Trimethoprim R plasmids isolated daring long-term treatment of urinary tract infection with co-trimoxazole

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1 Journal of Antimicrobial Chemotherapy (1979) 5, Trimethoprim R plasmids isolated daring long-term treatment of urinary tract infection with co-trimoxazole K. J. Towner, N. J. Pearson, W. R. Cattell* and F.O'Grady Department of Microbiology, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, England *St. Bartholomew's Hospital, London EC1, England Thirty out-patients treated for chronic urinary tract infection with long-term lowdosage co-trimoxazole over a period of five years were monitored for the emergence of trimethoprim resistance. Plasmids conferring high-level resistance to trimethoprim were isolated from 5-1 % of the isolates examined (five patients) and belonged to the P incompatibility group and the I incompatibility complex. The W group plasmid previously isolated from several London hospitals was not detected. Significant (8 tig/ml or more) resistance to trimethoprim, presumably resulting from chromosomal mutation, was detected in a further 16-7% of strains examined (12 patients). These strains did not require thymine for growth and may represent an important resistance mechanism in patients treated with long-term co-trimoxazole. Introduction Persistent or recurrent bacteriuria, even in patients with gross urological abnormalities, has been controlled with considerable success for a number of years using long-term low-dosage treatment with trimethoprim-sulfamethoxazole (TMP-SMZ) used under the generic name co-trimoxazole. Such treatment might be expected to favour the emergence of resistant organisms, particularly by means of the spread of resistance plasmids, but over a considerable period of time little trouble was encountered (O'Grady, Kelsey-Fry, McSherry & Cattell, 1973). Plasmids conferring resistance to trimethoprim and sulphonamides were first identified in bacteria causing infections at three London hospitals in 1971 (Fleming, Datta & Griineberg, 1972). All were members of the W incompatibility group and came from the Camden area (Datta & Hedges, 1972). In 1972, Escherichia coli strains carrying trimethoprim R plasmids were isolated from calves that had been fed large doses of a trirnethoprim-sulphonamide preparation for experimental purposes (Fleming, 1973). These plasmids were assigned to the I incompatibility complex rather than group W and the resistance pattern they conferred was to trimethoprim and streptomycin, but not to sulphonamides (Hedges, Datta & Fleming, 1972). A further study by Jobanputra & Datta (1974) described three different classes of trimethoprim R plasmids belonging to incompatibility groups I, P and W. These authors suggested that plasmids conferring /79/145+8 $1./ 1979 The British Society for Antimicrobial Chemotherapy Downloaded from at Pennsylvania State University on May 1, 216

2 46 K. J. Towner, N. J. Pearson, W. R. Cartel] and F. O'Grady resistance to trimethoprim were distributed in the normal intestinal flora of the community, but that their frequency of occurrence was low. Further evidence for the continuing diversity of trimethoprim R plasmids has been provided by recent studies in France and Italy (Acar, Goldstein, Gerbaud & Chabbert, 1977; Romero & Perduca, 1977) which described plasmids belonging to a number of incompatibility groups unlike those so far found in England. This paper reports the results of part of a study monitoring the emergence of resistance to trimethoprim amongst out-patients treated with long-term low-dose co-trimoxazole at St. Bartholomew's Hospital, London. Materials and methods Bacterial strains, plasmids and bacteriophage Standard E. coli K12 (Clowes & Hayes, 1968) strains used were J53.2 (F~, pro, met, rif), J62 (F~, pro, his, trp, lac) and AB2575 (Hfr, ilv). The strain of Acinetobacter calcoaceticus EBF65/65 (Towner & Vivian, 1977) used was C4121 (trp, his, rip). Plasmids of known incompatibility groups were obtained from Drs N. Datta and R. W. Hedges (Royal Postgraduate Medical School, London) and are listed in Table I. Bacteriophage used was the F-specific phage MS2 (Davis, Strauss & Sinsheimer, 1961). Trimethoprim-resistant bacteria Bacteria isolated from introital swabs, urine and faeces samples from 3 patients being treated for chronic urinary tract infection with long-term low-dosage TMP-SMZ were identified by standard laboratory techniques and their resistance to trimethoprim measured using multipoint inoculation onto agar plates containing doubling dilutions of the drug. Trimethoprim-resistant strains were tested for their ability to grow on lysed blood agar and minimal salts agar (Clowes & Hayes, 1968) supplemented with glucose, but without any added thymine since strains of naturally occurring resistant bacteria from patients treated with trimethoprim are sometimes found to be thymine-requiring (Maskell, Okubadejo, Payne & Pead, 1978). Transfer of trimethoprim resistance Overnight broth cultures of strains under test were mixed in equal proportions with E. coli K12 J53.2 and incubated in static conditions for 18 h at 37 C. They were then spread (-1 ml/plate) on Oxoid DST agar containing 4% lysed horse blood plus 8 ug/ml trimethoprim lactate (Burroughs Wellcome) and 1 ug/ml rifampicin (Lepetit). Neither donor nor recipient strains would grow on this medium. Colonies that developed were therefore E. coli K12 J53.2 which had received a plasmid conferring resistance to trimethoprim. This was confirmed after purifying the colonies by (i) checking that they required proline and methionine for growth by streaking on suitably supplemented minimal salts agar and (ii) by showing that trimethoprim resistance was transferable from the first K12 recipient to a second strain of K12, J62. In the cases where transfer of trimethoprim resistance was confirmed, single colonies were tested using antibiotic discs to determine whether other resistances had been co-transferred. Where transfer of several resistances had occurred, the experiment was repeated selecting for the transfer of each resistance independently. Transconjugants of each type were then tested to see whether the resistances were transferable separately (i.e. on different plasmids) or always together (i.e. on the same plasmid). Downloaded from at Pennsylvania State University on May 1, 216

3 Trimethoprim R plasmids 47 Classification of plasmids Transferable plasmids isolated in this study were classified as follows: (i) By fi character as described by Coetzee, Datta & Hedges (1972) using strain AB2575 (Hfr) and MS2 phage. (ii) By incompatibility. Plasmids were tested for their ability to transfer to and to co-exist stably in E. coli K12 containing the R plasmids of known incompatibility groups listed in Table I using standard methods (Coetzee, Datta & Hedges, 1972; Jobanputra & Datta, 1974). Where resistance markers of both donor and recipient cultures were identified in the same clone after transfer, the 'double' was tested as described by Coetzee et al. (1972) to see whether the two plasmids were transferable separately or always together. Table I. Standard plasmids belonging to known incompatibility groups Plasmid* RA1 R4a R386 Rl-16 R124 R27 R144 R621a R391 R387 R831 R446b N3 R724 RP4 R478 Rtsl S-a PICM Incompatibility group A C F, F,, F,v H I. IT J K L M N O P S T W Y Resistance determinantst TcSu ApKmSu Tc Ap Km Sm Su Cm Tc Tc TcKm Tc Km SmCm SmKm SmTc Tc Sm Su Tc Cm Sm Su ApTcKm TcKm Cm Km Km Cm Sm Su Cm *for plasmid references see Coetzee, Datta & Hedges (1972); Hedges (1975). fap, ampicfllin; Cm, chloramphenicol; Km, kanamycin; Sm, streptomycin; Su, sulphonamides; Tc, tetracycline. Downloaded from at Pennsylvania State University on May 1, 216 Results Trimethoprim-resistant strains During the study 2877 strains were examined, but a large number of these were either obtained by isolating several colonies from the same clinical sample or were considered to be the same strain recovered from serial samples from a patient over long periods of time. In order to avoid bias, Table II therefore shows only the identification and sensitivities of those strains which were considered to be different. All of these strains were able to grow both on lysed blood agar and minimal salts agar with added glucose; therefore resistance to trimethoprim was not the result of a requirement for thymine (Maskell, Okubajedo, Payne & Pead, 1978).

4 48 K. J. Towner, N. J. Pearson, W. R. CatteO and F. O'Grady Strains resistant to 8 ug or more trimethoprim/ml were tested for their ability to transfer this resistance to E. coli K12. Of the 43 different resistant strains in this group, 1 were found to transfer resistance. These strains were isolated from five of the 3 patients (Table HI). In each case the MIC transferred was > 124 ug trimethoprim/ml. Resistance to > 256 ug sulfamethoxazole/ml was also transferred, either on the same plasmid or on a separate co-transferable plasmid. In the cases where an identical resistance pattern was transferred from a number of strains isolated from the same patient, it was assumed in this study that only one plasmid was involved. Table IL Distribution of trimethoprim resistance in strains examined Identification E.coli Klebsiella sp. P. mirabilis A. calcoaceticus Citrobacter sp. Enterobacter sp. H.ahei Flavobacterium sp. Total Number of strains with MIC (tig/ml) < > Table IIL Strains with transferable trimethoprim resistance Patient A B C D E Strain identification 2 x E.coli 2 X E.coli 2 x Citrobacter sp. I x E.coli 2 X Citrobacter sp. lx E.coli Abbreviations as for Table I. Resistances transferred TpTcCm TpSu TpSu TpSu Tp Sm Cm Su Plasmid designation puni pun9 pun25 pun26 pun38 Classification of plasmids The five plasmids listed in Table HI were tested for their fi character. pun38 wasj?+; the remainder were found to be fi~. They were then tested for incompatibility with the plasmids of known incompatibility groups listed in Table I. Two major groups were identified amongst the five plasmids studied. (a) puni. This plasmid was capable of normal stable co-existence with members of all incompatibility groups tested with the exception of group P. Table IV shows the frequency of transfer of puni into a strain already containing the standard group P plasmid RP4. Only a slight reduction in the frequency of puni transfer occurred when the recipient strain contained RP4, but in the majority of cases RP4 was found to have been eliminated from puni recipients. In contrast, the reciprocal crosses, in which Downloaded from at Pennsylvania State University on May 1, 216

5 Trimethoprim R plasmids 49 Table IV. Incompatibility properties of plasmids isolated Transfer to Frequency/donor Characters of purified Plasmid host carrying cell transcipients punl punl RP4 RP4 RP4 punl PUN9* pun9 R144 R144 RJ44 pun9 3x1-' lxlo"' 3x1"' 1x1"' 1x1"* 2x1"' 1x1"' 5x1"' Results shown are the mean results from several experiments. Similar results were obtained for pun9, pun25, pun26 and pun38. 18/2 punl only 2/2 both present but unstable 4/2 pun9 only. 16/2 both present but unstable 2/2 both present but unstable transfer of RP4 was attempted into a strain already containing punl, showed a larger reduction in the frequency of RP4 transfer (in some experiments no RP4 transfer at all was observed), but no elimination of punl by RP4 was detected. In the cases where transfer occurred, but no elimination of the resident plasmid was observed, the 'doubles' isolated were tested for the continued stability of both plasmids. In every case, punl was stably maintained and transferred at a normal frequency (3 X 1~ 3 /donor cell) whereas RP4 was unstable and was only transferred from 'doubles' at a greatly reduced frequency (5x 1" '/donor cell). Further evidence that punl belonged to the P incompatibility group was provided by the demonstration that punl had the wide host range characteristic of P group plasmids (Olsen & Shipley, 1973). This was shown by transferring punl to A. calcoaceticus EBF65/65. Group P plasmids are the only group of plasmids which have so far been shown to be transferable from E. coli to A. calcoaceticus (Towner & Vivian, 1977; unpublished results). (b) pun9, pun25, pun26, pun38. These four plasmids were capable of normal stable co-existence with members of all incompatibility groups tested with the exception of those belonging to the I incompatibility complex. Table IV shows the frequency of transfer of pun9 into strains already containing the standard la group plasmid R144. A slight reduction in the transfer frequency of pun9 was obtained when R144 was present in the recipient strain, but, in contrast, a much larger reduction in the transfer frequency of R144 was observed for the reciprocal cross. Elimination of pun9 was never observed as a result of the acquisition of R144, but some elimination of R144 by pun9 was detected. Similar results were obtained for pun25, pun26 and pun38. Further evidence that the plasmids isolated belonged to the la group was provided by the finding that the transfer frequency of pun9 (or pun25, pun26, pun38) from 'doubles' with R144 was slightly reduced (2x 1" 4 /donor cell) while a much larger reduction was observed in the transfer frequency of R144 (1 x 1" '/donor cell). This contrasts with the behaviour of these plasmids with the Iy group plasmid R621a where, although occasional elimination of R621a was observed, no reduction in the transfer frequencies of the plasmids from 'doubles' was detected. Downloaded from at Pennsylvania State University on May 1, 216

6 5 K. J. Towner, N. J. Pearson, W. R. CatteU and F. O'Grady pun9, pun25 and pun26 conferred the same resistances and may be identical. However pun38 was different from these, conferring not only different resistances, but also a different./? character. The majority offi + plasmids belong to the F incompatibility complex, but other examples of fi + I group plasmids have been noted (Datta, 1975). pun38 was also observed to cause some elimination of the group O plasmid R724. This behaviour resembles that of th& fi + I group plasmid R8O5a (Falkow, Guerry, Hedges & Datta, 1974). O group plasmids share about 2% of their sequences (roughly 1 x 1* daltons) in common with typical I group plasmids and are considered to comprise part of the I incompatibility complex (Falkow, Guerry, Hedges & Datta, 1974). However, no reduction in the transfer frequencies of pun38 and R724 from 'doubles' was observed. Further inter-relationships between the plasmids described here and other known members of the I incompatibility complex are currently being investigated. Discussion The 3 patients studied had all been treated as out-patients for periods of up tofiveyears with long-term co-trimoxazole therapy. However, even under these highly selective conditions, only 5-1% of the isolates examined (5/3 patients) were found to carry transferable trimethoprim resistance. In two of these patients a plasmid was found only in a single stool isolate, whereas in the other three patients, apparently identical plasmids were demonstrated in isolates from both urinary tract and stool specimens. It would nevertheless appear that the frequency of trimethoprim R plasmids in nature is still fairly low. One plasmid belonging to incompatibility group P was found, but the majority of plasmids isolated in this study were assigned to the I incompatibility complex. This complex is not well-defined at present. Typical la group plasmids such as R144 are so strongly mutually incompatible that it is not possible to isolate strains carrying two such plasmids (Hedges & Datta, 1973). Others (e.g. R483 originally assigned to a separate group Ip") are capable of co-existing through many generations with typical la plasmids (Datta & Barth, 1976). The I plasmids in this study seemed to belong to this second group and it was somethimes difficult to demonstrate clearly incompatibility between them. Other plasmids conferring resistance to trimethoprim which belong to the I and P groups have been reported (Jobanputra & Datta, 1974). However, the plasmids described in this paper differ from those reported previously on the basis of the range of antibiotics to which they confer resistance, although it is quite possible that they may all be interrelated. Alternatively, genes determining trimethoprimresistancemay have been acquired by different plasmids of the same incompatibility group as separate events. In this context it is worth noting that pun38 may carry the transposon (Tn7) conferring resistance to trimethoprim and streptomycin which has been found on several plasmids isolated in South-east England (Barth & Datta, 1977). Of particular interest was the non-reciprocal exclusion and elimination behaviour of the plasmids isolated. This provides some further examples of the 'hierarchy' relationships recorded previously between different plasmids of the same incompatibility group (Datta & Barth, 1976). It was noted that the types of plasmids isolated did not include the W incompatibility group plasmid previously shown to be carried by Klebsiella sp. in several London hospitals (Jobanputra & Datta, 1974). There is no evidence that this plasmid has spread Downloaded from at Pennsylvania State University on May 1, 216

7 Trimetboprim R plasmlds 51 to out-patients attending St. Bartholomew's Hospital and no Klebsiellaisola.tes conferring resistance to > 124 (ig trimethoprim/ml were observed in this study. This may indicate that the W group plasmid is confined to an internal hospital environment and is not widely distributed amongst the general population. In contrast, trimethoprim R plasmids belonging to the I incompatibility complex seem to be more widely distributed and have been previously isolated from a number of different sources (Hedges et al., 1972; Jobanputra & Datta, 1974; Barth & Datta, 1977). A significant number of strains (16-7% 12/3 patients) were found which had a level of non-transferable trimethoprim resistance of 8 ug/ml or more. This resistance presumably resulted from chromosomal mutation, but by a mechanism different from that leading to a thymine reqiurement. None of the strains investigated in this study were found to require thymine, although such strains may not have been originally isolated owing to their failure to grow on the media normally employed for routine isolation. This chromosomal mechanism of resistance may prove to be of particular importance in patients treated with trimethoprim for long periods. The clinical course of the patients from whom resistant strains were isolated will be described in detail elsewhere, but episodes of urinary tract infection with trimethoprim resistant organisms were treated, usually for two weeks, with an agent to which the organism was sensitive. This agent was given in addition to co-trimoxazole and, following elimination of the resistant strain, long-term co-trimoxazole therapy was successfully continued. Acknowledgement We are indebted to Miss Anne McSherry for detailed information on the history of these patients. References Acar, J. F., Goldstein, F. W., Gerbaud, G. R. & Chabbert, Y. A. Plasmides de resistance au trim6thoprime: transferabilit6 et groupes d'incompatibilirt. Annales de Plnstitut Pasteur 128A: 41-7 (1977). Barth, P. T. & Datta, N. Two naturally occurring transposons indistinguishable from Tn7. Journal of General Microbiology 12: (1977). Clowes, R. & Hayes, W. Experiments in microbial genetics. Blackwell, Oxford and Edinburgh (1968). Coetzee, J. N., Datta, N. & Hedges, R. W. R factors from Proteus rettgeri. Journal of General Microbiology 72: (1972). Datta, N. Microbiology American Society for Microbiology, Washington, D.C. (1975), pp Datta, N. & Barth, P. T. Compatibility properties of R483, a member of the I plasmid complex. Journal of Bacteriology 125: (1976). Datta, N. & Hedges, R. W. Trimethoprim resistance conferred by W plasmids in Enterobacteriaceae. Journal of General Microbiology 72: (1972). Davis, J. E., Strauss, J. H. & Sinsheimer, R. L. Bacteriophage MS2: another RNA phage. Science 134: 1427 (1961). Falkow, S., Guerry, P., Hedges, R. W. & Datta, N. Polynucleotide sequence relationships among plasmids of the I compatibility complex. Journal of General Microbiology 85: (1974). Fleming, M. P. Trimethoprim resistance and its transferability in Escherichia coli isolated from calves treated with trimethoprim-sulphadiazine: a two year study. Journal of Hygiene 71: (1973). Fleming, M. P., Datta, N. & Grflneberg, R. N. Trimethoprim resistance determined by R factors. British Medical Journal i: (1972). Downloaded from at Pennsylvania State University on May 1, 216

8 52 K. J. TowDer, N. J. Pearson, W. R. CatteO and F. O'Grady Hedges, R. W. R factors from Proteus mirabilis and P. vulgaris. Journal of General Microbiology 87: (1975). Hedges, R. W. & Datta, N. Plasmids determining I pili constitute a compatibility complex. Journal of General Microbiology Tl: (1973). Hedges, R. W., Datta, N. & Fleming, M. P. R factors conferring resistance to trimethoprim but not sulphonamides. Journal of General Microbiology 73: (1972). Jobanputra, R. S. & Datta, N. Trimethoprim R factors in enterobacteria from clinical specimens. Journal of Medical Microbiology 7: (1974). MaskeLl, R., Okubadejo, O. A., Payne, R. H. & Pead, L. Human infections with thyminerequiring bacteria. Journal of Medical Microbiology 11: (1978). O'Grady, F., Kelsey-Fry, I., McSherry, A. & Cattell, W. R. Long-term treatment of persistent or recurrent urinary tract infection with trimethoprim-sulphamethoxazole. Journal of Infectious Diseases 128: S (1973). Olsen, R. H. & Shipley, P. Host range and properties of the Pseudomonas aeruginosa R factor R1822. Journal of Bacteriology 113: (1973). Romero, E. & Perduca, M. Compatibility groups of R factors for trimethoprim resistance isolated in Italy. Journal of Antimicrobial Chemotherapy 3 (Suppl. C): (1977). Towner, K. J. & Vivian, A. Plasmids capable of transfer and chromosome mobilization in Acinetobacter calcoaceticus. Journal of General Microbiology 11: (1977). {Manuscript accepted 8 August 1978) Downloaded from at Pennsylvania State University on May 1, 216