An eleven-year study of drug-resistance in Salmonella in the Netherlands*

Bull. Org. mond. Sante 11971, 45, 85-93 Bull. Wld Hlth Org. j An Eleven-Year Study of Drug Resistance in Salmonella in the Netherlands* A. MANTEN 1, P. A. M. GUINItE 2, E. H. KAMPELMACHER3 & C. E. VOOGD4 From 1959 to 1969 a total of 123 070 strains of salmonellae, representing nearly all the strains that have been isolated from animals and man, were collected in the Netherlands and tested for antibiotic resistance. In the course of the study, only a few strains of S. typhi and S. paratyphi B were found to be resistant to chloramphenicol, ampicillin, or tetracycline. From 1959 to 1966 there was a sharp increase in the prevalence oftetracycline resistance in both the human and the animal strains of S. typhimurium and S. panama; subsequently, however, the prevalence declined. During 1966-70 approximately one-third of the tetra- cycline-resistant human and animal strains of S. typhimurium were also resistant to ampicillin. This type of multiple resistance was only occasionally encountered in the other serotypes. In contrast to resistance to tetracycline and ampicillin, the incidence of S. typhimurium resistant to chloramphenicol remained low during the whole period of observation. In all other serotypes drug resistance remained at a low level. It is shown in this study that the greatly increased use of broad-spectrum antibiotics for medical and veterinary purposes and for animal feeding over the last decade in the Nether- lands has not led to the development of serious drug resistance from the medical point of view. An important factor involved in the low incidence of resistance to chloram- phenicol may be that R-factors in salmonellae usually lose this resistance determinant rapidly. In the Netherlands the annual mortality figures for all forms ofsalmonellosis decreased from 0.84 (per 100 000 inhabitants) in 1961 to 0.25 in 1969. Over the last two decades in most of the econo- mically developed countries, tens to hundreds of tons of antibiotics have been used annually for medical and veterinary purposes, and in foodstuffs and food preservatives. One of the hazards involved in this practice is that it may lead to the development of resistance in medically important bacteria. The dis- covery of extrachromosomal elements carrying drug resistance in bacteria has added a new dimension to the whole problem. In the Netherlands, the potential hazards for public health of antibiotic-resistant salmonellae have * From the National Institute of Public Health, Utrecht, The Netherlands. 1 Head, Laboratory of Chemotherapy. 2 Head, National Salmonella Centre. Head, Laboratory for Zoonoses. 'Research Officer, Laboratory of Chemotherapy. been recognized since 1955, and in 1958 a programme was started to screen all the strains of Salmonella isolated in that country for resistance to tetracycline and chloramphenicol. After 1966, resistance to ampicillin was also tested. It was relatively easy to establish the programme on a nation-wide basis, since it has always been the practice to send all Salmonella strains isolated to the National Institute of Public Health, Utrecht, for typing. The majority of the strains received by the Nation- al Salmonella Centre of our Institute are from human patients or from animals such as cattle, pigs, and poultry. A fairly large proportion (20-30 %) are, however, from other sources, such as laboratory and wild animals, slaughterhouses, meat-, bone-, and fish-meal, egg products, and sewage. The latter strains will not be dealt with in this paper. A more or less complete account of all the strains examined 2706 - 85 A. MANTEN AND OTHERS between 1959 and 1968 is to be found in a series of publications by Manten et al. (1961a, 1961b, 1962, 1964, 1966) and by Voogd et al. (1968, 1970). DEFINITION OF DRUG RESISTANCE AND METHOD OF ESTABLISHING RESISTANCE PATTERNS In vitro the genus Salmonella is usually fairly uniformly susceptible to tetracycline, chlorampheni- col, and ampicillin. In our laboratory, the vast majority of species and strains of drug-sensitive bacteria, when cultivated on antibiotic-supplemented slants of 5% sheep blood agar (pH 7.2-7.4), had minimum inhibitory concentration (MIC) values varying from 2 to 5 [kg of tetracycline per ml, 5 to 10 ,ug of chloramphenicol per ml, and 2 to 5 ,ug of ampicillin per ml. To detect resistant strains each strain was transfer- red to agar slants containing 25 jg of tetracycline per ml, 50 jug chloramphenicol per ml, or 25 ,ug of ampicillin per ml, amounts roughly corresponding to 10 times the normal MICs for Salmonella. In order to exclude any possibility of contamination with resistant bacteria of other species, the bacteria grown on one or more of the slants were identified again. If the same Salmonella serotype was found, the degree of resistance was determined by tube- dilution assays on 5% sheep blood agar slants using dilute suspensions of the organism in saline as inoculum. If the MIC at retesting appeared to be at least 10 times the normal value the strain was regarded as resistant. RESULTS During the period of observation (1959-69) alto- gether 123 070 strains of Salmonella were tested. They may be grouped into those of human origin (76 337 strains, or 62.0%), those obtained from slaughtered animals and poultry (28 936 strains, or 23.5 %), and those from various other sources (17 797 strains, or 14.5 %). The latter group will not be dealt with in any detail, since their relation to human health is generally more remote than that of the human and animal strains. Among the human strains, S. typhimurium has been the prevailing serotype over the whole period of observation. The second most frequently en- countered type was S. panama. These two serotypes have consequently been studied in great detail, as have S. typhi and S. paratyphi B, since the latter are the most dangerous serotypes in human salmonel- losis. The animal strains were isolated from cattle, pigs, and poultry. In the strains obtained from cattle, S. dublin was always the most prevalent type. In these animals S. typhimurium was also frequently found. The latter organism was predominant in pigs and poultry. Salmonella typhi and Salmonella paratyphi B Over the whole period of observation only a few strains of S. typhi and S. paratyphi B were found to be resistant to any of the three antibiotics examined. It is shown in Table 1 that of the 610 strains of S. typhi examined only 3 (0.5%) were found to be resistant to chloramphenicol, the main drug used in the treatment of typhoid fever. Two of these strains were also resistant to tetracycline. Ampicillin Table 1. S. typhi and S. paratyphi B: resistance to tetracycline (T), chloramphenicol (C), and ampicillin (A) during 1959-69 1No. of No. of strains resistant to: Organism strains examined T C A T+C T+A C+A T+C+A S. typhi 610 7 1 0 2 1 0 0 S. paratyphi B 1 295 15 3 1 15 3 0 0 resistance was encountered in only 1 strain, and resistance to tetracycline in 10 strains. Among the 1 295 isolates of S. paratyphi B, 18 (1.4o%) were resistant to chloramphenicol. Most of them (15) were also resistant to tetracycline. The chloram- phenicol-resistant strains were found mainly in 1959, a year in which the incidence of human infec- tions was also relatively high. In the 10 years after 1959, only 4 chloramphenicol-resistant strains were found. The number of tetracycline- and ampicillin- resistant strains was also low between 1959 and 1969. Salmonella typhimurium The patterns of drug resistance among strains of S. typhimurium varied in type. The strains that occurred most frequently were those resistant to tetracycline alone or to tetracycline and ampicillin. This was true of both the human and the animal strains. In contrast, chloramphenicol-resistant strains were found in only relatively small numbers. Because of an outbreak of salmonellosis in calves in 1968, 86 DRUG RESISTANCE IN SALMONELLA IN THE NETHERLANDS IlI Il ¢OO t uz ) S _ ~~~~~nuz I 0 0 0 0 0 0 CD .D .- 0 C. .D .D CD .' C. 0 0 D 9 o - 0 0 _ LO I- 0) ) 0 LO N N 0O C' _ N N ) C N _ n C LO - o- 0 r- CD CO C 0 tC C0 0 It w ) ) rC ` C CDC' C C 0) 0 0 N D 0 C- o o o o .': (I I C N CD t N N CD 0 N CD _- C _ _6000 0 0 0o _ o o O o 1111111~~~~~~~~~~~~~~~~~~~~~~~~~ CV-CO* 7 r'.. CD o DC DC CD CD C' _ 'a- N C') 1~ s 0 CD) £ V- D N - tD C') 0 CO 0 _- - N N C' C) C' N _- ) - C - D C) 0 _ CD 0 CD D _ C N D - C' * 0) 0 CD * N 0 C C 0) C 0 _ ) C' C t C' N N C C C' caused by a strain resistant to both chloramphenicol and tetracycline, the figure for that year is excep- tional. Strains resistant to tetracycline, chloram- phenicol, and ampicillin were also found, but usually only in small numbers (Table 2). From 1959 to 1963 there was a continuous increase in the number of tetracycline-resistant strains of S. typhimurium among those of both human and animal origin (see Fig. 1). In 1966, 40.4% and 32.2% of human and animal strains, respectively, of S. typhmurium were tetracycline-resistant. After this the proportions of tetracycline-resistant strains showed large fluctuations. Fig. 1 also shows that changes in the proportions of human and animal strains of S. typhimurium resistant to tetracycline follow a parallel course. This similarity probably demonstrates the close epidemiological relationship of animal and human strains of the serotype in question. In contrast to the findings with tetracycline, the incidence of chloramphenicol resistance remained very low in the human strains over the whole period of the survey. In 1963, it reached a peak of 1.62% and in 1969 it was no more than 0.42 %. The frequency of ampicillin resistance, however, was at first rather high (22.7% in 1966), but it subsequently decreased (Fig. 2.) More or less the same trends occurred in the strains of S. typhimurium isolated from animals. It is interesting that resistance to ampicillin in the animal strains varied in a similar way to tetracycline re- sistance, although ampicillin had not been used in animal medicines or in animal foodstuffs in the Netherlands at that time. Salmonella panama The total number of strains of S. panama, as well as the number of tetracycline-resistant strains, in- creased considerably between 1963 and 1965. In 1964 and 1965, the incidence of this type of resistance among the human strains reached levels of 42.6% and 43.0 %, respectively (Table 3). Maximum tetracycline resistance appeared later than the peak in the number of strains isolated from human patients. The incidence of ampicillin resistance in S. panama has always been low, in contrast to the position with S. typhimurium between 1966 and 1969. The incidence of chloramphenicol-resistance also re- mained low. Other serotypes A third serotype found among the human strains was S. stanley. Especially in the period from 1961 to 87 + + + 0 + H C) CC ..L 0 c m a, DC ._ c0._- 0M0 0) co _X 4) p (L 4o 0 a) 6 c- E Z Zn XX 0cc Cc 0n E ._ Cco C E I cn c C- cn CE C co cD E 0) E m . a) E CD 2 CD CD C._ E co 0 iD C._ CD CD 0 CD cn C.) cn coCt cD .) cD 0D cD 0O+ + 0 + H 0 0 I1) co -6 cD a. 'a O C C 6 m- E 0 X a) CD Dc 0 - N ) C CD C- 0) DCD CD CD CD CD CD CD CD CD CD CD _ DC DC DC DC DC DC DC DC DC DC I1- ~ -11 A. MANTEN AND OTHERS 20 m 10 CHLORAMPHENICOL 1959 '60 '61 '62 '63 '64 '65 '66 '67 '68 1969 Fig. 1. Increase in proportion of tetracycline-resistant strains of Salmonella typhi- murium between 1959 and 1969. 40 .c30 -HUMAN STRAINS 20- ANIMAL STRAINS 10 1959 '60 '61 '62 '63 '64 '65 '66 '67 '68 1969 Fig. 2. Resistance to tetracycline, ampicillin, and chloramphenicol among human strains of Salmonella typhimurium between 1959 and 1969. 1964, it caused many disease outbreaks in the Netherlands. This organism occurred only spora- dically in animals. Of the 76 337 human strains typed in the National Salmonella Centre during 1959-69, 7 685 (1 %) were S. stanley. Only 218 of them (2.9 Y.) were found to be resistant to any of the drugs used in this investigation. In the Netherlands, and also in other countries, S. dublin is the main cause of salmonellosis in cattle. This serotype also occasionally causes infection in other animals and in man. Only 268, or 2.6%, of the total number of 10 342 strains found, were resistant to the antibiotics tested. It is clear from the above figures that S. stanley and S. dublin did not readily become resistant. In this respect they appear to differ from S. typhimu- rium and S. panama. Table 4 shows that the many other serotypes obtained from man and animals also showed only a low tendency to become resistant to antibiotics. They fall in the same group as S. stanley and S. dublin. 88 DRUG RESISTANCE IN SALMONELLA IN THE NETHERLANDS 1o o o 0 00 00 '-0 0 I I o 00 0 00 C) 0 0 0 0 0 CD 0)0)co N N _ 0 0 0 0 0 0 N 0 0 0) 0 6 0 0 6 0 0 N co N 0 6 0 6 0 0 0 6 0 6 0 C' 0 N CD N C 0 C' _ 0) C') c-I r~- U) C' I- C' r- C' v-w I I nC o " I I O O a 0. 0. UCo. LN U)ND'-- 0 O N 'q O N N N meq it I N eq N .4 o N _- _N C'- 0) r, LO CO CY) 0 '- N X U) CD (D 0) 0) 0) 0) v- V- - - - r- CY) CD 0) U) CD - N 0) CD C- C- 0 (D (D CD CD (D0) 0 0) 0 v- v- t- U) co CO E C 0C E E co ECL CL 0 E Co CD -o ) ._ -5 0 C) 4- 0.. Co C.) 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MANTEN AND OTHERS Of the total of 25 278 strains of human origin col- lected in the period from 1959 to 1969, 625 (2.5%) were resistant to tetracycline and 129 (0.51 %) were resistant to chloramphenicol; 109 (1.7%) of 6 345 strains were resistant to ampicillin. Of 18 755 animal strains the corresponding figures were 547 (2.9%) for tetracycline and 24 (0.13%) for chlor- amphenicol; 80 (0.97%) of 8 256 strains were resistant to ampicillin. DISCUSSION Salmonellosis in man is usually a self-limiting disease that is confined to the intestines, though infants and elderly people may succumb during the acute phase of the infection. S. typhimurium is the prevailing species incriminated in this type of enteritis, but a great many other serotypes may be involved as well. For example, in the Netherlands S. panama and S. stanley have been responsible for many disease outbreaks in the human population during the past decade. Among the salmonellae, S. typhi and S. paratyphi B stand out because of their high pathogenicity and host-specificity to man. In typhoid and para- typhoid fever the disease agents enter the intestines, and then penetrate their walls, leading to generalized systemic infections. Occasionally, other Salmonella types may also enter the blood and tissues and cause serious disease, but in healthy adults this occurs but rarely. In all cases of generalized systemic salmonellosis in man, chloramphenicol is the therapeutic agent of first choice (Woodward et al., 1952; Geddes, 1964; Sleet et al., 1964; Dawking & Hornick, 1967). Its high activity in typhoid fever and similar diseases is probably due to its excellent penetration into tissues and cells harbouring the bacteria (Showacre et al., 1961). A second important antibiotic is ampicillin, which up to now has been the only agent suitable for the treatment of human carriers of Salmonella (Christie, 1964; Whitby, 1964; Muinnich et al., 1964; Simon & Miller, 1966). The tetracyclines, notwith- standing their high in vitro activity on salmonellae, are practically devoid of any therapeutic action in typhoid fever as well as in salmonelloses of all other kinds. The same applies to streptomycin, gentami- cin, paromomycin, the polymyxins, nalidixic acid,' and the sulfonamides (Woodward et al., 1952; 1 1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridine-3- carboxylic acid. Dawkins & Hornick, 1967). Treatment of human salmonellosis of the self-limiting type with antibiotics, as has recently been done in an epidemic caused by S. typhimurium, may lead to prolongation of the carrier state and thus increase the opportunity of spread of infection and of resistant bacteria (Aser- koff & Bennett, 1969). Consequently, the treatment of Salmonella enteritis with antibiotics, including chloramphenicol and ampicillin, in previously healthy subjects appears, in general, not to be justified. Seen in this light, the sharp increase of tetracycline resistance in S. typhimurium and S. panama observed from 1960 to 1966 in the Netherlands, as well as similar findings in other countries (Ramsey & Edwards, 1961; McWorther et al., 1963; Yurack, 1964; Anderson & Datta. 1965; Chabbert & Baudens, 1966; Lebek, 1967; Hofmann et al., 1967; Schroeder et al., 1968), would not appear to be of much significance for public health. It is obvious, however, that the situation with respect to ampicillin resist- ance and especially chloramphenicol resistance must be further analysed. The incidence of ampicillin resistance in the human strains of S. typhimurium (the only serotype in which resistance to this antibiotic appeared frequently) dropped from 22.7% in 1966 to 4.6% in 1968 and then rose to 9.7% in 1969. It is improbable that these fairly large variations were due to differences in the annual therapeutic use of ampicillin in the Netherlands. As shown in Fig. 2, the changes in resistance to tetracycline and to ampicillin from year to year run more or less parallel, since a fairly constant propor- tion of the tetracycline-resistant strains was also resistant to ampicillin. The same was true for the animal strains of S. typhimurium (Table 2). The use of tetracyclines in human and veterinary medicine has probably led to the emergence of R-factors carrying determinants for resistance to both tetra- cycline and ampicillin, and the use of tetracyclinos in animal husbandry may have contributed to such an effect. It is certain that the relatively high incidence of ampicillin resistance in the human strains of S. typhi- murium observed in some years cannot be associated with the use of ampicillin in veterinary medicine or as a supplement in animal foodstuffs, since up to 1970 in the Netherlands it had not been used in these fields. The tetracyclines, on the other hand, have found wide application both in veterinary medicine and, in amounts usually not exceeding 10 ppm, in DRUG RESISTANCE IN SALMONELLA IN THE NETHERLANDS animal foodstuffs. It has been found in animals that oral administration of the tetracyclines, even in small quantities, may lead to a multiplication of R-factors in Escherichia coli (Smith & Halls, 1966; Walton, 1966; Bulling et al., 1968). Furthermore, R-factors with determinants for more than one drug may become prominent in this way and, under certain conditions, be transferred en bloc to other Enterobacteriaceae, including S. typhimurium. It may be presumed, therefore, that the use of tetra- cycline in animal foodstuffs has also contributed to the incidence of ampicillin resistance in the latter organism, though the part it has played is probably small. It has been said already that chloramphenicol is the only reliable drug for the treatment of typhoid and paratyphoid fevers. Resistance of the causative agents of these diseases to chloramphenicol would create a major problem in treatment. As shown in Table 1, chloramphenicol resistance was found only rarely in S. typhi and in S. paratyphi B between 1960 and 1969. In the former pathogen, it occurred in only 3 strains of a total of 610. In 1959, during disease outbreaks due to S. paratyphi B, 14 (3.5 °/) of 401 strains were resistant to chloramphenicol. In the following 10 years, only 4 chloramphenicol-resistant strains (out of a total of 894) were found. Drug resistance in typhoid and paratyphoid fever has thus not really been a problem in the Netherlands so far. Also in the majority of strains of S. typhitnurium and the other serotypes isolated, the incidence of resistance to chloramphenicol was low and remained so throughout the period of observation. In 1963, among the human strains of S. typhimurium, the incidence of chloramphenicol resistance was at its highest level of 1.62 %, whereas in 1969 it was no more than 0.42 %. The figures for other salmonellae were similar. Chloramphenicol is not used as a supplement in animal foodstuffs. However, the drug is fairly extensively used in veterinary practice. The question therefore arises as to why the incidence of resistance to chloramphenicol among animal and human strains of salmonellae remained at such a low level, at least in the Netherlands, whereas the figures for tetracycline resistance increased so considerably in both groups of strains. At least two factors were involved. From 1959 to 1969 the use of the tetra- cyclines in both human and veterinary medicine increased in the Netherlands, and following a partial raising of the governmental regulations on the addition of antibiotics to animal foodstuffs in 1959, the use of oxytetracycline and chlortetracycline also increased. Although the exact figures for the annual national consumption of chloramphenicol and the tetracyclines are not known to us, there is no doubt that the former antibiotic has always been used on a much smaller scale than the tetra- cyclines. A more important factor involved in the low incidence of chloramphenicol resistance is the spontaneous loss of this type of resistance which frequently occurs in Salmonella. In contrast, tetra- cycline resistance is retained much longer. The phenomenon underlying the disappearance of re- sistance in Enterobacteriaceae is the segregation of resistance determinants in R-factors. In species of Salmonella the rate of this segregation has been found to be usually high (and especially so in chlor- amphenicol resistance) as compared with that in E. coli, for instance (Lebek, 1963; Watanabe et al., 1964). If the use of antibiotics for non-medical purposes were to lead to a significant increase in the rate of mortality from human salmonellosis, this would be a strong argument against such use. Table 5 shows the annual rates of mortality from all forms of salmonellosis, including typhoid and paratyphoid Table 5. Annual mortality from salmonellosis (all forms) in the Netherlands, 1959-69 ' Year 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 Mortality per 100 000 inhabitants 0.63 0.41 0.84 0.60 0.39 0.32 0.28 0.35 0.30 0.32 0.25 ' Data kindly supplied by Dr H. Bijkerk, Inspector of Commu- nicable Diseases, the Netherlands. - 91 92 A. MANTEN AND OTHERS fever, during the period 1959-69. In the Nether- lands, the annual mortality rate decreased from 0.84 (per 100000 inhabitants) in 1961 to 0.25 in 1969. It appears from the above analysis that the veterinary and non-medical use of antibiotics in the Netherlands, has not so far led to any significant emergence of drug resistance in Salmonella (including S. typhi and S. paratyphi B). RESUME J-ThDE, FOURSUlVIE PENDANT 11 ANNtES, DE LA PHARMACORtSISTANCE DE SALMONELLA, AUX PAYS-BAS De 1959 a 1969, on a recherche la resistance aux antibiotiques de 123 070 souches de Salmonella, soit la quasi-totalite des souches isol6es chez 1'homme et chez I'animal aux Pays-Bas. Durant cette periode, seules quelques rares souches (respectivement 0,5 et 1,4%) de S. typhi et de S. para- typhi B ont fait preuve d'une resistance au chloram- phenicol. Le nombre de ces serotypes resistants a I'ampicilline et a la tetracycline etait egalement tres faible. De 1959 ai 1966, la proportion des souches, d'origine humaine ou animale, de S. typhimurium (le s6rotype le plus fr&quemment rencontre aux Pays-Bas) et de S. panama resistantes a la tetracycline s'est fortement accrue. En ce qui regarde les souches humaines de S. typhimurium, elle est passee de 2,2% en 1959 a 40,4% en 1966. Elle s'est cependant abaissee par la suite. Pendant les quatre dernieres annees de 1'etude, un tiers environ des souches de S. typhimurium, humaines et animales, resistantes a la tetracycline 1'etaient egalement a l'ampicilline. Ce type de r6sistance multiple n'a ete trouve qu'occasionnellement chez les autres serotypes. Contrastant avec la resistance 'a la tetracycline et a I'ampicilline, la resistance de S. typhimurium au chlo- ramphenicol ne s'est manifestee que chez un petit nombre de souches: 1,62 % des souches humaines en 1963, 0,42% en 1969. Chez tous les autres serotypes, la phar- macoresistance est restee peu frequente. Le chloramph6nicol est le medicament le plus utilise pour traiter les salmonelloses. Il ressort de la presente etude que l'usage croissant des antibiotiques a large spectre en medecine humaine et veterinaire, en elevage et en agriculture aux Pays-Bas pendant la demiere decennie n'a pas entrainW l'apparition d'une pharma- coresistance genante du point de vue therapeutique. Aux Pays-Bas, le taux annuel de la morbidite par salmonellose (toutes formes) a decru de 0,84 pour 100 000 en 1961 a 0,25 pour 100 000 en 1969. REFERENCES Anderson, E. S. & Datta, N. (1965), Lancet, 1, 407 Aserkoff, B. & Bennett, J. V. (1969) N. Engl. J. Med., 281, 636 Bulling, E., Scholz, M. & Malla, D. S. (1968) Berl. Munch. tierdrztl. Wschr., 81, 151 Chabbert, Y. A. & Baudens, J. G. (1966) In: Hobby, G. L., ed., Antimicrobial agents and chemotherapy-1965, Ann Arbor, Michigan, American Society for Micro- biology Christie, A. B. (1964) In: Brumfitt, W. & Williams, J. 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