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Scholars
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African Journal of Pig Farming ISSN 2375-0731 Vol. 2 (4), pp. 001-003, April, 2014. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

Short Communication 

 

Antimicrobial susceptibility and genetic relatedness of 

Salmonella enterica subsp. enterica serovar Mbandaka 

strains, isolated from a swine finishing farm in Greece 
 

G. Filioussis1,2*, E. Petridou3, A. Johansson2, G. Christodoulopoulos1 and S. K. Kritas3
 

 
1
Department of Veterinary Medicine, University of Thessaly, Karditsa, Greece. 

2
Institute of Veterinary 

Bacteriology, University of Bern, Switzerland. 
3
Laboratory of Microbiology and Infectious Diseases, 

School of Veterinary Medicine, University of Thessaloniki, Thessaloniki, Greece. 

 
Accepted 11 November, 2013 

 
The current study investigated the antimicrobial susceptibility of Salmonella enterica subsp. enterica serovar 

Mbandaka (Salmonella Mbandaka) isolated from finishing swines in Greece. Pulsed-field gel electrophoresis (PFGE) 

was used to examine the genetic relatedness of the isolates. The study was carried out for 1 year as part of a project 

focusing on antimicrobial resistance of salmonellae recovered from asymptomatic pigs. A total of 400 finishing pigs 

stabled in 20 swine farms in central Greece were included in the study. Fecal samples taken directly from the rectum, 

one sample from each pig, were cultured for Salmonella spp. Five of the 400 tested finishing pigs, originating from the 

same herd, were asymptomatic carriers of Salmonella Mbandaka. All five isolates were resistant to tetracycline, four 

were resistant to trimethoprim/sulfamethoxazole, and three to ampicillin and amoxicillin/clavulanic acid. In contrast, 

all five isolates were susceptible to cefuroxime and ceftriaxone, as well as to nalidixic acid, ciprofloxacin, and 

levofloxacin. All five isolates had indistinguishable PFGE patterns. The present study confirms the existence of a 

nontyphoid Salmonella serotype, Salmonella Mbandaka in asymptomatic carrier pigs in Greece. Further, the 

Salmonella Mbandaka isolates were found to be resistant to several antimicrobials. 

 

Key words: Salmonella enterica subsp. enterica serovar Mbandaka, antimicrobial susceptibility, swine, Pulsed-field gel 

electrophoresis (PFGE), zoonotic. 

 
INTRODUCTION 

 
Salmonella spp. is recognized worldwide as important 

pathogens in the intestinal tracts of both animals and 

humans. Infected animals are usually asymptomatic carriers, 

a fact that has a major effect on the spread of infection. The 

number of human cases of salmonellosis caused by the 

nontyphoid Salmonella enterica subsp. enterica serovar 

Mbandaka (Salmonella Mbandaka) increased during the 

1990s. Related outbreaks of human infection characterized 

by diarrhoea have been reported in the United Kingdom 

(Reid et al., 1993), Italy (Fantasia et al., 1989), Australia 

(Scheil et al., 1998), and the United States (Gill et al., 2003). 

Pigs infected with Salmonella  
 
 
 
*Corresponding author. E-mail: gfiliu@vet.uth.gr. Tel: 
00312106664688Fax: 00302107496632. 

 
 
 
Mbandaka, as well as pig products, have been implicated 
throughout pork production and processing in cases of 
human infection and clinical disease (Davies, 1998). Pou-try 
has also been considered an important source of Sal-
monella Mbandaka for humans (Hoszowski and Wasyl, 
2001).  

A prominent reason for concern with regard to gastro-
enteritis-causing bacteria is the recognized emergence of 
antimicrobial resistance among key species. Over the past 
decade, particularly in developing countries, the increase in 
resistance of animal origin nontyphoid salmo-nellae to 
broad-spectrum antibiotics such as cephalo-sporins, 
tetracycline, and quinolones has been extremely worrisome 
(Streit et al., 2003). As a result, attempts are being made to 
trace salmonellosis outbreaks to contami-nated sources, and 
numerous typing methodologies have been used. 

Information concerning the prevalence of S. enterica 



 
 
 

 

serotypes in Greek finishing swine herds has previously 
been published (Leontides et al., 2002; Wong et al., 
2003). In this study, we report the antimicrobial sus-
ceptibility and the genetic relatedness of five Salmonella 
Mbandaka isolates originating from healthy finishing 
swines in a Greek herd. 

 

MATERIALS AND METHODS 
 
Study design 
 
Asymptomatic carriers have a major effect on the spread of 
Salmonella infections to humans (Wong et al., 2003). Therefore our 
study, carried out from March 2003 to October 2004, was focused 
on antimicrobial resistance and genetic variation of salmonellae 
recovered from asymptomatic pigs. Samples were collected from 20 
swine finishing farms, representing 10% of the swine finishing farms 
in central Greece. Twenty finishing pigs from each farm were 
sampled (almost 5% of the stabled animals), and 400 fecal samples 
were collected. Faecal samples were taken directly from the 

rectum, one sample from each pig. Samples were transported (4
o
C) 

within 4 h to the laboratory. 

 

Sampling and cultivation 
 
25 g of each faecal sample was diluted in sterile plastic bags with 
225 ml of buffered peptone water (BPW, Merck KGaA, 64271 

Darmstadt, Germany) and incubated in 37
o
C for 24 h. After enrich-

ment (41.5
o
 C for 24 h) in Modified Semi-solid Rappaport - 

Vassiliadis ( MSRV, Merck KGaA, 64271 Darmstadt, Germany), 
Salmonella spp. was isolated on Xylose-Lysine-Deoxycholate (XLD 
, Merck KGaA, 64271 Darmstadt, Germany) and Hektoen agar 
(EMD, Merck KGaA, 64271 Darmstadt, Germany, according to 
6579:2002 ISO. Suspect colonies were sub cultured on nutrient  
agar (Merck KGaA, 64271 Darmstadt, Germany ) and were 
characterized biochemically using Triple Sugar Iron agar (TSI, 
Merck KGaA, 64271 Darmstadt, Germany), Lysine broth, Urea 
broth, Simmons Citrate agar and Tryptophan broth (Merck KGaA, 
64271 Darmstadt, Germany). Serotyping was carried out by the 
National Veterinary Services Laboratories (Halkida, Greece) 
according to the Kaufmann-White scheme (Popoff and Le Minor, 
1997). 

 
Antimicrobial susceptibility testing 
 
Antimicrobial susceptibility of Salmonella Mbandaka isolates to nine 
antimicrobials (Ampicillin, Amoxicillin/clavulanic acid, Cefuroxime, 
Ceftriaxone, Nalidixic acid, Ciprofloxacin, Levofloxacin, Tetracycline 
and Trimethoprim/sulfamethoxazole) was determined by dilution 
antimicrobial susceptibility tests in accordance to the M100-S16 
method and the interpretive criteria of the Clinical and Laboratory 
Standards Insitute (CLSI, 2006). The MIC was determined as the 
lowest concentration of antimicrobial agent that inhibited visible 
growth. Escherichia coli ATTC 25922 and Klebsiella pneumoniae 
ATCC 700603 were used as quality control strains. 

 

PFGE 
 
The genetic relatedness of the isolates was investigated by pulsed-
field gel electrophoresis (PFGE) as described in detail by Gautom 

(1997). After cleavage with XbaI, electrophoresis was performed at 
6 V/cm with 1.0% AgaroseNA agar (Amersham Biosciences, 

Uppsala, Sweden) by using the CHEF-DR II system (Bio-Rad Labo- 

 
 
 
 

 
ratories, Richmond, California, USA). Running conditions for XbaI-
digested DNA were 2.2 to 64 s for 20 h. Lambda ladder with a size 
range of 0.13 to 194 kb (Low Range PFG Marker, New England 
Biolabs Inc.), were used as molecular weight standard. The DNA 

bands were visualized on a UV transilluminator and Polaroid 
photographs of the gels were visually examined. 

 

RESULTS AND DISCUSSION 
 

Epidemiological data show that salmonellosis is a serious 
problem worldwide in pigs, cattles, horses, poultry, and 
even exotic animals. Nontyphoid salmonellae are among 
the leading causes of food-borne disease in humans and 
are therefore of increased importance in public health. In 
the present study, five (1.25%) of the 400 tested finishing 
pigs were asymptomatic carriers of salmonellae, which 
were further serotyped as Salmonella Mbandaka. All five 
pigs originated from the same herd; the herd included 
180 sows all of the same breed. The prevalence was 
lower than previously reported in other European 
countries (Wong et al., 2003). No other Salmonella 
serotypes were identified in these animals. Therefore the 
present findings confirm the existence of the particular 
nontyphoid Salmonella serotype in asymptomatic carrier 
swines in Greece and therefore indicate a potential risk 
for trans-mission of the bacterium from swine to humans, 
directly or in meat products.  

Concerning the susceptibility to antimicrobial agents all 
five isolates were found to be resistant to tetracycline 
(MIC90 8 g/ml) four to trimethoprim/sulfamethoxazole 
(MIC90 0.5 g/ml), and three to ampicillin and amoxi-
cillin/clavulanic acid ( MIC90 16 g/ml). In contrast, all five 
isolates were susceptible to cefuroxime and ceftria-xone, 
as well as to nalidixic acid, ciprofloxacin, and levo-
floxacin. Given the zoonotic potential of Salmonella 
Mbandaka, research is needed to determine which 
antimicrobials might be efficacious against this pathogen. 
In the present study, susceptibility to some third-genera-
tion cephalosporins (cefuroxime, ceftriaxone) and quino-
lones (nalidixic acid, ciprofloxacin, levofloxacin) was 
detected in all five isolates. This susceptibility can be 
partially explained by the limited use of these antibiotics 
in Greek farms (data not shown). However, an increasing 
resistance in Salmonella Mbandaka strains to some 
cephalosporin -lactams (cefotaxime, ceftazidime and 
aztreonam) has been observed by other researchers 
(Szych et al., 2001). The resistance of Salmonella 
Mbandaka isolates to tetracycline, ampicillin, amoxicil-
lin/clavulanic acid, and trimethoprim/sulfamethoxazole 
has previously been reported (Streit et al., 2003) and 
reflects the use of these antimicrobials as growing factors 
in swine finishing farms.  

Salmonella Mbandaka sensitivity to antibiotics was exa-
mined in vitro in this study. In vivo confirmation of these 

results is necessary because persistent antibacterial 
effects at subinhibitory concentrations (postantibiotic 
effects), which facilitate removal of affected bacteria by 
host defense mechanisms, have been 



 
 
 

 

M 1 2 3 4 5  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 1. PFGE patterns of 5 Salmonella Mbandaka isolates. 

The patterns were obtained by the PFGE protocol with XbaI 
digestion. Lane M, lambda ladder with a size range of 45.5 to 

727.5 kb (High Range PFG Marker, New England Biolabs Inc.). 
Lanes 1 – 5, the five Salmonella Mbandaka isolates analyzed. 

 

 

demonstrated for penicillins, cephalosporins, macrolides, 
tetracyclines, aminoglycosides, and several other 
antibac-terial agents (Aiello S.E, 1998). In the case of 
increasing resistance to antibiotics, other methods such 
as identi-fication and elimination of carrier pigs might 
prevent the spread of this Salmonella serotype.  

The XbaI PFGE patterns of the Salmonella Mbandaka 
isolates are presented in Figure 1. All five isolates had 
indistinguishable PFGE patterns. The results are not 
surprising as all the isolates were from the same farm. 
Hoszowski and Wasyl (2001) also demonstrated a high 
genetic relatedness among Salmonella Mbandaka strains 
from poultry and human origin. However, no studies con-
cerning the genetic relatedness among swine and human 
isolates have been published so far.  

No studies have yet been published concerning isolation of 

Salmonella Mbandaka in any case of human disease in 

 
 
 
 

 

Greece. Therefore, we did not have the opportunity to 
include data relating to human isolates in our study. How-
ever, human cases in Europe were mainly attributed to 
contact with asymptomatic carrier pigs. It is thus recom-
mended that this particular Salmonella serotype may be 
considered in investigations of future cases of animals 
and humans salmonellosis. 

 

ACKNOWLEDGEMENTS 
 
We would like to thank H. Simpson for technical support 

 
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