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
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Vet-240.p65Pesq. 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.
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(1977) have shown that the same resistance pattern is expressed Izumiya H., Terajima J., Matsushita S., Tamura K. & Watanabe H. 2001.
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