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337
*Corresponding author: Mailing address: Department of
Microbiology, Faculty of Medicine, Slovak Medical
University, Limbova 14, 833 03 Bratislava, Slovakia. Tel:
＋
421-2-59370726, Fax:
＋
421-2-59370327, E-mail: lubica.
majtanova
＠
szu.sk
337
Jpn. J. Infect. Dis., 66, 337-340, 2013
Short Communication
Antimicrobial Resistance of Human
Salmonella enterica
Serovar Typhimurium U302 Strains:
Prevalence of R-Type ASSuT in Slovakia, 2006–2011
L'ubica Majt áanov áa
1
*,JurajMajtáan
1,2
, and Viktor Ma jt áan
1
1
National Reference Centre for Phage Typing of Salmonella, Faculty of Medicine,
Slovak Medical University, Bratislava; and
2
Institute of Zoology, Slovak Academy of Science, Bratislava, Slovakia
(Received February 21, 2013. Accepted April 22, 2013)
SUMMARY:
Salmonella enterica
serovar Typhimurium is a common cause of non-typhoid salmonello-
sis in humans. Since 2006, an increase in the human infections caused by U302 phage type in Slovakia
has been documented and, from 2006 to 2011, a total of 291 U302 human strains were isolated. An-
timicrobial susceptibility testing demonstrated that these strains had a high overall antimicrobial
resistance and 244 (83.8
z
) of the tested strains were multidrug-resistant (MDR). The most prevalent
resistance was to ampicillin, streptomycin, sulfisoxazole, and tetracycline (R-type ASSuT), which was
verified in 87 (29.9
z
) strains. The annual rate of this resistance type varies, but the largest number of
these strains were identified in 2009 and 2010. The classical pentaresistance phenotype (R-type
ACSSuT), characteristic of the DT104 phage type, was found only in 40 (13.7
z
) U302 strains. These
results suggested that although the prevalence of DT104 phage type has decreased, ASSuT as well as
ACSSuT resistance markers continue to circulate. Therefore, continual surveillance of the occurrence of
these and similar MDR phage types is necessary.
Salmonella
is one of the most important pathogens in
both humans and animals worldwide, as they cause gas-
trointestinal infections and septicemia. It comprises
more than 2,600 serovars (1) with different host adapta-
tions and degrees of virulence, and most are considered
as potential human pathogens, although only a rela-
tively small number of serovars have been associated
with human infections. Among these,
Salmonella en-
terica
serovar Typhimurium is the second most frequent
type of
Salmonella
serovar isolated from Slovakia.
For epidemiological purposes, differentiation of the
strains of this serovar is essential and phage typing has
been a useful phenotypic definitive method of choice in
many countries for decades.
In 2006, the Slovak National Reference Centre for
Salmonella
phage typing (Slovak Medical University,
Bratislava, Slovakia) for the first time identified a mul-
tidrug-resistant (MDR) U302 phage type among
S.
Typhimurium strains isolated from human infections.
In recent years, MDR
S.
Typhimurium DT104, which
usually has chromosomal-encoded pentaresistance
against ampicillin, chloramphenicol, streptomycin, sul-
fonamides, and tetracyclines has spread internationally,
and is now prevalent in Slovakia (2–4). Apart from
causing severe illnesses, there are concerns regarding the
emergence of MDR
S.
Typhimurium in various phage
types as well as other serovars because of the reduction
in the number of available treatment options, which can
lead to treatment failure (5).
In the present study, we assessed the prevalence of
resistance to ampicillin, streptomycin, sulfonamide,
and tetracycline (R-type ASSuT) in
S.
Typhimurium
phage type U302 strains isolated from human cases
from 2006 to 2011.
During this period, a total of 1,779
S.
Typhimurium
strains were isolated from sporadic human cases and
sent to the National Reference Centre for
Salmonella
phage typing (Table 1). Isolated strains were phage
typed using phage preparations supplied by the Health
Protection Agency (Colindale, London, UK) according
to the methods of Anderson et al. (6). On the basis of
the phage typing results, phage type U302 strains were
selected and isolated from feces, except for two strains
isolated from urine and one from a purulent wound.
A total of 291 U302 strains were tested for an-
timicrobial susceptibility using the standard disk diffu-
sion method according to the protocol of the Clinical
and Laboratory Standards Institute (7). Resistance
against the following antimicrobial agents was assessed:
ampicillin (10
m
g), ceftriaxone (30
m
g), chloramphenicol
(30
m
g), gentamicin (10
m
g), ciprofloxacin (5
m
g), sul-
fisoxazole (250
m
g), tetracycline (30
m
g), streptomycin
(10
m
g), trimethoprim (5
m
g), trimethoprim-sulfameth-
oxazole (1.25/23.75
m
g), and nalidixic acid (30
m
g).
Escherichia coli
ATTC 25022 was used as the reference
strain.
S.
Typhimurium is the second most common serovar
isolated from Slovakia, exceeded only by
S.
Enteritidis
(8). Subtyping of
S.
Typhimurium by phage typing is
a well-established method and plays a vital role in the
detection of outbreaks and identification of connection
338
Table 1. Distribution of 291 human
S.
serovar Typhimurium
isolates identified as U302 phage type during 2006–2011
Year Isolates phage-typed
No. Isolates identified as U302
No. (
z
)
2006
2007
2008
2009
2010
2011
Total
190
223
279
382
453
252
1,779
25 (13.2)
51 (22.9)
48 (17.2)
73 (19.1)
55 (12.1)
39 (15.5)
291 (16.4)
Table 2. Antimicrobial resistance among 291 human
S.
serovar Typhimurium U302 strains
isolated during 2006–2011
Resistance
type
No. of resistant isolate
2006 2007 2008 2009 2010 2011 Total (
z
)
ACSSuTSxTTMP 1012004(1.4)
ASSuT SxT TMP NA 0 0 1 0 1 0 2 (0.7)
ACSSuTNA 03214111(3.8)
ACSSu SxT TMP 0 0 0 1 0 0 1 (0.3)
ASSuT SxT TMP 0 0 1 6 0 0 7 (2.4)
CSSuT SxT TMP 0 0 0 0 1 0 1 (0.3)
ACSSuT 310895540 (13.7)
SSuT SxT TMP 6 2 0 1 0 0 9 (3.1)
ASuT SxT TMP 0 0 0 3 0 1 4 (1.4)
ASSuT TMP 0 0 0 0 0 1 1 (0.3)
ASSuT NA 0 0 0 0 6 0 6 (2.1)
ASSuT 4 14 7 26 21 15 87 (29.9)
AGST 0000101(0.3)
SuT SxT TMP 0 0 1 0 0 1 2 (0.7)
ACSSu 0 0 1 0 0 1 2 (0.7)
ASSu NA 0 0 0 0 0 1 1 (0.3)
ASSu 6 11 12 10 2 1 42 (14.4)
SSuT 4 2 3 2 0 0 11 (3.8)
AST 000430 7(2.4)
SuSxT TMP 0 0 0 1 0 0 1 (0.3)
ASuT 0 0 0 0 1 1 2 (0.7)
ACSu 000002 2(0.7)
ASu 100000 1(0.3)
SSu 0 7 2 3 3 0 15 (5.2)
AS 00612615(5.2)
ST 001010 2(0.7)
SuT 0 0 0 2 0 0 2 (0.7)
AT 000003 3(1.0)
S 0110215(1.7)
T 0010001(0.3)
A 0010113(1.0)
Total 255049725441291(100.0)
The most prevalent resistance types are indicated as bold letters.
A, ampicillin; C, chloramphenicol; S, streptomycin; Su, sulfisoxazole; SxT, trimethoprim-
sulfamethoxazole; T, tetracycline; TMP, trimethoprim; NA, nalidixic acid.
338
between seemingly sporadic cases. It also enables the
monitoring of new phage types or phage types with new
characteristics.
Here we report the emergence of the MDR U302
phage type, which was identified in the National Refer-
ence Centre for
Salmonella
phage typing for the first
time in 2006. The distribution of a total of 291 human
S.
Typhimurium isolates identified as U302 phage types
during 2006–2011 is shown in Table 1. Although the
number of U302 human strains in individual years
differed among the total number of isolates, it was the
most frequent phage type identified in 2007–2009. Since
1990, MDR
S.
Typhimurium DT104 has emerged as a
worldwide health concern in humans and food animals.
In Slovakia, this phage type was identified for the first
time in 1997 and since then has become the most preva-
lent phage type (4).
S.
Typhimurium U302 is a phage type of epidemio-
logical importance because of its multi-resistant charac-
ter. Pritchett et al. (9) identified a DNA sequence
unique to the DT104 and U302 phage types and
confirmed a close relationship between them. Although
U302 phage type strains were sporadically isolated in
Slovakia, other infectious outbreaks caused by strains
of this phage type have been reported. Ethelberg et al.
(10) described a prolonged restaurant-associated out-
break in Denmark and neighboring countries during
July and August 2003, which comprised 67 laboratory-
339339
confirmed cases; however, the actual number of
patients was estimated to be more than 390. This par-
ticular strain was isolated from a buffet, which was
probably contaminated by an assistant chef who was
found to excrete the epidemic strain. Another
widespread outbreak caused by the epidemic U302
strain involved 45 cases in southern Ontario (Canada)
over a 3-month period in 2005. The most likely hypothe-
sis supported by the findings of an epidemiologic inves-
tigation was that the illness was associated with salami,
mortadella, and/or prosciutto made by a single
producer (11).
Antibiotic resistance dramatically increased in
S.
Typhimurium, which is characterized by an especially
high incidence of MDR strains in Europe due to a clonal
and international spread of phage type DT104 that was
initially linked to cattle farms (2,12). The aim of this
study was to examine the antimicrobial susceptibility of
the human U302 phage type strains that belonged to the
most frequent phage types in recent years. Among 291
isolates tested, 31 resistance profiles were identified
(Table 2).
Among the tested strains, 47 (16.2
z
) were resistant
against one or two antimicrobial agents and up to 244
(83.8
z
)wereMDR.MDR
Salmonella
are of worldwide
interest because they reduce the number of therapy op-
tions in human and veterinary medicine. The most
prevalent resistance is to ampicillin, streptomycin, sul-
fisoxazole, and tetracycline (R-type ASSuT), which was
identified in 87 isolates (29.9
z
) during 2006–2011. The
aforementioned infectious outbreak described by Ethel-
berg et al. (10) was caused by a phage type U302 strain
with the same resistance type. The annual rate of
this resistance type varied, but the largest numbers were
identified in 2009 and 2010. Although a close relation-
ship between the DT104 and U302 phage types has been
described, the characteristic pentaresistance of DT104
to ampicillin, chloramphenicol, streptomycin, sul-
fonamide, and tetracycline (R-type ACSSuT) was found
only in 40 (13.7
z
) U302 strains. However, we did not
investigate antibiotic resistance genes among the U302
tested strains. Walker et al. (13) demonstrated that the
majority of MDR U302 strains possessed the same anti-
biotic resistance genes as MDR DT104. The ACSSuT
resistance type predominantly belonged to phage types
DT104, DT120, and DT20a (14–16), but Reis et al. (17)
also found this resistance type in human DT193 phage
type strains. An almost identical number of U302
strains (14.4
z
) in the present study belonged to the
ASSu resistance type.
In the 1970s, various
Salmonella
outbreaks erupted in
the United Kingdom and other European countries
provoked by DT204, DT204c, and DT193, which ex-
hibited multidrug resistance with the exception of the
DT193 phage type, but they have not been recorded in
Slovakia (5,18,19).
The results of the present study confirmed that all of
the tested
S.
Typhimurium U302 strains were suscepti-
ble to third-generation cephalosporin and fluoroquino-
lone, which should therefore be considered crucial
components of therapy for
Salmonella
infections in
humans. However, only 20 strains (6.9
z
) were resistant
to nalidixic acid, which is a precursor to fluoroquino-
lones.
Apart from causing severe illnesses, there are con-
cerns regarding the emergence of MDR
Salmonella
strains of various phage types because they are potential
donors of resistance genes to other pathogens or com-
mensals in the gastrointestinal tract (20,21). Further-
more, it is widely accepted that the development of
MDR bacteria is promoted by the use of antimicrobial
drugs in food animals (18). Our results suggested that
although there was a change in prevalent phage types,
the MDR markers continued to circulate. Lastly, with
regard to lysogenic conversion, there is need for con-
tinual surveillance of the occurrence of these and similar
MDR phage types.
Acknowledgments
This work was by Ministry of Health of the
Slovak Republic under the project Molecular Analysis of Antibiotic
Resistance of Nontyphoid Salmonella Serovars.
Conflict of interest
None to declare.
REFERENCES
1. Guibordenche, M., Roggention, P., Mikoleit, M., et al. (2010):
Supplement 2003–2007 (No. 47) to the White-Kaufmann-
Le Minor scheme. Res. Microbiol., 161, 26–29.
2. Threlfall, E.J. (2000): Epidemic
Salmonella
typhimurium
DT104–a truly international multiresistant clone. J. Antimicrob.
Chemother., 46, 7–10.
3. Helms, M., Ethelberg, S., Mølbak, K., et al. (2011): International
Salmonella
Typhimurium DT104 infections, 1992–2001. Emerg.
Infect. Dis., 69, 454–456.
4. Majtanova, L., Majtan, J. and Majtan, V. (2011): Trends in
phage types of
Salmonella enterica
serovars Enteritidis and
Typhimurium isolated in Slovakia from 1995 to 2009. Diagn.
Microbiol. Infect. Dis., 69, 454–456.
5. Threlfall, E.J. (2002): Antimicrobial drug resistance in
Salmo-
nella
: problems and perspectives in food- and water-borne infec-
tions. FEMS Microbiol. Rev., 26, 141–148.
6. Anderson, E.S., Ward, L.R., de Saxe, M.J., et al. (1977): Bac-
teriophage–typing designations of
Salmonella
typhimurium. J.
Hyg., 78, 297–300.
7. Clinical and Laboratory Standards Institute (CLSI) (2007): Per-
formance standards for antimicrobial susceptibility testing. 17th
informational supplement. CLSI, Wayne, Pa.
8. Gavacova, D., G äoczeova, J. and Sirotna, Z. (2012): Salmonel-
loses—what brought year 2011. Abstract 17. The Scientific Con-
ference of Slovak National Reference Centers for Surveillance of
Infectious Diseases (in Slovak).
9. Pritchett, L.C., Konkel, M.E., Gay, J.M., et al. (2000): Identifi-
cation of DT104 and U302 phage types among
Salmonella enteri-
ca
serotype Typhimurium isolates by PCR. J. Clin. Microbiol.,
38, 3484–3486.
10. Ethelberg, S., Lisby, M., Torpdahl, M., et al. (2004): Prolonged
restaurant-associated outbreak of multidrug-resistant
Salmonella
Typhimurium among patients from several European countries.
Clin. Microbiol. Infect., 10, 904–910.
11. Public Health Agency of Canada (2006): Outbreak of
Salmonella
Typhimurium phage type U302 in Ontario, spring 2005. Can.
Commun. Dis. Rep., 32, 1–7.
12. Chaslus-Dancla, E., Lafont, J.P. and Martel J.L. (2000): Spread
resistance from food animals to man: the French experience. Acta
Vet. Scand., 93 (Suppl.), 53–60.
13. Walker, R.A., Lindsay, E., Woodward, M.J., et al. (2001): Vari-
ation in clonality and antibiotic-resistance genes among mul-
tiresistant
Salmonella enterica
serotype
typhimurium
phage-type
U302 (MR U302) from humans, animals, and foods. Microb.
Drug. Resist., 7, 13–21.
14. Baggesen, D.L., Sandvang, D. and Aarestrup, F.M. (2000):
Characterization of
Salmonella enterica
serovar Typhimurium
DT104 isolated from Denmark and comparison with isolates
from Europe and the United States. J. Clin. Microbiol., 38,
1581–1586.
15. Chang-You, Y., Chou, S.J., Yeh, C.M., et al. (2008): Prevalence
and characterization of multidrug-resistant (type ACSSuT)
340340
Salmonella enterica
serovar Typhimurium strains in isolates from
four gosling farms and a hatchery farm. J. Clin. Microbiol., 46,
522–526.
16. Majtanova, L. and Majtan, V. (2009): Molecular characterization
of the multidrug-resistant phage types
Salmonella enterica
serovar Typhimurium DT104, DT20a and DT120 strains in the
Slovakia. Microbiol. Res., 164, 157–162.
17. Reis, E.M.F., Rodrigues, D.P., Freitas-Almeda, A.C., et al.
(2011): Prevalence of R-type ACSSuT in strains of
Salmonella
serovar Typhimurium DT193 isolated from human infections in
Brazil. Rev. Panam. Salud. Publica., 29, 387–392.
18. Rabsch, W., Tsch äape, H. and B äaumler, A.J. (2001): Non-
typhoidal salmonellosis: emerging problems. Microbes Infect., 3,
237–247.
19. Threlfall, E.J., Ward, L.R. and Rowe, B. (1978): Spread of mul-
tiresistant strains of
Salmonella
Typhimurium phage types 204
and 193. Br. Med. J., 2, 997.
20. McEwen, S.A. and Fedorka-Cray, P.J. (2002): Antimicrobial use
and resistance in animals. Clin. Infect. Dis., 34, 93–106.
21. Harbottle, H., Thakur, S., Zhao, S., et al. (2006): Genetics of
antimicrobial resistance. Anim. Biotechnol., 17, 111–124.
