Pesq. Vet. Bras. 24(2):57-60, abr./jun. 2004
Antimicrobial resistance and R-plasmid in Salmonella spp
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: firstname.lastname@example.org
Salmonella 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 SalmonellaTyphimurium 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 per-
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-
Salmonella 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@example.com
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
et al.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 V
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, by
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 isolated from
Table 2. Degree of resistance transfer marks to E. coli
clinically healthy swine, and also originating from the
LR1 (K12 55) by Salmonella spp strains
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 K12 55
(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.
Regarding S. 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
Salmonella (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. Typhimurium was
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 Typhimurium and
(3.75 and 3.45 Kb) on transconjugant Sm of S. Typhimurium
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 V517.
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
procedures were set forth, the outstanding being the one
Heffernan H.M. 1991. Antibiotic resistance among salmonbella from human
developed by Kado & Liu (1981), which is the most convenient
and other sources in the New Zealand. Epidemiol. Infect. 106:17-23.
for extraction in view of plasmids with high molecular weight.
Hofer E. & Reis E.M.F. 1994. Salmonella serovars in food poisoning episodes
Another explanation for this phenomenon could be the outcome
recorded in Brazil from 1982 to 1991. Revta Inst. Med. Trop. 36:7-9.
of integration of the resistance-plasmid in the receptor’s
Holmberg S.D., Wachsmuth I.K., Hickman-Brenner F.W. & Cohen M.L. 1984.
Comparison of plasmid profile analysis, phage typing, and antimicrobal
chromosome (Madigan et al. 1997); or, still, because of the poor
susceptibility testing in characterizing Salmonella typhimurium isolates
stability during the storage period between tests.
from outbreaks. J. Clin. Microbiol. 19:100-104.
The ease of in vitro transfer has led to the conclusion that
Ishiguro N., Goto J. & Sato G. 1980. Genetical relationship between R
similar pattern of resistance to antibiotics in different intestinal
plasmids derived from Salmonella and Escherichia coli obtained from a
bacteria are mediated by the same resistance plasmid (R factor)
pig farm, and its epidemiological significance. J. Hyg. Camb. 84:365-
and has the same origin (Cherubin 1981). In contrast, Avril et al.
(1977) have shown that the same resistance pattern is expressed
Izumiya H., Terajima J., Matsushita S., Tamura K. & Watanabe H. 2001.
by different episomes, and in the same way in different Salmonella
Characterization of multidrug-resistant Salmonella enterica serovarTyphimurium isolated in Japan. J. Clin. Microbiol. 39:2700-2703.
serovars. Whenever a Salmonella bearer is being treated with
Kado C.I. & Liu S.T. 1981. Rapid procedure for detection and isolation of
antibiotics and the microorganism develops a multi-resistance,
large and small plasmids. J. Bacteriol. 145:1365-1373.
similar resistance pattern may be found in the patient’s intestinal
Lázaro N.S., Tibana A. & Hofer E. 1997. Salmonella spp. in healthy swine
flora (Aserkoff & Bennett 1969).
and in abbatoir environments in Brazil. J. Food Prot. 60:1029-1033.
The genic transfer phenomenon observed in the samples
Ling J.M., Koo I.C., Kam K.M. & Cheng A.F. 1998. Antimicrobial
which are the object of our study emphasizes the relevance of
susceptibilities and molecular epidemiology of Salmonella enterica serotype
those factors in propagating resistant bacteria in different
Enteritidis strains isolated in Hong Kong from 1986 to 1996. J. Clin.
ecological niches, besides the progressive limitations in the
therapeutics using antimicrobials mostly when the level of
Lírio V.S., Silva E.A. & Stefani S. 1998. Freqüência de 17 sorotipos de
Salmonella isolados de alimentos. Revta Hig. Alimentar 12:36-42.
Madigan M.T., Martinko J.M. & Parker J. 1997. Microbial genetics, p. 305-
356. In: Brock T.D., Madigan M.T., Martinko J.M. & Parker J. (ed.) Biologyof Microrganisms. 8th ed. Prentice-Hall Inc., New Jersey.
Alaniz R.O., Ibarra M.L.R., Barbosa B.T.R. & Morales A.L.J. 1997. Resistencia
Millemann Y., Lesage M.C., Chaslus-Dancla E. & Lafont J.P. 1995. Value of
a antimicrobianos de cepas de Salmonella aisladas de fuentes animales.
plasmid profiling, ribotyping, and detection of IS200 for tracing avian
isolates of Salmonella typhimurium and S. enteritidis. J. Clin. Microbiol.
Asensi M.D. & Hofer E. 1994. Serovars and multiple drug resistant Salmonella
sp. isolated from children in Rio de Janeiro, Brazil. Revta Microbiol.
National Commitee for Clinical Laboratory Standards (NCCLS) 1998.
Performance Standards for Antimicrobial Disk Susceptibility Tests 18:1-
Aserkoff B. & Bennett J.V. 1969. Effect of antibiotic therapy in acute
salmonellosis on the fecal excretion of salmonellae. New England J.
Rivera M.J., Rivera N., Castillo M., Rubio M.C. & Gómes-Lus R. 1991.
Molecular and epidemiological study of Salmonella clinical isolates. J.
Avril J.L., Dabernat H.J., Gerbaud G.R., Horodiniceanu T., Lambert-Zechovsky
N., LeMinor S., Mendez B & Chabbert Y.A. 1977. Groups d’incompatibilité
Sambrook J., Fritsch E.F. & Maniatis T. 1989. Molecular cloning: a laboratory
des plasmides R chez les souches de Salmonella epidémiques. Ann.
manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY,
Barbour S.D. 1967. Effect of nalidixic acid on conjugational transfer and
Sant’Ana Y.X., Cassali G.D., Barbosa A.J.A., Zucchi T.M.A.D. & Chartone-
expression of epissomal Lac genes in Escherichia coli K12. J. Mol. Biol.
Souza E. 1995. Salmonella typhimurium plasmids simultaneously coding
for tetracycline resistance, colicin production and pathogenicity. Revta
Birnboim H.C. & Doly J. 1979. A rapid alkaline extraction procedure for
screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523.
Simmons K.W., Wooley R.E.& Brown J. 1988. Comparison of virulence
Carvalho C.L. & Hofer E. 1989. Antimicrobial resistance among Salmonella
factors and R plasmids of Salmonella spp. isolated from healthy and ill
serovars isolated from different sources in Brazil during 1978-1983.
swine. Appl. Environ. Microbiol. 54:760-767.
Antonie van Leeuwenhoek J. Microbiol. Serology 35:349-359.
Smith H.W. 1969. Transfer of antibiotic resistance from animal and human
Cherubin C.E. 1981. Antibiotic resistance of Salmonella in Europe and the
strains of Escherichia coli to resident E.coli in the alimentary tract of man.
United States. Rev. Infect. Dis. 3:1105-1126.
Costa G.A., Hofer E., Costa M.D.M., Silva J.A.R., Santos J.V. & Doria J.D.
Threlfall E.J. & Frost J.A. 1990. A Review - The identification, typing and
1972. Isolation of Salmonella from pigs lymph nodes slaughtered at the
fingerprinting of Salmonella: laboratory aspects and epidemiological
abattoir of Salvador, Bahia. Mem. Inst. Oswaldo Cruz 70:417-431.
applications. J. Appl. Bacteriol. 68:5-16.
Dias J.C.A.R. & Hofer E. 1985. Bactérias Gram negativas resistentes a
Timoney J.F. 1978. The epidemiology and genetics of antibiotic resistance
antimicrobianos em alimentos. Mem. Inst. Oswaldo Cruz 80:411-421.
of Salmonella typhimurium isolated from diseased animals in New York. J.
Hampton M.D., Threlfall E.J., Frost J.A., Ward L.R. & Rowe B. 1995. Salmonella typhimurium DT 193: differentiation of an epidemic phage
Vinhas S.A. & Almeida D.F. 1984. Plasmid mediated antibiotic resistance
type by antibiogram, plasmid profile, plasmid fingerprint and Salmonella
and colicinogeny among Salmonella in Rio de Janeiro, Brazil. Anais Acad. plasmid virulence (spv) gene probe. J. Appl. Bacteriol. 78:402-408.
Hansen M.S., Rogers M.S., Emge S. & Jacobs N.J. 1964. Incidence of
Zebral A.A., Freitas C.A. & Hofer E. 1974. The occurrence of Salmonella
Salmonella in hog colors as affected by handing pratices prior to slaughter.
in lymphnodes of seemingly normal swine slaughtered at abattoir of
J. Am. Vet. Med. Assoc. 145:139-140.
Santa Cruz, Rio de Janeiro, GB. Mem. Inst. Oswaldo Cruz 62:223-236.
Pesq. Vet. Bras. 24(2):57-60, abr./jun. 2004
Calculating Confidence Intervals for the Number Needed to Treat Ralf Bender, PhD Department of Epidemiology and Medical Statistics, School of Public Health, University of Bielefeld, Bielefeld, Germany ABSTRACT: The number needed to treat (NNT) has gained much attention in the past years as a useful way of reporting the results of randomized controlled trials with a binaryoutcome. Def
Troika Dialog Research Russia Oil and Gas Desknote Onaco asset swap Yesterday, TNK and Sibneft issued a joint statement confirming conversion of the latterís 38% stake in Orenburgneft and 3% stake in Onaco into TNK Intl stock. According to the agreement, Sibneft will receive an 8.6% stake in TNK Intl, together with an option to sell this stake to TNK Intl shareholders