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The Pathogenic Potential of Different Pulsed-Field Gel Electrophoresis Types of

Listeria monocytogenes Strains Isolated from Food in Northeast Bosnia and
Herzegovina

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VECTOR-BORNE AND ZOONOTIC DISEASES
Volume 11, Number 9, 2011
ª Mary Ann Liebert, Inc.
DOI: 10.1089/vbz.2010.0208

The Pathogenic Potential of Different Pulsed-Field Gel

Electrophoresis Types of Listeria monocytogenes Strains
Isolated from Food in Northeast Bosnia and Herzegovina

zić,1 Mirsada Hukić,2 Giovanna Franciosa,3 and Paolo Aureli 3

Snjezana Hod

Abstract

Listeria monocytogenes is often present in meat and meat products that are sold in the area of northeast Bosnia and
Herzegovina. The major objective of this study was to examine the virulence of L. monocytogenes strains isolated
from these types of food in that geographic area. Polymerase chain reaction was used to detect eight genes
responsible for virulence of this pathogen, namely, prfA, inlA, inlB, hly, plcA, plcB, actA, and mpl. All examined
isolates were confirmed to possess the eight virulence genes. Ten different pulsed-field gel electrophoresis
(PFGE) macrorestriction profiles were recognized among 19 L. monocytogenes strains after restriction with two
different endonucleases (ApaI and AscI). The pathogenicity of three different PFGE types of L. monocytogenes was
confirmed through in vivo tests, which were performed on female white mice (Pasteur strain), and it ranged from
3.55 · 108 LD50 to 1.58 · 1010 LD50. All of the three different PFGE types of L. monocytogenes were regarded as
moderately virulent in relation to the reference strain L. monocytogenes Scott A. This result might be one of the
reasons for the absence of reported listeriosis in northeast Bosnia and Herzegovina, despite the high degree of
food contamination with this pathogen.

Key Words: Listeria monocytogenes—PFGE types—virulence genes.

Introduction Ingestion of L. monocytogenes is very common considering

the omnipresent distribution of these bacteria and the high

L isteria monocytogenes is an ubiquitous, Gram-

positive bacterium and can be isolated from almost all ca-
tegories of food (Kozak et al. 1996). It is pathogenic for both
domestic and wild animals and also for humans, by causing the
serious disease listeriosis. The routes of transmission of listeri-
osis are different, and most commonly, it is transmitted trans-
placentally from mother to fetus and through ingestion of food
contaminated with L. monocytogenes (Farber and Peterkin 1991).
The primary site of entry of L. monocytogenes is the digestive
tract: the pathogen penetrates very fast into the intestinal
epithelium, where it is phagocytosed by macrophages (in
humans, this phase is asymptomatic). Intracellular prolifera-
tion occurs, with destruction of macrophages and a conse-
quential septicemia (Cousens and Wing 2000). Certain
segments of human population such as the elderly, infants,
pregnant women, people with reduced immunity, and indi-
viduals undergoing immunosuppressive therapy have an
increased risk of infection.
incidence of contamination of raw and industrially processed
food. Yet, the incidence of human listeriosis is very low,
usually about 2–8 sporadic cases per annum on a million of
inhabitants in Europe and the United States of America (Re-
court et al. 2000). Despite the high percentage, 23.3% (42/180)
of raw meat samples (Hod zić and i Hukić 2004) and 11.1%
(10/90) of meat product samples contaminated by L. mono-
cytogenes in the area of Bosnia and Herzegovina (Hod zić and i
Hukić 2006), no cases of human listeriosis were reported in the
past 10 years in the same area (Department of Health Statis-
tics, 2009).
Even though occurrence of listeriosis may be under-
estimated, another plausible reason for such conflicting proofs
may be the variability of virulence of L. monocytogenes. In fact,
although L. monocytogenes is defined as a pathogen at the level
of species, within this species a high degree of genetic diver-
sity and an extremely variable pathogenic potential are
present. Main genes responsible for virulence of this pathogen

1
Faculty of Science, University of Tuzla, Tuzla, Bosnia and Herzegovina.
2
The Institute of Clinical Microbiology, Clinical Center University of Sarajevo, Sarajevo, Bosnia and Herzegovina.
3
Istituto Superiore di Sanita, Rome, Italy.

1279
1280 HODŽIĆ ET AL.

are located on the bacterial chromosome (Vazquez-Boland Polymerase chain reaction (PCR) experiments to detect eight
et al. 2001). Molecular analyses of known virulence genes may L. monocytogenes virulence genes ( prfA, inlA, inlB, hly, plcA,
be useful (Nexmann-Larsen et al. 2002), although they cannot plcB, actA, and mpl) were performed according to the method
replace experiments on animals for the determination of the described by Franciosa et al. (2005).
pathogenic potential of L. monocytogenes.
The major objectives of this article were to examine the Genotyping
genetic diversity and pathogenic potential of L. monocytogenes
L. monocytogenes strain genotyping was performed by ap-
isolates from meat and meat products in northeast Bosnia and
plication of pulsed-field gel electrophoresis (PFGE), according
Herzegovina.
to the PulseNet standardized protocol (Graves and Swami-
nathan 2001), by using CHEF-DRII apparatus (Bio-Rad La-
Materials and Methods
boratories, Hercules, CA). The restrictive endonuceases ApaI
Bacterial isolates (Roche Diagnostics, Penzberg, Germany) and AscI (New
England BioLabs, Beverly, MA) were used.
The research included 20 isolates of L. monocytogenes (Table
Similarity of the macrorestriction profiles was calculated
1). L. monocytogenes Scott A was used as a control virulent
using the program Molecular Analyst Software Fingerprint-
strain, which is associated with outburst of invasive listeriosis
ing (Bio Rad Laboratories, Hercules, CA). Dendrogram was
(Fleming et al. 1985). The L. monocytogenes control avirulent
formed by applying a correlation coefficient dice and un-
strains used in this research were strain American Type Cul-
weighted pair group method by means of arithmetic mean
ture Collection (ATCC) 43248, isolated from guinea pig; strain
according to the same program. A degree of homology q80%
ATCC 15313, isolated from rabbit; and strain L.285, isolated at
(2% tolerance of position) between the genetic profiles of L.
the Institute of Health in Rome from a meat sample imported
monocytogenes isolates was selected for defining one cluster.
from Brazil. Listeria innocua isolated from beef collected from
retail in Tuzla Canton was also included in the study.
Biologic experiments on the mouse model
Isolation and identification of the Listeria species was per-
formed by applying the method ISO 11290–1/204 (Anon- The pathogenicity of selected PFGE types of L. mono-
ymus 2004), and identification was confirmed by a cytogenes was performed on the model of immunocompetent
biochemical test 10300 API Listeria (BioMerieux Italia, Flor- females of white mice (Pasteur strain [Department of Phar-
ence, Italy). The serotyping was performed using a commer- macology and Toxicology at the Veterinary Faculty in Sar-
cial kit (Listeria Antiserum kit; Denka Seiken, Tokyo, Japan). ajevo], 6–8 weeks old, weighing about 20 g in 5 days). The
pathogenicity of selected PFGE types of L. monocytogenes was
PCR detection of virulence genes examined on 20 mice as follows: Inocula of 106, 107, 108, and
109 cells/mL were prepared in physiological solution for each
Preparation of bacterial cells and isolation of DNA were
selected L. monocytogenes PFGE type, according to Takeuchi
performed as previously described (Franciosa et al. 1998).
et al. (2003). Groups of five mice were intraperitoneally in-
jected, each with 0.1 mL of the different inocula.
Table 1. Serotypes and Pulsed-Field Gel L. monocytogenes Scott A and L. innocua were used as pos-
Electrophoresis Types of Listeria itive and negative controls of pathogenicity, respectively, by
monocytogenes Isolates the protocol described earlier. As a further negative control, a
group of five mice were also used, which was intraperitone-
Listeria monocytogenes PFGE types AscI ally inoculated with 0.1 mL of physiological solution.
isolates Food Serotype and ApaI
All animal procedures were performed in accordance with
51 PSM 1/2c 1 the European Directive 86/609/EEC on protection of animals
70 GSM 4b 2 used for experimental and other scientific purposes.
71 GSM 4b 2 Isolates of L. monocytogenes that caused at least one death in
93 PSM 4b 3 the course of 5 days were considered to be pathogenic. The
206 PSM 1/2b 4 isolates that did not cause a single death in the course of 5
153 GGP 1/2a 5 days were considered nonpathogenic (Takeuchi et al. 2003).
179 GSM 1/2a 6 The pathogenic potential of the L. monocytogenes isolates
62 PSM 1/2a 7 was determined by calculation of lethal dosage for 50% of the
174 GSM 1/2a 8
inoculated animals (LD50) through graphic linear interpola-
61 GGP 1/2a 8
158 GGP 1/2a 9 tion (Reed and Muench 1938) and by determining the relative
286 GSM 1/2a 9 virulence (%) in relation to the reference strain of L. mono-
292 GSM 1/2a 9 cytogenes Scott A, according to Dongyou (Dongyou 2004).
59 GSM 1/2a 10
237 SSM 1/2a 10 Results
194 SGP 1/2c 10
298 PSM 1/2c 10 Serotyping
172 GSM 1/2c 10 Serologic testing was performed in 20 isolates of L. mono-
175 GSM 1/2c 10 cytogenes (Table 2). Four serotypes were determined: 1/2a in
195 SGP 1/2c nontyped
50% (10/20) of the isolates, 1/2b in 5% (1/20) of the strains, 1/
GGP, beef product; GSM, raw beef; PFGE, pulsed-field gel 2c in 30% (6/20) of the isolates, and 4b in 15% (3/20) of the
electrophoresis; PSM, raw chicken; SGP, pork product; SSM, raw pork. isolates (Table 1).
L. monocytogenes IN NORTHEAST BOSNIA AND HERZEGOVINA 1281

Table 2. Comparison of the Virulence of Listeria monocytogenes Isolates of Different Pulsed-Field

Gel Electrophoresis Types on White Mouse–Pasteur Strain

Mortality
Death/ Relative
Dose per 0 I II III IV V reference virulence
Strain mouse (CFU) Day Day Day Day Day Day strain (%) LD50

L.m.70 PFGE type 2 1.5 · 109 2 2 5/10 50 4.74 · 108

1.5 · 108 1
1.5 · 107
1.5 · 106
L.m.59 PFGE type 10 2 · 109 1 1 5/10 50 3.55 · 108
2 · 108 3
2 · 107
2 · 106
L.m.206 PFGE type 4 5 · 109 2 1 3/10 30 1.58 · 1010
5 · 108
5 · 107
5 · 106
L.m.Scott A reference strain 3.5 · 109 4 1 10/10 100 1.11 · 108
3.5 · 108 1 4
3.5 · 107
3.5 · 106
Listeria innocua negative control 3.5 · 109 0/10 0
3.5 · 108
3.5 · 107
3.5 · 106
Control Physiological solution 0/10 0

CFU, colony forming unit.

Virulence genes of L. monocytogenes Five days after inoculating L. monocytogenes isolate 70

PCR results indicated that all examined L. monocytogenes
isolates possessed the eight virulence-related genes ( prfA,
inlA, inlB, hly, plcA, plcB, actA, and mpl) considered in this
study.
PFGE types of L. monocytogenes
The analysis of the PFGE macrorestriction profiles obtained
with two different endonucleases, AscI and ApaI, distin-
guished 10 different PFGE types among the tested 19
L. monocytogenes isolates (Table 1). Each PFGE type represents
a separate clone of L. monocytogenes. L. monocytogenes serotype
1/2a displayed the broadest genomic variation: in fact, 10
isolates of serotype 1/2a were grouped into six different PFGE
types. Five L. monocytogenes isolates of serotype 1/2c were
grouped in two different PFGE types. Within L. monocytogenes
isolates of the serotype 4b, two different clones were identi-
fied. The only L. monocytogenes strain of serotype 1/2b pro-
duced a single PFGE type.
Virulence of L. monocytogenes
Three different PFGE L. monocytogenes types, each repre-
sentative of serotypes 1/2a, 1/2b, and 4b, were selected for
the virulence assay. Results are summarized in Table 2.
All three L. monocytogenes isolates (70, 206, and 59) caused
the death of some inoculated mice within 5 days of the ex-
periment and were considered to be pathogenic. Death of
mice was caused by microbial inocula of 109 and 108 cells/mL,
respectively, whereas the microbial inocula of 106 and 107 did
not kill any mice.
(PFGE type 2, serotype 4b), a total of five animals died,
of which four animals (4/5) were infected by inoculum of
1.5 · 109 cells/mL, and one animal (1/5) received a dose
of 1.5 · 108 cells/mL.
Five days upon inoculation of L. monocytogenes isolate 59
(PFGE type 10, serotype 1/2a), a total of five animals died, of
which one animal (1/5) was infected by an inoculum of 2 · 109
cells/mL and four animals (4/5) received a dosage of 2 · 108
cells/mL.
Five days upon inoculation of L. monocytogenes isolate 206
(PFGE type 4, serotype 1/2b), a total of three animals died, of
which two animals (2/5) were infected by an inoculum
of 5 · 109 cells/mL, and one animal (1/5) that received a dose
of 5 · 108 cells/mL.
Five days upon inoculation of L. monocytogenes Scott A
(positive control), a total of 10 animals died, of which five
animals (5/5) were infected by an inoculum of 3.5 · 109 cells/
mL and five animals (5/5) received a dose of 3.5 · 108 cells/
mL.
All animals inoculated with L. innocua (negative control)
were alive after 5 days.
The five animals inoculated with the physiological solution
were also alive after 5 days.
Relative virulence was calculated in relation to the refer-
ence strain L. monocytogenes Scott A, which caused a 100%
death rate in two dosing groups: 109 and 108 cells/mL. LD50
calculated for strain L. monocytogenes Scott A was 1.11 · 108
cells/mL. LD50 for L. monocytogenes isolate 70 (PFGE type 2) was
4.74 · 108 cells/mL, and the derived relative virulence was
50%. LD50 for L. monocytogenes isolate 59 (PFGE type 10)
was 3.55 · 108 cells/mL and relative virulence is 50%. LD50 for
1282
L. monocytogenes isolate 206 (PFGE type 4) was 1.58 · 1010
cells/mL and relative virulence is 30%.
Discussion
The L. monocytogenes species include strains with variable
pathogenic potential. Although many strains of L. mono-
cytogenes are very pathogenic and sometimes lethal to humans
and animals, others are relatively avirulent and do not cause a
great damage in the host. An obvious association has been
noticed between the antigenic properties and pathogenicity of
L. monocytogenes. The proof lies in the fact that of the 13 known
serotypes of L. monocytogenes, only 3 (1/2a, 1/2b, and 4b)
causeed more than 90% of human and animal cases of liste-
riosis (Low et al. 1993). Our research, which was based on
L. monocytogenes isolates from foods, showed that most of
them (14 of 20) belonged to serotypes 1/2a, 1/2b, and 4b, thus
representing a potential health risk.
Many typing methods, including the very discriminatory
genotyping method of PFGE, have confirmed the presence
of genetic clusters in L. monocytogenes. Results of our PFGE
analyses showed some genetic diversity among the differ-
ent serotypes of the L. monocytogenes tested, in accordance
with other researchers’ observations (Bibb et al. 1998). In
addition, consistent with data from the literature (Bibb
et al. 1998), certain serotypes were genetically more di-
versified than others, with serotype 1/2a being the most
diversified.
Virulence of L. monocytogenes strains depends on expres-
sion of several genes responsible for the pathogen capability
to penetrate the host cells and to proliferate and expand. The
entry process includes the expression of two proteins, inter-
nalin A and B, coded by two genes inlA and inlB. The other
gene locus includes genes involved in the functions essential
for intracellular survival and proliferation of the bacterium:
these genes code listeriosyn O, phospholipase C, and actin. All
of these cell surface proteins are regulated coordinately by
means of a pleiotrophic transcriptional activator coded by
prfA.
In this study, the above mentioned virulence genes were
detected in all L. monocytogenes isolates analyzed, conforming
with previous results (Franciosa et al. 2005). Moreover, all of
the isolates were equally pathogenic after intraperitoneal in-
oculation in mice.
However, the control avirulent strain ATCC 43248, isolated
from a guinea pig, possessed the virulence genes inlA, inlB,
hly, plcA, plcB, actA, and mpl, but it lacked the prfA gene.
Despite the presence of many genes associated with virulence,
as confirmed by PCR, defective regulation by prfA results in
avirulence of strain ATCC 43248 for mice. Control avirulent
strain L 285, isolated from a meat sample originating from
Brazil, is avirulent in mice, although PCR analysis confirms
the presence of all examined genes (Franciosa et al. 2005). The
control avirulent strain originating from the infected rabbit,
ATCC 15313, was initially hemolytic but has become nonhe-
molytic and avirulent after the next laboratory screening
(Kathariou and Pine 1991). Further PCR analysis has shown
that this strain had lost genes responsible for virulence
(Franciosa et al. 2005).
Analysis of virulence in mice provides a reference standard
for in vivo determation of the pathogenic potential of
L. monocytogenes. By applying criteria for calculation of the
HODŽIĆ ET AL.
relative virulence described by Liu (2004), the L. mono-
cytogenes isolates tested in this study may be classified as
moderately virulent, which may be one of the reason for the
absence of listeriosis in Bosnia and Herzegovina.
The results of this study indicate that Listeria is an im-
portant emerging pathogen in northeast Bosnia and Herze-
govina, thereby presenting potential threat for public health
and also for the neighboring countries because of livestock
market. Serious epidemiology studies among humans are
necessary to give insight on the significance of listeriosis for
the population in Bosnia and Herzegovina. However, less-
virulent isolates in the northeast part of the country may
suggest that less-severe cases may predominate, with certain
percentage of unrecognized cases, which are consequently
underreported.
Acknowledgment
The authors express gratitude to the Veterinary Faculty of
the University of Sarajevo, Department of Pharmacology, for
enabling this biological experiment.
Disclosure Statement
No competing financial interests exist.
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