A case of typhoid fever caused by Salmonella typhi occurred in Geneva. The patient was probably infected in Mexico City. The strain isolated from this patient corresponds with the description of the Mexican S. typhi strain, since it is a degraded Vi-strain resistant to chloramphenicol, streptomycin, sulphonamides and tetracyclines. It carried an fi− transferable R factor with a CSSuT resistance pattern. It can be accepted that this case forms part of the Mexican outbreak of chloramphenicol-resistant typhoid fever which has already been observed in visitors to Mexico from England and the United State

Pitton, J. S.

Waldvogel, F. A.

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J. Hyg., Camb. (1973), 71, 509Printed in Great Britain509Typhoid fever imported from Mexico to Switzerland. Studieson R factor mediated chloramphenicol resistanceBY F. A. WALDVOGELInfectious Disease Unit, Department of Medicine, CantonalHospital, GenevaAND J. S. PITTONUniversity Institute of Medical Microbiology, University of Geneva, Geneva{Received 11 January 1973)SUMMARYA case of typhoid fever caused by Salmonella typhi occurred in Geneva. Thepatient was probably infected in Mexico City. The strain isolated from this patientcorresponds with the description of the Mexican S. typhi strain, since it is a de-graded Vi-strain resistant to chloramphenicol, streptomycin, sulphonamides andtetracyclines. It carried an fi~ transferable R factor with a CSSuT resistancepattern. It can be accepted that this case forms part of the Mexican outbreak ofchloramphenicol-resistant typhoid fever which has already been observed invisitors to Mexico from England and the United States.INTRODUCTIONThe fact that drug resistance can be transferred between enterobacterial strainsby conjugation has important epidemiological and clinical implications. Althoughresistance transfer apparently occurs less frequently in vivo than in vitro, it hasbeen demonstrated in man and animals (reviews: Watanabe, 1963; Anderson,1968). Clinical isolates capable of transferring multiple drug resistance to otherstrains during mixed cultivation have been described in epidemic shigellosis(Ochiai, Yamanaka, Kimura & Sawada, 1959), in urinary tract infections (Smith &Armour, 1966), in nosocomial infections (Gardner & Smith, 1969) and in Salmonellainfections (Anderson & Lewis, 1965a, b; Gill & Hook, 1966).Recent reports from Mexico have described a protracted outbreak of typhoidfever caused by a strain of Salmonella typhi resistant to chloramphenicol becauseit carries a transferable resistance factor (W.H.O. Weekly Epidemiological Record,1972). The recent account by Anderson & Smith (1972) of typhoid infection in twoBritish visitors to Mexico, and the occurrence of a similar case at our hospital ina patient returning from Mexico City, prompted us to investigate the resistancepattern of the strain of S. typhi isolated from this case and to document itsidentity with the Mexican strain.https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0022172400046490Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 15:54:56, subject to the Cambridge Core terms of use, available at510 F. A. WALDVOGEL AND J. S. PITTONCASE REPORTA 22-year-old girl was admitted to the Hospital on 6 June 1972 with a 6 days'history of fever, cough, nausea and vomiting. She had been perfectly well until6 weeks before admission, when she visited South and Central America. While inMexico City, 3 weeks before her admission to hospital in Switzerland, she sufferednausea, vomiting and diarrhoea of 24 hr. duration, for which she received a shortcourse of chloramphenicol. The symptoms abated until her return to Switzerland.Six days before admission she developed fever up to 40° C, anorexia, nausea andvomiting, followed by an unproductive cough.On admission the physical signs included a temperature of 40° C, a heart rateof 88/min., a short atypical systolic murmur and a slightly tender abdomen with-out peritoneal signs.1 Routine laboratory findings included a hematocrit reading of 45%, a whiteblood cell count of 4000/mm.3 with a slight shift to the left, a blood urea nitrogenof 14 mg./lOO ml., normal electrolytes and normal liver function tests. All bloodcultures and two stool cultures taken on the first 2 days after admission yieldedS. typhi resistant to chloramphenicol but sensitive to ampicillin by the standarddisk method.Treatment was instituted on the 3rd hospital day with 4 g. of ampicillin bymouth, and the patient recovered. Antibiotic treatment was continued for 3 weeks.The TO and TH agglutinin titres were 1/400 and 1/200 respectively on admission;they rose to 1/800 and 1/400 after 1 week and had returned to 1/100 and 1/501 month later. However, no significant Vi agglutinin titre was detected during orafter the first treatment period.Because of intermittently positive stool cultures for S. typhi during the 2 weeksafter completion of the antibiotic treatment, a second 6 weeks' course of ampicillinwas given. Careful monitoring of the stools showed all specimens tested to benegative for 8. typhi; they have remained negative since then.LABORATORY STUDIESBacteriology. The resistance pattern of 9 isolates of 8. typhi (7 from stools and2 from blood cultures) was studied, using the standard disk diffusion method. Allwere resistant to chloramphenicol and belonged to a degraded Vi-strain.Minimal inhibitory concentrations {MIC). Small inocula (0-1 ml. of an overnightbroth culture diluted 10~3) were added to 5 ml. of broth (Bacto Penassay Broth,Difco) containing serial dilutions of drugs. The MIC values chosen were the lowestdrug concentrations which inhibited visible growth after overnight incubation at37° C.Transfer of drug resistance. The nine chloramphenicol-resistant isolates wereexamined for the presence of R factors by the methods of Anderson & Lewis(1965a, b), using Escherichia coli K12F+, K12F~ and K12HfrH as recipient strains.Drug-resistant recipient colonies from these mating mixtures were isolated onplates containing the appropriate antibiotic, purified on MacConkey Agar (Difco),and their full resistance pattern was determined by a disk-diffusion method. Thehttps:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0022172400046490Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 15:54:56, subject to the Cambridge Core terms of use, available atTyphoid fever imported from Mexico to Switzerland 511fi (fertility inhibition) character was examined by the method described by Pitton& Anderson (1970), using phages fi2 and R17 (male-specific) and <j>2 (female-specific). A second series of crosses was carried out, using resistant E. coli K12 asdonor strains and S. typhi phage type A and S. typhimurium phage type 36 asrecipient strains.Mechanisms of resistance. The acetylating ability for chloramphenicol was de-termined by the method of Piffaretti (Piffaretti & Pitton, 1970).Stability of the plasmid in its original strain of S. typhi. This was determined bythe replica plating technique (Lederberg & Lederberg, 1952).RESULTSAntibiotic sensitivity testsThe nine isolates were resistant to chloramphenicol (C), streptomycin (S),sulfafurazole (Su) and tetracyclines (T) but sensitive to penicillins and cephalo-sporins as well as to kanamycin, gentamicin, neomycin and colomycin by thestandard disk method. The MIC's of antibiotics for the wild-type strains were120/tg./ml. for chloramphenicol, more than 200/<g./ml. for streptomycin, 100/ig./ml. for tetracycline, 3 /^g./ml. for neomycin and gentamicin, and 6 /ig./ml. forkanamycin. The MIC's for chloramphenicol were 160/tg./ml. in the K12 lines,100/ig./ml. in S. typhi A, and 160/tg./ml. in S. typhimurium.Transfer of drug resistanceThe nine primary isolates transferred chloramphenicol resistance to E. coli K12at a frequency of about 10~4. All the K12 colonies selected on chloramphenicolshowed the stable resistance pattern CSSuT. In the second series of crosses, allresistances were transferred]at the same frequency, about 10~5, into the two standardsalmonellas, 8. typhi Vi-type A and 8. typhimurium type 36 (Anderson, 1966).This CSSuT resistance factor is ft~ in K12HFrH lines when tested with the fi2 andR17 phages. In K12F+, however, the R factor displaces the F factor, giving rise toF~ cells. It thus shows one-sided incompatibility with F, since it is stable in, andseems not to affect the fertility of, Hfr strains, where the F factor is integrated intothe chromosome. The same properties have been observed independently in this iR factor by E. S. Anderson & H. R. Smith and by D. H. Smith (personal communica-tions). This factor belongs to the 'H group' (Anderson & Smith, 1972; Grindley,Grindley & Anderson, 1972). It does not modify the sensitivity of K12F~ linestowards the <fi2 phage or produce phage restriction in 8. typhi or S. typhimurium.All the strains studied (the nine wild-type 8. typhi, the K12F+ and F~ deriva-tives, and the 8. typhi and 8. typhimurium progeny obtained in the second crosses)acetylated chloramphenicol.The CSSuT complex is probably not very stable in the wild-type strains, sincethe frequency of spontaneous loss of the resistance was about 3 x 10~3 for C and10-3 for T.https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0022172400046490Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 15:54:56, subject to the Cambridge Core terms of use, available at512 F. A. WALDVOGEL AND J. S. PITTONDISCUSSIONAll cultures of the chloramphenical-resistant strain of S. typhi isolated fromthis case have identical properties. They all react with the Vi-typing phages asdegraded Vi-strains; and they carry the same fi~ transferable R factor with aCSSuT resistance pattern. These results, as well as the clinical and epidemiologicalhistory of our patient, establish the identity of these isolates with those implicatedin the outbreak of typhoid fever in Mexico (CDC Weekly Report, 1972a, b). SinceAnderson & Smith (1972) have recently studied two C-resistant S. typhi culturesinvolved in this epidemic, it was of interest to determine whether our studieswould show that the strain isolated in Switzerland was genetically indistinguishablefrom that found in Britain. No differences appeared: the Swiss strain belongs tothe same Vi-type and carries the same R factor as those isolated in England.These findings suggest that the Mexican epidemic of typhoid fever was causedby a single strain of S. typhi carrying the same R factor throughout. This has theresistance pattern CSSuT, belongs to group H, and displaces the F factor when thelatter is in the extrachromosomal state.An interesting finding in this case is the relatively high rate of spontaneous lossof C and T resistance in our S. typhi. This may explain the puzzling fact that oneof the stool cultures performed after completion of the first course of antibiotictreatment yielded drug-sensitive S. typhi. This sensitive line had the same charac-teristics - phage type, biochemical and serological properties - as the resistantisolates.We are indebted to Professor B. Courvoisier for allowing us to study this clinicalcase and to Dr Marie Sitavanc for isolation and microbiological identification ofour strains. The expert technical assistance of Miss Janine Corlet is gratefullyacknowledged. Our thanks are also due to Dr E. S. Anderson for his help in thegenetic study of this R factor and in preparing this report.REFERENCESANDERSON, E. S. (1966). Influence of the A transfer factor on the phage sensitivity of salmon -ellae. Nature, London 212, 795-9.ANDERSON, E. S. (1968). The ecology of transferable drug resistance in the enterobacteria.Annual Review of Microbiology 22, 131-80.ANDERSON, E. S. & LEWIS, M. J. (1965a). Drug resistance and its transfer in Salmonellatyphimurium. Nature, London 206, 579-83.ANDERSON, E. S. & LEWIS, M. J. (19656). Characterization of a transfer factor associatedwith drug resistance in Salmonella typhimurium. Nature, London 208, 843-9.ANDERSON, E. S. & SMITH, H. R. (1972). Chloramphenieol resistance in the typhoid bacillus.British Medical Journal iii, 329-31.CENTER FOR DISEASE CONTROL WEEKLY REPORT (1972a). Typhoid fever - Mexico. Morbidityand Mortality 21, 177-8.CENTER FOR DISEASE CONTROL WEEKLY REPORT (19726). Typhoid fever contracted inMexico. Morbidity and Mortality 21, 193-4.GARDNER, P. & SMITH, D. H. (1969). Studies on the epidemiology of resistance (R) factors.Annals of Internal Medicine 71, 1-9.GILL, F. A. & HOOK, E. W. (1966). Salmonella strains with transferable antimicrobial resist-ance. Journal of the American Medical Association 198, 1267-9.https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0022172400046490Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 15:54:56, subject to the Cambridge Core terms of use, available atTyphoid fever imported from Mexico to Switzerland 513GRINDLEY, N. D. F., GRINDLEY, J . N. & ANDERSON, E. S. (1972). R factor compatibilitygroups. Molecular and General Genetics 119, 287-97.LEDBRBERG, J . & LEDERBERG, E. M. (1952). Replica plating and indirect selection of bacterialmutants. Journal of Bacteriology 63, 399-406.OCHIAI, K., YAMANAKA, T., KIMTJRA, K. & SAWADA, O. (1959). Studies on inheritance of drugresistance between Shigella strains and Escherichia coli strains. (In Japanese.) Nippon IjiShimpo 1861, 34-46.PIFFARETTI, J . C. & PITTON, J . S. (1970). Chloramphenicol acetylation in whole cells ofEscherichia coli carrying R-factors characterization and kinetic studies. Chemotherapy 15,84-98.PITTON, J . S. & ANDERSON, E. S. (1970). The inhibitory action of transfer factors on lysisof Escherichia coli K12 by phages fi2 and 02- Genetical Research 16, 215-24.SMITH, D. H. & ARMOUR, S. E. (1966). Transferable R factors in enteric bacteria causinginfection of the genitourinary tract. Lancet ii, 15-18.WATANABE, T. (1963). Infective heredity of multiple drug resistance in bacteria. Bacterio-logical Reviews 27, 87-115.WORLD HEALTH ORGANIZATION WEEKLY EPIDEMIOLOGICAL RECOBD (1972). Typhoid fever.47, 268-9.https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0022172400046490Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 30 May 2017 at 15:54:56, subject to the Cambridge Core terms of use, available at

Typhoid fever imported from Mexico to Switzerland. Studies on R factor mediated chloramphenicol resistance

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