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Food Microbiology 42 (2014) 66e71

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Food Microbiology

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Short communication

Listeria ivanovii ATCC 19119 strain behaviour is modulated by iron andacid stress

Catia Longhi a,*, Maria Grazia Ammendolia b, Maria Pia Conte a, Lucilla Seganti a,Francesca Iosi b, Fabiana Superti b

aDepartment of Public Health and Infectious Diseases, Sapienza University, P.le A. Moro 5, 00185 Rome, ItalybDepartment of Technology and Health, National Health Institute, Via Regina Elena 299, 00185 Rome, Italy

a r t i c l e i n f o

Article history:Received 8 April 2013Received in revised form7 February 2014Accepted 18 February 2014Available online 28 February 2014

Keywords:Listeria ivanoviiIron and acid stressVirulence

* Corresponding author.E-mail address: catia.longhi@uniroma1.it (C. Longh

http://dx.doi.org/10.1016/j.fm.2014.02.0150740-0020/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

It has been suggested that the rarity of human listeriosis due to Listeria ivanovii reflects not only hosttropism factors but also the rare occurrence of this species in the environment, compared with Listeriamonocytogenes. In the present study we evaluate the effects on the reference strain L. ivanovii ATCC 19119behaviour of two combined stresses, low iron availability and acid environment, that bacteria canencounter in the passage from saprophytic life to the host. In these conditions, L. ivanovii evidenced adifferent behaviour compared to L. monocytogenes exposed to similar conditions. L. ivanovii was not ableto mount an acid tolerance response (ATR) even if, upon entry into the stationary phase in iron-loadedmedium, growth phase-dependent acid resistance (AR) was evidenced. Moreover, bacteria grown in ironexcess and acidic pH showed the higher invasion value in Caco-2 cells, even though it was not able toefficiently multiply. On the contrary, low iron and acidic conditions improved invasion ability in amnioticWISH cells.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Within the Gram-positive genus Listeria, Listeria monocytogenesand Listeria ivanovii have been recognized as pathogenic (Vazquez-Boland et al., 2001). Although these bacteria have virulence prop-erties in common, they differ in pathogenicity. L. monocytogenescauses severe infections in newborns, pregnant women, immuno-compromised individuals, and animals, whereas L. ivanovii pos-sesses restricted host specificity, since it infects mostly ruminants.Human cases of L. ivanovii infections are extremely rare but it hasbeen demonstrated that it could represent a potential risk ofinfection in immunocompromised hosts (Cummins et al., 1994;Snapir et al., 2006; Guillet et al., 2010).

Both L. monocytogenes and L. ivanovii are facultative intracellularparasites capable to cross the intestinal barrier and to proliferatewithin macrophages and a variety of normally nonphagocytic cells,such as epithelial and endothelial cells, and hepatocytes (Vazquez-Boland et al., 2001; Cossart and Toledo-Arana, 2008), and to inducecell-to-cell spread. Most of the virulence genes responsible for theintracellular life cycle are clustered within a 9 kb locus that is

i).

located in the same chromosomal position in both species(Dominguez-Bernal et al., 2006). Pathogenic Listeriae, similarly toother pathogens, in the passage from saprophytic life to the host arefaced with different stressful environments: food-related stressesand environmental conditions can modulate bacterial surfacesproperties and influence the survival of the microorganism duringgastrointestinal passage in the host or through foeto-placentaltransmission in pregnant women (Gahan and Hill, 1999; Bereksiet al., 2002; Chavant et al., 2002; Giotis et al., 2007; Barmpalia-Davis et al., 2009; Stopforth et al., 2005); on the other hand hostimposes stress conditions including the acidic pH of the stomach,the deleterious effects of volatile fatty acids produced in the gut, thelow pH inside the host cell vacuoles, and the decrease of ironavailability in host tissues.

Listeria spp. have evolved different strategies for survival at lowpHs, including the stationary phase dependent acid resistance (AR)and the logarithmic-phase-dependent acid tolerance response(ATR) (Gahan et al., 1996; Chorianopoulos et al., 2011). The adaptiveATR plays an important role in the survival of L. monocytogenes in avariety of foods and in the ability of this pathogen to cause illness;induction of ATR also protects L. monocytogenes against the effectsof different environmental stresses (Lou and Yousef, 1997;Chorianopoulos et al., 2011; Saklani-Jusforgues et al., 2000; Conteet al., 2000a, 2002; Koutsoumanis and Sofos, 2004).

C. Longhi et al. / Food Microbiology 42 (2014) 66e71 67

Iron is essential for the growth of most bacteria and iron-limiting conditions can be encountered in both the natural envi-ronment and during host infection (McLaughlin et al., 2011). Free-living pathogenic bacteria have evolved mechanisms to acquireiron from a variety of sources: both pathogenic Listeriae use a va-riety of siderophores, catechol siderophore-like compounds, andcatecholamines, existing in the environment or secreted in thehuman body, to acquire iron (Coulanges et al., 1998; Lungu et al.,2009). In L. monocytogenes, iron deficient environments havebeen shown to up-regulate the expression of the principal viru-lence regulator PrfA and significantly increase the production of thehaemolysin listeriolysin O promoting phagosomal escape, and theactin polymerisation protein ActA which plays a role in cell-to-cellspread (Conte et al., 1996, 2000b). It has been hypothesized that therequirement for iron has played a part in driving the evolution of anintracellular life-cycle for L. monocytogenes as the bacterium canutilize the iron-saturated protein ferritin stored in the cytosol ofhost cells (McLaughlin et al., 2011).

Up to date most of the studies concerning the relationship be-tween environmental stress sensing and pathogenicity have beenperformedwith L. monocytogenes. The aim of this research is to gainfurther information on the relationship between the ability ofL. ivanovii to sense and respond to acid or/and iron stress and itsinvasion and replication capabilities in epithelial cells.

2. Materials and methods

2.1. Bacterial strain and culture conditions

An L. ivanovii strain from American Type Culture Collection,Manassas, VA (ATCC 19119) of animal origin was utilized in thisstudy. This strain was biochemically controlled by the API Listeriakit (Bio Mérieux) according to the manufacturer’s instructions.Haemolysis on Muller Hinton agar (Oxoid) supplemented with 5%sheep blood was used as additional test. Bacteria were maintainedas stock cultures in 15% glycerol-brain heart infusion broth (BHI)(Oxoid) at �80 �C. When needed, aliquots of frozen bacteria werethawed and routinely grown in BHI at 37 �C, pH 7.2, andmaintainedfor few passages onto tryptone soy agar plates (TSA) (Oxoid), oronto selective Oxford agar plates (Oxoid). Iron limitation and ironexcess conditions were obtained by culturing bacteria in BHI asalready described (Conte et al., 1996).

2.2. Adaptation of L. ivanovii strain to acid and measurement of ATR

L. ivanovii strainwas preliminarily tested for tolerance to low pH(pH 3.5). L. ivanovii cells cultured in iron stress or iron excess wereanalyzed for influence of acid stress as follows: overnight culturesof bacteria in iron-deficient or iron-loaded BHI were diluted (1:30)into fresh medium and grown at 37 �C until the A600 of the culturereached approximately 0.15 (early log phase). Duplicate centrifugedsamples were resuspended in an equal volume of BHI, adjusted topH 5.1 with 1 M lactic acid (acid-adapted bacteria), or to pH 7.2(non-adapted bacteria), and incubated for 1 h and 2 h at 37 �C. Todetermine the ATR, cells were harvested by centrifugation and re-suspended in iron-deficient or iron-loaded BHI acidified to pH 3.5with 3 M of lactic acid. Survival was determined performing platecounts at intervals (0, 1, and 2 h) by serial dilutions of samples inphosphate-buffered saline (PBS, pH 7.2) and plating onto TSA agarplates.

AR response was determined by centrifuging overnight cultureof bacteria grown in iron-deficient or iron-loaded BHI for 15 min at1500 g. Then bacteria were resuspended in BHI acidified to pH 3.5with 3 M of lactic acid for 1 h at 37 �C. Bacteria survival wasdetermined as above described.

2.3. Haemolytic activity

Bacteria were grown overnight at 37 �C in iron-deficient or iron-loaded BHI. Duplicate samples were centrifuged, the pellets wereresuspended in an equal volume of BHI adjusted to pH 5.1 or 7.2,and incubated for 1 h at 37 �C. Samples of 1 ml of the cultures werethen centrifuged at 10,000 g for 10 min at 4 �C and the clear su-pernatants were supplemented with 10 mM dithiotreitol. Haemo-lytic activity assays were performed with sheep erythrocytes onmicrotiter plates as already described (Dominguez Rodriguez et al.,1986). The haemolytic activity titre was expressed in minimalhaemolysis units (MHU) defined as the reciprocal of the highestdilution at which haemolysis was detected.

2.4. Cell lines

Cells derived from a human colon carcinoma (Caco-2) or humanamniotic tissue (WISH) were cultured in Eagle’s minimum essentialmedium with Earle’s salts (EMEM, EuroClone), supplemented with10% (v/v) heat-inactivated foetal calf serum (FCS, JRH Biosciences),and 2 mM glutamine. All incubations were carried out in a 5% CO2

atmosphere at 37 �C. Cells were used 48 h after seeding.

2.5. Invasion and intracellular growth assays

The invasion assays were carried out by infecting semiconfluentcell monolayers grown in 24-well plates (Nalge Nunc International),with logarithmically grown bacteria, cultured in different culturalconditions, as abovedescribed, at anMOIof100bacteria/cell for 1h at37 �C. After this incubationperiod, the cells were extensivelywashedand 1 ml of fresh medium containing 50 mg/ml of gentamicin wasaddedtoeachwell andmaintained for1hat37 �C.The cellswere thenlysedwith ice-cold 0.1% Triton-X 100 and plated onTSA to determineviable intracellular bacteria. Invasion efficiencywas expressed as thepercent of the inoculated CFU that were internalized.

For intracellular growth assays, incubation of cells ingentamicin-containing medium was prolonged for an additionalperiod of 4 h at 37 �C followed by lysis and CFU counts. Intracellulargrowth was expressed as replication index (RI), corresponding tothe number of CFU at 4 h post-infection divided by the number ofCFU at time 0 (1 h post infection).

2.6. Transmission electron microscopy (TEM)

To visualise bacteriaecell interaction, semiconfluent cellmonolayers were infected with L. ivanovii (MOI 100). One hourpost-infection, mock- and bacteria-infected cells were harvested,washed in PBS, and fixed in 2.5% glutaraldehyde in 0.1 M cacodylatebuffer (pH 7.2) at room temperature for 1 h. Cells were then post-fixed in 1% osmium tetroxide in cacodylate buffer, dehydratedthrough graded series of ethanol solutions and, finally, embeddedin Agar 100 epoxy resin. Thin sections were stained with lead cit-rate and uranyl acetate and examined with a Philips 208S electronmicroscope at 80 kV.

2.7. Fluorescent imaging of F-actin

Fluorescent imaging of F-actin was performed according asalready described (Ammendolia et al., 2007). Cells, grown in 8-chamber culture slides (Lab-Tek, Nunc) for 48 h at 37 �C in 5% CO2

atmosphere, were incubated with logarithmically grown bacteriafor 1 h at 37 �C at anMOI of 100 bacteria per cell, as above described.At different time intervals after the addition of gentamicin-containing medium, cells were fixed by incubating with 3.7%formaldehyde in PBS for 5min at room temperature. Then cellswere

Table 1Invasion efficiency, intracellular multiplication in Caco-2 and WISH cells, and hae-molysis of L. ivanovii ATCC 19119 strain grown in iron loaded or iron depleted BHI atpH 7.2 or pH 5.1.

Growth conditions % of invasion RI MHU

Caco-2 WISH Caco-2 WISH

Iron-loadedBHI pH 7.2

7.0 � 1.2 8.0 � 1.0 14 � 2.2 7.0 � 0.9 512

Low iron contentBHI pH 7.2

4.8 � 0.8 15 � 3.2 9.0 � 1.2 5.0 � 0.5 128

Iron-loadedBHI pH 5.1

21.0 � 3.8 5.0 � 1.0 3.0 � 2.0 4.0 � 1.0 32

Low iron contentBHI pH 5.1

4.0 � 0.6 22 � 4.1 11.0 � 3.8 5.0 � 0.5 64

Viable intracellular bacteria were determined by plate counts. Invasion wasexpressed as the percentage of the initial inoculum of bacteria that were gentamicinresistant 1 h post-infection. Intracellular multiplication was expressed as replicationindex (RI) corresponding to the number of CFU at 4 h post infection divided by thenumber of CFU at time 0 (1 h post infection). Assays were performed in triplicate.The haemolytic titre was expressed in minimal haemolysis units (MHU), corre-sponding to the reciprocal of the highest dilution at which a complete haemolysiswas detected. Data reported are from a representative experiment of four inde-pendent experiments.

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washed with PBS and permeabilized by incubation with 100 ml ofPBS containing 0.1% Triton X-100 for 10 min at room temperature.After washings in PBS, cells were incubated in the dark for 20min atroom temperaturewith 100 ml of TRITC-phalloidin (Sigma ChemicalCo.) diluted into PBS. After extensive washings in PBS, glass slideswere mounted in glycerol and observed using a Leica microscopeDM5000B equippedwith a Photometrics LeicaDFC340FCamera andLeica Application suite 2.4.0.R1 software.

2.8. Statistical analysis

All tests were carried out in triplicate and the results wereaveraged. Data were expressed as the mean � S.D. The statisticalsignificance between different experimental conditions wasdetermined by the paired Student’s t-test (P values of <0.05 wereconsidered significant).

3. Results

3.1. Acid resistance response of L. ivanovii grown under differentiron availability

A first set of experiments was carried out to assess the survivaland growth of L. ivanovii ATCC 19119 strain in BHI in relation to pHand iron availability. As shown in Fig. 1, maximal growth rates ofL. ivanovii were achieved at pH 7.2, whereas the incubation ofbacteria in media acidified to pH 5.1 caused an inhibition of bac-terial replication independently to iron concentrations. L. ivanoviicells exponentially growing in iron-deficient or iron-loaded BHIwere rapidly killed when the pH was reduced to 3.5 even if wereexposed to a sublethal pH 5.1. These results indicate that this strain,in all cultural condition tested, was not able to mount an ATR.However, L. ivanovii was able to acquire, upon entry into the sta-tionary phase in iron-loaded medium, growth phase-dependentacid resistance (AR) (data not shown).

3.2. Haemolytic activity in acid-adapted and non-adaptedL. ivanovii grown under different iron availability

The ability of acid-adapted and non-adapted L. ivanovii cells,grown in iron-excess or iron-limiting conditions, to lyse sheep

Fig. 1. Survival curves and ATR of Listeria ivanovii (ATCC 19119) grown in iron-loaded orrepetitions. Bars represent standard errors.

erythrocytes was tested (Table 1). Results from quantitative hae-molytic assays showed that L. ivanovii was a strong producer ofhaemolysin, even than at a different extent in the different condi-tions, ranging the MHU titres from 32 to 512. The highest MHUvalue was observed for bacteria grown in iron loaded BHI pH 7.2.

3.3. Invasion of Caco-2 and WISH cells by acid-adapted and non-adapted L. ivanovii grown under different iron availability

The capability of acid-adapted and non adapted L. ivanovii cells,grown in iron-supplemented or iron-depleted media, to enter andto multiply in Caco-2 and WISH cells was assayed (Table 1). Whennon acid-adapted L. ivanovii was grown in iron-excess conditions,the percentage of internalized bacteria in Caco-2 was significantlyenhanced respect to iron-limited condition of growth (7.0 � 1.2instead of 4.8 � 0.8, respectively); higher percent of internalizationwas observed when acid-adapted L. ivanovii were grown in iron-supplemented media (21.0 � 3.8) even if bacteria, differently tothat previously observed for L. monocytogenes by Conte et al.

iron-depleted BHI at pH 7.2 or pH 5.1. Each value is the mean of three independent

C. Longhi et al. / Food Microbiology 42 (2014) 66e71 69

(2000a), was not able to efficiently replicate in this cell line (RI3.0 � 2.0).

Differently to that observed for Caco-2 cells, results obtained inWISH cells showed that, independently of acid pre-adaptation ofcultures, bacteria grown in iron-depleted media invaded amnioticcells at a significant higher efficiency as compared to bacteriagrown in iron-loaded media (15 � 3.2 instead of 8.0 � 1.0 at pH 7.2;22 � 4.1 instead of 5.0 � 1.0 at pH 5.1), while intracellular multi-plication of bacteria was almost similar in all the experimentalconditions tested.

3.4. Intracellular behaviour of L. ivanovii: electron and lightmicroscopy

As already reported for L. monocytogenes, L. ivanovii showed anenhanced percentage of internalized bacteria in Caco-2 cells ifgrown in iron-loaded and low pH conditions (Conte et al., 2000a,2002). Concerning WISH cells, it has been reported that L. ivanoviiis able to adhere, invade, lyse the phagosomal membrane, poly-merize host cell actin, and spread from cell to cell more efficientlythan L. monocytogenes (Ammendolia et al., 2007). In the presentstudy, the invasive behaviour of L. ivanovii, grown in differentconditions, in WISH cells, was explored by transmission electronmicroscopy. As already demonstrated in invasion assays, 1 h afterinfection the most pronounced invasion could be observed whenL. ivanovii was grown in iron stress at acidic pH (Fig. 2A) comparedto that observed for bacteria cultured in iron loadedmedium at acidpH (Fig. 2B): bacterial cells, in both conditions, appeared inside

Fig. 2. Electron micrographs of thin sections of WISH cells 1 h post-infection withL. ivanovii (ATCC 19119) cultured in iron loaded BHI pH 5.1 (A) or iron deprived BHI pH5.1 (B).

vacuoles in the cytoplasm. Fluorescence microscopy, performed 3 hpost infection, showed that the bacteria grown in iron loadedmedium at pH 5.1 were able to polymerize actin around them(Fig. 3A) and intracellular movement was documented by presenceof long actin tails. At the same time of infection, most of L. ivanoviicells, cultured in iron deprived medium at pH 5.1, were free in thecytoplasm (Fig. 3B) but few bacterial cells had already polymerizedactin around them.

4. Discussion

Pathogenic Listeriae can respond rapidly to changing environ-mental conditions, as demonstrated by their ability to transitionfrom a saprophyte to an orally transmitted intracellular pathogen.Although a number of studies addressing the stress hardeningphenomenon and its relationship with virulence ofL. monocytogenes have been reported (Koutsoumanis et al., 2003),there are still gaps in data concerning L. ivanovii, a pathogenicListeria with low virulence in humans but with potentially fataloutcome (Snapir et al., 2006). The ecology of L. ivanovii suggeststhat the rarity of human listeriosis due to this species reflects notonly host tropism but also its rare occurrence in the environment(Guillet et al., 2010).

In our study, the reference strain L. ivanovii ATCC 19119 exposedto iron and acid stresses, evidenced a different behaviour comparedto L. monocytogenes exposed to similar conditions. L. ivanovii,exponentially growth in both iron-deficient and iron-loadedmedia,was not able to exhibit a significant adaptive acid toleranceresponse following exposure to mild acid, which is instead capableof protecting L. monocytogenes cells from severe acid stress; be-sides, the growth of L. ivanovii strain in medium in relation to thepH and iron level showed that the incubation of bacteria in mediaacidified to pH 5.1 caused an inhibition of bacterial replication. Thegeneral restricted resistance at low pH probably could represent alimitation for bacterial survival in the environment and in partic-ular in acid foods; however, the results obtained showed thatL. ivanoviiwas able to acquire, upon entry into the stationary phasein iron loaded medium, growth phase-dependent acid resistance(AR) that probably provide it the possibility to survive to low pH forshort periods of time. Moreover, electron microscopic observationson bacteria grown at the stress conditions considered in thisresearch, evidenced that morphological changes did not occur inthe case of L. ivanovii structures respect to L. monocytogenes inwhich virulent filamentous forms have been observed (data notshown) (Rowan et al., 2000).

L. ivanovii cells grown in iron deficiency and neutral pH condi-tions showed a reduced invasiveness in Caco-2 cells but, differentlyto that previously reported for L. monocytogenes grown in thesimilar cultural conditions (Conte et al., 1996), an enhance ofintracellular multiplication rate was not observed. However,although L. ivanovii grown in iron excess and pre-exposed to acidicpH showed the higher invasion value in Caco-2 cells, successively itwas not able to efficiently multiply.

A different behaviour was also observable in the case of amniotichuman cells in which, as already demonstrated by Ammendoliaet al. (2007), L. ivanovii invades and multiplies more efficientlythan L. monocytogenes. In the present study we showed that bac-terial growth in low iron and acid pH conditions, improved theinvasion ability of WISH cells as demonstrated also by electron andfluorescence microscopy. Intra-amniotic infection appears withreduced iron concentration due to high levels of lactoferrin, an ironbinding protein with antimicrobial properties (Pacora et al., 2000).This context with low iron concentration could represent a helpfulfactor for bacterial infection even if L. ivanovii survival in WISH cellcytoplasm is limited respect to that of L. monocytogenes

Fig. 3. L. ivanovii (ATCC 19119) infected WISH cells 3 h post-invasion. Bacteria werecultured in iron deprived BHI pH 5.1 (A) or in iron loaded BHI pH 5.1 (B). Cells werestained with TRITC-phalloidin.

C. Longhi et al. / Food Microbiology 42 (2014) 66e7170

(Ammendolia et al., 2007). Moreover, the higher haemolytic ac-tivity observed in iron loaded and neutral pH condition can supportevidence of a different behaviour and role of haemolysin inL. ivanovii respect to L. monocytogenes, in which iron deficientconditions have been shown to up-regulate its expression and thelow pH is required for its activity in vitro (Conte et al., 1996). AsL. ivanovii ivanolysin O, differently to listeriolysin O, possess anoptimum pH of 6.5 (Jones et al., 1996), L. ivanovii may need thecooperative action of ILO and SmcL to reach cytoplasmiccompartment as also supposed by González-Zorn et al. (1999).

Notwithstanding is difficult to generalize because our study isperformed with only one L. ivanovii reference strain, all togetherour data further support the different virulence behaviour alreadyobserved by other authors between L. ivanovii and L. monocytogenes(Guillet et al., 2010).

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