Pesq. Vet. Bras. 24(2):57-60, abr./jun. 2004
Antimicrobial resistance and R-plasmid in Salmonella
Norma S. Lázaro2*, Anita Tibana3, Dália P. Rodrigues4, Eliane M.F. Reis4, Bianca R.
.- Lázaro N.S., Tibana A., Rodrigues D.P., Reis E.M.F., Quintaes B.R. & Hofer E. 2004.
Antimicrobial resistance and R-plasmid in Salmonella
spp from swine and abattoirenvironments. Pesquisa Veterinária Brasileira 24(2):57-60
. Depto Epidemiologia e Saúde Pública,Instituto de Veterinária, UFRRJ, Seropédica, RJ 23890-000, Brazil. E-mail: [email protected]
serovars isolated from swine are of particular interest not only because of the
pathogenic potential for this animal species, but also due to its relevance with regard to publichealth. On basis of the profile of resistance to antimicrobials, 13 Salmonella
strains were selectedwhich belonged to the serovars Muenster (7), Derby (4), Typhimurium (1), and Braenderup (1). Theywere isolated from healthy swine as well as from the abattoir environment in the state of Rio deJaneiro. All strains of Salmonella
were subjected to bacterial conjugation, and the E. coli
K12 NalrLac+ F standard strain was used as receptor, with the purpose to verify the ability to transfer theresistance marks. Gene transfer phenomenon was detected in seven strains, and except Salmonella
Typhimurium which transconjugated to Sm, Tc and Su, the remaining ones were characterized bytransferring mark Su only. By plasmidial analysis of strains used and their respective transconjugants,63 Kb plasmid was found, which was probably related to S
. Typhimurium resistance.
INDEX TERMS: Salmonella
, swine, antimicrobial resistance, R-plasmid.
RESUMO.- [Resistência a antimicrobianos e plasmidios
e, com exceção de Salmonella
Typhimurium que transconjugou
R em Salmonella
spp isoladas de suínos e do ambiente
para Sm, Tc e Su, as demais se caracterizaram por transferir
de abatedouro.] Sorovares de Salmonella
isolados de suinos
somente o marco Su. Na análise plasmidial das amostras doado-
são de particular interesse não só pelo potencial patogênico
ras e suas respectivas transconjugantes foi revelado um plasmídio
para esta espécie animal, como também pela sua relevância em
de 63 Kb, provavelmente relacionado com a multirresistência
Saúde Pùblica. Com base no perfil de resistência aos antimi-
crobianos foram selecionadas 13 amostras de Salmonella
TERMOS DE INDEXAÇÃO: Salmonella,
suínos, resistência antimicro-
tencentes aos sorovares
Muenster (7 amostras), Derby (4),
Typhimurium (1) e Braenderup (1), isoladas de suinos sadios e doambiente de abatedouro no Estado do Rio de Janeiro. As amos-
tras foram submetidas a conjugação bacteriana, utilizando comoreceptora E.coli
K12 55 Nal r Lac+ F -, com a finalidade de verifi-
serovars other than those related to disease are being
car a capacidade da transferência de marcos de resistência. O
identified in clinically healthy swine by the time of slaughter
fenômeno de transferência gênica foi detectado em 7 amostras
(Costa et al. 1972, Zebral et al.
1974, Lázaro et al. 1997). This facthas implications on Public Health, in so far as a considerablenumber of such serovars are also isolated from outbreaks of
human salmonellosis (Hofer & Reis 1994, Lirio et al. 1998).
Its significance does not only lie on the attributes of virulence,
Accepted for publication on August 20, 2003.
but also on the capability of resistance to antimicrobials shown
2 Depto Epidemiologia e Saúde Pública, Instituto de Veterinária, UFRRJ,
by some strains, as well as of the transfer of this feature through
Seropédica, RJ 23890-000, Brasil. *Author for correspondence. E-mail:[email protected]
plasmids (Ishiguro et al. 1980, Simmons et al. 1988, Heffernan
3 Instituto de Microbiologia Prof. Paulo de Góes, UFRJ, Rio de Janeiro.
The quick and widespread drug-resistance mediated by
Laboratório de Zoonoses Bacterianas, Depto Bacteriologia, IOC/
plasmidial genes in Salmonella
isolates has been reported
worldwide. During the past few decades, various countries
as recipient Escherichia coli
K12 55, F- Lac+ Nalr, susceptible to all
have witnessed a significant increase in human isolates of
drugs except nalidix acid. Transconjugants were seleted on MacConkey
multiresistant salmonellae (Holmberg et al. 1984, Carvalho
agar (Oxoid) containing the antimicrobials to which the standard strain
& Hofer 1989, Rivera et al. 1991, Asensi & Hofer 1994, Ling
was originally sensitive. The resistance patterns of transconjugant
1998), as well as animal isolates (Hampton et al. 1995,
strains was confirmed through the disc diffusion method according toNCCLS (1998), and considering the original profile of the corresponding
Millemann et al. 1995, Alaniz et al. 1997, Izumiya et al.
Plasmidial analysis. The analysis of the plasmidial contents
Of particular interest is the fact that most plasmids acquire
regarding the original strains and their respective transconjugants
their resistance genes through transposons, whether from an
was performed by the alkaline lysis method of Birnboim & Doly (1979),
other plasmid in the strain, from the chromosome or plasmids
modified by Sambrok et al.
(1989). The plasmidial DNAs of E. coli
carried by other bacterial strains which are present in the host
517 and 29R861 were included as molecular weight controls.
(Threlfall & Frost 1990). In the light of this, research was carriedout which concentrated essentially on R factors of Salmonella
means of conjugation tests (Timoney 1978, Vinhas & Almeida
Table 2 shows the conjugation positivity in seven (53.84%) out of
1984, Simmons et al. 1988, Sant’Ana et al. 1995).
the 13 donor strains, which evidences the total transference of
In view of the complexity of the factors associated with the
model Su, Tc, Sm on S
. Typhimurium and partial transfer of mark
dissemination of resistant Salmonella
strains, this paper has the
Su in regard to serovars Derby and Muenster.
purpose of assessing the capability to transfer marks of resistanceto antimicrobials by means of bacterial conjugation as well as byanalysis of the plasmidial profile of Salmonella
Table 2. Degree of resistance transfer marks to E. coli
clinically healthy swine, and also originating from the
LR1 (K12 55) by Salmonella
environment where these animals were slaughtered, in the state
Strains Resistance Transconjugants Transfer Transferred
For the conjugation experiments 7 samples were selected belonging
to serovar Muenster; 4 samples of serovar Derby, one belonging to
serovar Typhimurium , and one to serovar Braenderup. These were
isolated from swine and the abattoir environment; they were resistant
and/or multiresistant to sulphonamide (Su), streptomycin (Sm) and
Conjugation experiments. For the determination of R factors
in the samples under study, the methodology used was the one
Su= Sulfonamide, Tc= Tetracyclin, Sm= Streptomycin.
described by Dias & Hofer (1985). Conjugation was performed using
Regarding the transfer degree determined in salmonellae by
view of the growth of transconjugant samples in dilutions 10o,
Table 1. Profile of resistance of strains utilized as
10-1, and 10-2, it was found that from the seven transconjugants,
the isolation of transconjugating up to dilution 10-2 was obtained
only with S.
Typhimurium; the others were characterized by
The antimicrobial susceptibility tests confirmed the transfer
of resistance marks in all of the transconjugant strains.
In Table 3 are listed plasmids transferred by the conjugation
process between Salmonella
(donor) and (receptor) E. coli
(LR1). Despite the donor serovars Muenster and Derby, which
showed resistance transfer to mark Su for E. coli
, the analysis of
plasmidial DNA in transconjugant samples did not reveal plasmids
which were evidenced in the donor samples.
Typhimurium (strain no. 76), marks Tc, Sm, and
Su were transferred, and the analysis of transconjugants (Fig. 1)
revealed the presence of plasmids showing approximate sizes(Kb) of 63-3.75 and 3.45 Kb for transconjugant Sm (T76Sm x
aSpecimen corresponding to animal nº Ex.: MES4 = swine 4 mesenteric
LR1), and only the 63 Kb plasmid on transconjugants Tc and Su
lymph node; MES= mesenteric lymph node; ING= inguinal lymph node;
(T76&c x LR1, and T76Su x LR1). It is noteworthy that the
Ton= tonsil; TE= scalding tank; ET= evisceration table. For the
antimicrobial susceptibility test in order to confirm transfer of R
environmental samples the number follows the origin corresponding tothe order of collection (visit) , e.g. ET8= 8th visit.
factors revealed simultaneous resistance to marks Sm, Tc, and
bSm= streptomycin; Su= sulfonamide; Tc= tetracycline.
Su in the three transconjugants resulting from S. Typhimurium
Pesq. Vet. Bras. 24(2):57-60, abr./jun. 2004
Antimicrobial resistance and R-plamid in Salmonella
spp from swine and abattoir environments
Table 3. Antibiotic-resistance and plasmids R transferred via conjugation between
(donor) and E. coli
, K12 55 Nalr Lac+ F (receptor)
aSu= Sulfonamide, Tc= Tetracycline, Sm= Streptomycin.
related to one of the following features: the genic-chromosomicfeature of these determinants; the non-existence of the transferfactor; the interference of nalidixic acid with selective plates fortransconjugants; the inability of plasmid reception of the standardstrain used (Barbour 1967); or even plasmid loss during handlingof the samples.
The hypothesis is also admitted that these strains carry
thermo-sensitive plasmids R encoding a resistance to Tc and Sm,which are effectively transferred at 25° C. Factors related toresistance to tetracycline seem to be effectively transferredoutside the body, thereby decreasing transfer at 37°C (Timoney1978). This may account for the difficulty of in vivo
transfer onthe part of plasmids bearing resistance of animal origin into theflora residing in man’s intestine (Smith 1969). A wide distributionof thermo-sensitive plasmid in salmonellae isolated from swinewas noted by Ishiguro et al. (1980) in Japan.
Analyzing Table 3 it was found that only S.
capable of transferring R- plasmid to E. coli
. The close associationbetween marks Su, Tc, Sm, along with the finding of simultaneoustransfer to S. typhimurium
transconjugants, suggests that suchgenic expression may be determined by the same plasmid (63Kb), although there have been detected two additional plasmids
Fig. 1. Plasmid profile of resistant Salmonella
(3.75 and 3.45 Kb) on transconjugant Sm of S.
transconjugant lines: 1- E. coli
39R861; 2- S
. Typhimurium; 3,4 and
(T76Sm x LR1). This feature is found in the literature in so far a
5- transconjugants Su, Sm and Tc; 6- E coli
. K 1255; 7- E. coli
non-conjugating plasmid may be transferred to a receptor cellby cooperative action of a conjugative plasmid when they arepresent in the same cell. With regard to plasmids with relatively
high molecular weights and encoding resistance to
The information contained in the literature points out the transfer
antimicrobials, in Salmonella
Typhimurium these appears to exist
of genes among bacteria by means of conjugation, in varied
a 40 Kb plasmid associated to resistance to amoxicilin,
environments as well as in the intestinal tracts of humans and
streptomycin, tetracycline, chloranphenicol, and sulfametoxazol-
trimetroprin (Hansen et al. 1964), and another 80 Kb plasmid
Although laboratorial experiments do not exactly reproduce
encoding resistance to marks Ap, Sm, Su, Tc (Hampton et al.
– even under simulated conditions – the complexity existing in
transfer processes occurring in vivo
, they may constitute an
As to the absence of plasmids in transconjugants of S
important tool for making such inferences.
Muenster and S.
Derby, the hypothesis can scarcely be admitted
The negative result in the conjugation of the six Salmonella
that the determinant of resistance to mark Su is encoded by a
Muenster strains, as well as the absence of transconjugants for
large plasmid, which has not been demonstrated through the
marks Sm in S. Muenster (no. 8), and Sm, Tc in S
. Derby, may be
methodology employed. For this purpose, various methods and
Pesq. Vet. Bras. 24(2):57-60, abr./jun. 2004
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Pesq. Vet. Bras. 24(2):57-60, abr./jun. 2004
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