ANTIBIOTIC-RESISTANT ESCHERICHIA COLI CAUSING URINARY-TRACT INFECTION IN GENERAL PRACTICE: RELATION TO FÆCAL FLORA

315 from livestock to man. 6,10,11 Resistant E. coli, appearing in our patients after therapy, was never the original predominant serotype with acquired resistance, neither was there overgrowth of resistant serotypes previously present in small numbers: there was always colonisation of the bowel with a new resistant serotype. The resistant colonisers could have originated in food and indirectly from livestock,12 but serotypes and R factors of E. coli derived from livestock cannot be distinguished from those derived from man. The effect of tetracycline in replacing the original intestinal E. coli with new drug-resistant strains must be viewed in the light of the unexpectedly frequent changes in E. coli serotypes in all our patients. Many studies have shown that predominant serotypes of E. coli in the human intestine normally remain resident for many months or even years. The frequent changes seen in our patients probably resulted from disturbance of the bowel flora by the antibacterial drugs, even in the absence of selection of resistant strains. The other possibility, that patients with urinary symptoms have an unusually unstable intestinal flora, could have been tested had we included an untreated control group, but that we considered unjustified. We thank our colleagues in general practice, especially Dr. C. Hodes, Dr. N. C. Mond, and Dr. A. Spiers for their kind collaboration, and Dr. C. C. Spicer, Medical Research Council Computer Unit, for statistical analysis of the resistance scores. Requests for reprints should be addressed to N. D., Department of Pathology, Royal Postgraduate Medical School, London

W.12. REFERENCES

Lewis, M. J. Lancet, 1968, i, 1389. Salzman, T. C., Klemm, L. Antimicrobial Agents and Chemotherapy—1966; p. 212. New York, 1967. 3. Datta, N. Br. med. J. 1969, ii, 407. 4. Moorhouse, E. C. ibid. p. 405. 5. Brumfitt, W., Faiers, M. C., Reeves, D. S., Datta, N. Lancet, 1971, i, 315. 6. Joint Committee on the Use of Antibiotics in Animal Husbandry and Veterinary Medicine: report. H.M. Stationery Office, 1969. 7. Knothe, H. Dt. med. Wschr, 1963, 88, 1469. 8. Watanabe, T. Bact. Rev. 1963, 27, 87. 9. Datta, N. Br. med. J. 1969, ii, 697. 10. Anderson, E. S. Ann. Rev. Microbiol. 1968, 22, 131. 11. Smith, H. W. Nature, 1968, 218, 728. 12. Shooter, R. A., Cooke, E. M., Rousseau, S. A., Breaden, A. L. Lancet, 1970, ii, 226. 1. 2.

ANTIBIOTIC-RESISTANT ESCHERICHIA COLI CAUSING URINARY-TRACT INFECTION IN GENERAL PRACTICE: RELATION TO FÆCAL FLORA W. BRUMFITT D. S. REEVES

MARY C. FAIERS NAOMI DATTA Edgware General Hospital, Edgware, Middlesex, and Royal Postgraduate Medical School, London W.12

Of thirty-seven women, seen in general practice with Escherichia coli urinary seven infections, (19%) were infected with strains resistant to one or more antibacterial drugs: resistance was R-factor determined in at least six. Carriage of predominantly resistant intestinal E. coli was correlated with resistant urinary infection: in five of the seven patients E. coli in the urine had the same serotype and resistance pattern as that predominating in the Summary

fæces. It did not seem that R+ strains of E. coli, when carried in the intestine, were either more or less likely than R- strains to infect the urinary tract. Introduction

URINARY-TRACT infections in general practice are usually caused by Escherichia coli, and the infecting strains are most commonly derived from the patient’s bowel. E. coli resistant to antibiotics is now common in the normal human intestine. Resistance, either single or multiple, in these organisms is determined by R factors (resistance factors), acquired genetic elements often transmissible from one bacterium to another.2-4 The frequency of R factors in enteropathogenic bacteria, and probably also in the normal bowel flora, has risen steeply during the past decade.5 It is believed that, before 1960, strains of E. coli causing urinary infection in domiciliary practice were nearly always sensitive to antibiotics and sulphonamides, although we have found no contemporary publications to support or refute this view. If the frequency of drug resistance in intestinal E. coli has risen, a corresponding increase in resistance in strains causing urinary-tract infection is likely. The investigation described here was designed to find out, first, how many urinary-tract infections in patients living at home are caused by drug-resistant strains of E. coli, and, second, whether resistant, (R+) strains of E. coli are either more or less likely than sensitive (R-) ones to infect the urinary tract, given their relative prevalence in the patients’ fseces. Patients and Methods Patients with symptoms of urinary-tract infection were immediately by their doctor to us at Edgware General

sent

Hospital. There were fifty-three patients, all female; twenty-nine had a previous history of urinary-tract infection at some time from 2 months to 15 years earlier, and twenty-four had no previous history. Two specimens of urine and one of fxces were taken for culture before treatment was started. Urine culture was quantitative, and infection was diagnosed only if both specimens yielded 105 or more bacterial colonies per ml. urine. Isolates of E. coli were tested for

sensitivity to ampicillin (A), streptomycin (S), tetracycline (T), chloramphenicol (C), kanamycin (K), sulphonamide (sulphafurazole) (Su), nalidixic acid (N), and nitrofurantoin (F) using impregnated discs (Oxoid), with E. coli K12 as the sensitive control. In addition, the minimal inhibitory concentrations (M.L.c.) of all the above drugs, except nitrowere determined for each isolate: serial dilutions of drug, incorporated in plates of solid medium (Oxoid D.S.T. agar with 3% lysed horse blood), were seeded with a standardised bacterial inoculum, yielding approximately 20 colonies on drug-free control plates, and incubated for 18 hours. The M.l.c. was taken as the lowest concentration to prevent the appearance of visible growth. The 0 and H antigens of E. coli isolates were identified as described by Datta et a1.6 Fxcal specimens were examined as described by Datta et a1.6 Using drug selection, resistant bacteria could be detected and isolated when present as 1 per 10,000 of the total yield. Resistant E. coli isolated from urine or faeces were tested for transfer of resistance in mixed culture to E. coli K12.6 If resistance in E. coli cultures from urine failed to transfer, an actively transmissible R factor was introduced into the

furantoin,

316 culture and a further test made to demonstrate mobilisation of the original resistance.7 Patients were allocated at random to one of three thera-

peutic regimens--oral ampicillin, tetracycline,

or

TABLE II-CORRELATION BETWEEN RESISTANT E. COLI IN FACES AND URINE IN DOMICILIARY PRACTICE

sulpha-

dimidine.6The clinical course of the patients was followed, and further specimens of urine and fxces were collected and cultured 4-7 days and 5 weeks after therapy. Results

Of fifty-three patients with symptoms, forty-four had a urinary infection; thirty-seven with E. coli, five with Proteus mirabilis, and two with Staphylococcus albus. Resistant E. coli Of the 37 E. coli strains, 7

(19%) were resistant to drugs (table i). Except with sulphafurazole, M.LC.S for sensitive strains did not differ by one or more

than two-fold from those of the control culture. Resistant strains were 8-128 times more resistant. The M.l.c. of sulphafurazole for the control E. coli

more

TABLE I-RESISTANCE PATTERNS IN E. COLI ISOLATED FROM URINE IN DOMICILIARY PRACTICE

8 jjt-g. per ml. For the 5 strains recorded as resistant it was over 500 g. per ml. There were 2 strains whose M.i.c. for sulphafurazole was more than twice that for the control organism but less than 100 {jLg. per ml.; these were recorded as sensitive. Of the seven patients with resistant urinary strains, three had previous history of urinary symptoms, a proportion similar to that in patients with sensitive strains. was

R Factors 6 of the 7 resistant strains from urine transferred resistance to E. coli K12 either directly or on introduction of an actively transmissible R factor. Relation between Resistant E. coli in Urine and Fc,--cal Flora The thirty-seven patients whose urine yielded E. coli were classified according to the numbers of resistant E. coli in their fasces (table 11) as follows. Where resistant strains were isolated from the selection plate, but not found among 10 separate colonies from a nonselective plate, the ratio of resistant to sensitive E. coli in the fxces was 0-001-9%. One to five resistant colonies among 10 separately picked ones indicated that 10-50% of the fascal E. coli was resistant; six to ten resistant colonies indicated 60-100%. Resistant E. coli was isolated from urine from five of eight patients with 60%or more resistant intestinal E. coli but from only two of twenty-nine patients with a smaller proportion, a significant association (P<0-01). Furthermore, in these five cases the strain from the urine had the same 0 and H antigens as well as the same drug-resistance pattern as E. coli predominating

in the fxces. Where the resistance pattern of E. coli from urine was different from that of the predominant fascal strain, the 0 and H antigens were also different.

Response to Therapy Of forty-four patients with positive urine cultures, thirty-nine had sterile urine 1 week after the end of Lack of response in three of the five failures could be explained by resistance of the infecting organism to the drug prescribed. There were: one infection with Proteus mirabilis (resistant to T) treated with tetracycline; one R+ E. coli resistant to T and Su treated with tetracycline; and one R+ E. coli resistant to A, S, T, and Su, treated with sulphonamide. Although allocation to treatment groups was random, no other patients happened to be treated with a drug unsuitable according to in-vitro sensitivity tests. The two other failures of treatment (one with ampicillin and one with sulphonamide) were unrelated to drug resistance. treatment.

Discussion

Among the group of patients studied, thirty-seven had urine infected with E. coli. 7 E. coli strains (19%) were resistant to one or more antibacterial drugs. Resistance was R-factor determined in at least 6 of the 7 strains. Although only a few patients were investigated, it was clear that those patients whose faecal E. coli was predominantly resistant were most likely to be infected with their own resistant serotype. This was so if they contracted a urinary infection, but there was no evidence that fsecal carriage of resistant E. coli in itself predisposed to urinary infection. Resistant E. coli was isolated with equal frequency from the faeces of patients with infected urine and patients with symptoms but without bacteriuria. The frequency was also equal to that found previously in patients before admission to hospital for non-urgent surgery, a group in whom urinary infection was also absent. It seems that R-factor-determined drug resistance is associated neither with special pathogenicity in the urinary tract, nor with any loss of ability to cause infection. Thus our findings do not disturb the " prevalence " theory which holds that the strains of E. coli responsible for urinary infections in domiciliary practice are those prevalent in the bowel, rather than ones having any special pathogenicity.1 The incidence of drug resistance underlines the importance of accurate sensitivity testing in bacterial infections. Where laboratory facilities are available, resistance is not a serious clinical problem. There remains a choice of therapeutic agents active against the resistant strains. Further spread of R factors in

317

the reservoir of intestinal bacteria, and especially further accumulation of resistance genes to a wider range of antibacterial drugs, could make treatment of acute urinary-tract infections more difficult. Requests for reprints should be addressed to N. D., Department of Pathology, Royal Postgraduate Medical School, London W.12. REFERENCES 1.

Gruneberg, R. N., Leigh, D. A. Brumfitt, W. in Urinary Tract Infection (edited by F. O’Grady and W. Brumfitt) p. 68. London, 1968.

T. Bact. Rev. 1963, 27, 87. Anderson, E. S. Ann. Rev. Microbiol. 1968, 22, 131. Novick, R. P. Bact. Rev. 1969, 33, 210. 5. Datta, N. Br. med. J. 1969, ii, 407. 6. Datta, N., Faiers, M. C., Reeves, D. S., Brumfitt, W., Ørskov, I. Lancet, 1971, i, 312. 7. Anderson, E. S. Nature, 1965, 208, 1016.

2. 3. 4.

N. P. MALLICK J. S. CHOPRA* Department of Medicine, University of Manchester

M. C. STONE

Leigh Infirmary

Serum-lipoprotein patterns were deterby "MNC analysis (membrane filtration, nephelometry, and serum-cholesterol estimation) in 33 patients with nephrotic syndrome, "

mined

of whom were in spontaneous or steroid-induced remission. 17 of the patients had abnormal lipoprotein patterns, in 82% of which increased concentrations of both Sf 0-20 (&bgr;) and Sf 20-400 (pre-&bgr;) lipoproteins were found. This observation is contrasted with the much lower prevalence of this variety of hyperlipoproteinæmia (8.5% of abnormal patterns) previously reported in a randomly selected group from the same geographical area. Because of the and association between hyperlipoproteinæmias ischæmic heart-disease, it is advisable to examine the lipoprotein patterns of all subjects with nephrotic some

syndrome and

to treat

any

significant abnormality.

This course of action is now feasible with the simple and inexpensive form of lipoprotein analysis used in this study. Introduction

AN association between nephrotic syndrome and hyperlipidxmia has been recognised for more than fifty years, but, despite the well-documented association between hyperlipidaemias (hyperlipoprotein-

smias)

and ischsemic heart-disease

(I.H.D.),2-4

an

increased prevalence of I.H.D. in nephrotic syndrome has only recently been described.55

Serum-lipoproteins in nephrotic syndrome were using zone electrophoresis,6analytical ultracentrifugation2 and preparative ultracentrifugation.7 Analytical ultracentrifugation, although it provides a detailed quantitative analysis of the lipoprotein spectrum, is not suitable for general use. Stone and Thorp B,9 have described a simple earlier studied

* Present address: Department of (Haryana), India.

and renal

biopsy.

Subjects Ørskov, F.,

HYPERLIPOPROTEINÆMIAS IN NEPHROTIC SYNDROME

Summary

serum-cholesterol estimation. 9,10° We report here the use of this technique to determine the prevalence of the different types of hyperlipoproteinaemia in 33 patients with nephrotic syndrome and relate our findings to the severity of the disease process judged by serum-albumin concentra-

tion, the degree of proteinuria,

Watanabe,

Clinical Research Unit,

quantitative analysis of Sf 0-20 (j3), Sf 20-400 (pre-P), >400 lipoproteins (chylomicrons) using analysis ", which comprises membrane filtration, nephelometry (light-scattering photometry), and

and Sf " MNC

Medicine, Medical College, Rohtak

We have studied 33 unselected patients with nephrotic syndrome (31 due to glomerulonephritis and 2 due to S.L.E.), of whom 18 were males aged 19-59 years and 16 were females aged 15-73 years. Some of the patients were still nephrotic whilst others were in spontaneous or steroidinduced remission, but all patients had at some time been found to have proteinuria > 3 g. per 24 hours. They were all receiving a high-protein and restricted-sodium diet, with diuretics when required, and 11 patients were also receiving corticosteroid therapy.

Materials and Methods Serum-albumin and Urine Protein Serum-albumin was estimated by the method of Northam et al.11 and urine protein by the method of Varley.12 Individual urine proteins were not estimated.

Lipoprotein Analysis Blood was drawn after a 14-hour overnight fast for the determination of the lipoprotein pattern. The concentrations of the lipoprotein fractions were estimated from the results of MNC analysis. In 30 patients at least two determinations of the lipoprotein pattern were made at twoweekly intervals, but only a single analysis was made in each of the remaining 3 patients. Serum- total-cholesterol (T.C.) was estimated by the method of Connerty et al. 13 The accuracy and reproducibility of the method is described elsewhere.10o Membrane filtration and nephelometry were carried out by the method of Stone and Thorp.8.9 Very-low-density

lipoproteins (V.L.D.L.)

and

chylomicrons

were

separated by

filtration of diluted serum through microporous membrane filters (0,1 µm. pore diameter), and the lipoproteins in each fraction were quantified by measurement of light-scattering intensity in the micronephelometer described by Thorp et al.14 The concentration of three lipoprotein fractionsSf 0-20 (&bgr;-lipoproteins), Sf 20-400 (pre-&bgr;-lipoproteins), and Sf > 400 (chylomicrons)-were calculated from the nephelometric measurements and the value of T.c., using the equations described by Stone et al.,9,10 who have shown a high correlation between these estimated values and those obtained by analytical ultracentrifugation. The concentrations of serum-triglycerides (T.G.) were calculated using the equations described elsewhere.9

Terminology The terminology used to identify the three lipoprotein fractions estimated by MNC analysis is based on differences in the size of the lipoproteins in each fraction. 15 These are called small, medium, and large (S, M, and L) particles and have been shown9 to correspond to &bgr;-lipoproteins (Sf 0-20), pre-&bgr;-lipoproteins (Sf 20-400), and chylomicrons (Sf > 400) respectively. The type of lipoprotein 10 from the concentrations of these pattern is determined three fractions using the cut-off levels defined by Stone et a1.1o from measurements made in 216 healthy young subjects. A " significantly abnormal " lipoprotein pattern