simulated GI conditions was evaluated. It was demonstrated that

L. monocytogenes was able to survive in extreme acid conditions. However, it did not

survive when submitted to sequential GI transit, i.e., presence of bile salts. Although GI

conditions do not simulate the physiological environment of digestion in the human GI

tract, they allow us to understand the microorganism’s fate in the digestive environment.

From a food safety point of view the results of this study renders many of the

conventional food manufacturing processes ineffective against L. monocytogenes cells, as a

certain number of this species will survive at very extreme pH conditions.

It was also concluded in the present study that antibiotic-resistant isolates of L.

monocytogenes are not more GI-, acid- or alkaline-tolerant than antibiotic-susceptible

isolates, but, at the same time, are not less GI-, acid- or alkaline-tolerant either. It is

important to point out that an unequal number of AR and AS isolates was used in this

study and this may have contributed to bias in the results. The continuing increase in the

prevalence of AR L. monocytogenes in foods justifies further investigation of the stress

responses of such strains, in particular for multiple antibiotic-resistant strains.

The response of thirty-five L. monocytogenes isolates was shown to be strain- and

stress-dependent and no relation between food and clinical isolates was observed.

36

5. Proposals for future work

The present work demonstrated that L. monocytogenes is able to survive under

extreme acid and alkaline conditions, but not through sequential GI tract, i.e. the activity of

bile salts after the combined action of acid and pepsin. Also, the presence or absence of

antibiotic resistance genes did not modulate the resistance response of the isolates

examined towards the stress conditions investigated. In order to investigate and understand

the mechanisms that underly these answers is important to comprehend the bacterial

strategies that effectively allow resistance development as well as overcoming some

limitations inherent to the technique used to simulate GI tract digestion. Therefore,

suggestions for future work include:

1. Improvement in the artificial digestive systems, extending their potential and

approach the in vivo human situation more closely. Always bearing in mind that it

is still impossible to mimic fully the overall digestive parameters in vivo in a single

in vitro model;

2. Evaluation of genotypic and phenotypic characteristics of L. monocytogenes

isolates, concerning:

x Resistance, adaptation and cross-adaptation to conventional food preservatives;

x Resistance to different stress conditions (e.g., salt, pH, etc.);

x Acquisition of antibiotic resistance genes by L. monocytogenes and possible

linkage to resistance to food processing stresses.

37

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