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Copyright © 1973 American Society for Microbiology Printed in U.S.A.
Hospital waste water contains relatively low contain many of these organisms (19-22). Rou-
numbers of pathogenic microorganisms (W. 0. K. tine monitoring of R+ bacteria in sewage may
Grabow and E. M. Nupen, Water Res., in press). serve to evaluate the incidence of R factors in
Such effluents have not been investigated for the general population and to detect changes in
their content of drug-resistant bacteria which the resistance pattern of prevailing R factors (22).
cause increasing problems in antimicrobial This report compares numbers and some
therapy (1, 6). Selection of resistant organisms properties of drug-resistant coliforms in the
can be expected in a hospital environment where sewage of Pietermaritzburg (population, 113,000)
drugs are used on a large scale (4, 8, 13, 24). in South Africa and in the sewage of the Eden-
The known hazard of drug resistance was dale General Hospital (1,650 beds) near that
originally limited. UJntil recently, only bacteria city. The hospital waste water is not discharged
with nontransferable resistance to one or a few into the city sewers. The purpose of the study was
drugs were known (7-9, 27). The currently more to evaluate the load of R+ bacteria in sewage
alarming type of resistance is mediated by R and to determine whether selection for these
factors (extrachromosomal genetic elements) organisms occurs in the hospital environment.
which are transferable by conjugation among This information is important for consideration
Enterobacteriaceae and other gram-negative or- of measures to prevent the spread of R+ bac-
ganisms such as Aeromonas species, Yersinia teria (19-22). Counts of microorganisms in the
pestis, Y. pseudotuberculosM, and Vibrio cholerae above effluents have been determined (Grabow
(16, 25, 27). These factors usually confer re- and Nupen, in press).
sistance to high concentrations of up to eight
drugs simultaneously (27). R factors harbored MATERIALS AND METHODS
by nonpathogenic intestinal bacteria such as Sewage samples. Hospital sewage and city
Eswherichia coli can be transferred to drug- sewage were sampled simultaneously at about
susceptible pathogens such as Salmonella or 3-week intervals from January to June 1972 (a
Shigella (10, 27). Bacteria which carry R factors total of nine samples from each source) by the
(R+ bacteria) are present in large numbers of methods of Grabow and Nupen (in pre8s).
patients (1, 4, 6, 14) and healthy individuals Isolation of resistant bacteria. Saline
(17, 22, 29). R+ coliform bacteria have been iso- (0.85%) dilutions of sewage were plated on Mac-
lated from rivers (19) and coastal bathing waters Conkey agar (Difco) to obtain the total coliform
(20) polluted with sewage plant effluents which count and on plates of the same medium contain-
175
176 GRABOW AND PROZESKY ANTIMICROB. AG. CHEMOTHER.
ing one drug (Table 1) to isolate resistant coli- RESULTS
forms (19, 22). Unless stated otherwise, incuba-
tion was at 37 C. In experiments with sulfon- Total coliform counts and numbers of drug-
amide, MacConkey agar was replaced by a me- resistant coliforms in hospital and city sewage
dium modified to overcome interference from are listed in Table 1. These results include both
inhibitors (28). This medium consisted of Mueller- transferable and nontransferable resistance be-
Hinton medium (Difco) which contained the cause the concentrations of drugs were low (13).
selective ingredients of MacConkey agar. Counts Counts of coliforms resistant to Bactrim (25
were done in duplicate.
Transfer of resistance. Nalidixic acid-resist- .ug of trimethoprim plus 125 ug of sulfamethoxa-
ant mutants of an F- fecal E. coli strain, E25, and a zole per ml), gentamicin (10 ug/ml), nalidixic
Salmonella typhi strain, N, were used as recipients. acid (25 ug/ml), and nitrofurantoin (200 ,ug/ml)
The former was kindly provided by D. R. Woods never exceeded 0.1 % of the total coliform count.
(29), and the latter was isolated from a patient Table 1 shows that the hospital waste water
with typhoid fever. Overnight broth cultures of a contained a higher percentage of coliforms re-
drug-resistant coliform (0.1 ml) and a recipient sistant to each drug except cephaloridine. Differ-
(1 ml) were mixed in 10 ml of Nutrient Broth No. ences between hospital and city sewage were
2 (Oxoid) for conjugation. After overnight incu- greatest for coliforms resistant to kanamycin,
bation, 0.1 ml of each cross was plated on Mac- neomycin, or tetracycline. In both hospital and
Conkey agar which contained nalidixic acid (30
pg/ml) and the drug (concentrations in Table 2) city sewage, resistance was mainly to chlor-
to which the potential donor was resistant. amphenicol, streptomycin, or sulfonamide.
recipient ability. Resistance markers were oc- sistance to the 5 drugs concerned. The slightly
casionally lost during R factor transfer from lower number in city sewage is due to its low
coliforms to S. typhi N. content of coliforms with nontransferable re-
Calculations based on results in Tables 1 and sistance to streptomycin or tetracycline.
2 show that 26% of all coliform bacteria in the The most frequent patterns of resistance trans-
hospital discharge carried R factor resistance to ferred by R+ organisms to E. coli E25 appear in
one or more of five drugs (Table 3). This figure Table 4. The resistance spectrum of coliforms
was 4% for city sewage. In both effluents, the with nontransferable resistance was not in-
counts of R+ coliforms resistant to chlorampheni- vestigated. Table 4 shows that in both effluents
col were the highest and those resistant to R factors which confer multiple resistance to
tetracycline were the lowest. The index of R+/R-
resistant organisms was, however, the same for ampicillin, chloramphenicol, streptomycin, sul-
both drugs. Table 3 also shows that hospital fonamide, and tetracycline were encountered
and city sewage did not differ significantly in most frequently. In hospital sewage, an average
counts of coliforms with nontransferable re- 49% of R+ coliforms transmitted this pattern
TABLE 2. Number of coliform bacteria with transferable drug resistance in hospital and city sewage
Hospitala city"
TABLE 5. Incidenice of fecal Escherichia coli among drug-resistant coliform bacteria isolated
from hospital and city sewage
Hospital City
No. No. of fecal No. No. of fecal No. No. of fecal No. No. of fecal
tested E. coli tested E. coli tested E. coli tested E. coli
Ampicillin .50 8 (16%) 56 24 (43%) 86 4 (5%) 16 9 (56%)
Chloramphenicol 43 11 (26%) 53 17 (32%) 53 20 (38%) 27 20 (74%)
Streptomycin. 73 13 (18%) 26 10 (38%) 96 9 (9%) 9 1 (11%)
Sulfonamide . 80 10 (13%) 29 9 (31%) 95 9 (9%) 14 0 (0%/)
Tetracycline . 35 5 (14%) 63 17 (27%) 71 24 (34%) 39 20 (51%)
Average .56 9 (17%) 45 15 (34%) 80 13 (19%) 21 10 (38%)
VoL. 3, 1973 DRUG-RESISTANT COLIFORMS IN SEWAGE 179
drugs (7). Since polluted water plays a major 5. Davies, J. E., and R. Rownd. 1972. Transmissible
role in the transmission of microorganisms (20, multiple drug resistance in Enterobacteriaceae.
Science 176:758-768.
23), improved purification of sewage should re- 6. Editorial. 1966. Infectious drug resistance. N. Engl.
ceive special attention. J. Med. 275:277.
In contrast to the notable difference in num- 7. Finland, M., W. F. Jones, and M. W. Barnes.
bers of R+ organisms, the counts of coliforms 1959. Occurrence of serious bacterial infectione
since introduction of antibacterial agents. J.
with nontransferable resistance did not differ Amer. Med. Ass. 170:2188-2197.
significantly in hospital and city sewage (Table 8. Gill, F. A., and E. W. Hook. 1965. Changing pat-
3). This may explain the exceptionally low counts terns of bacterial resistance to antimicrobial
of cephaloridine-resistant coliforms in the hos- drugs. Amer. J. Med. 39:780-795.
9. Hayes, W. 1968. The genetics of bacteria and their
pital discharge, because it is the only drug among viruses, 2nd ed. Blackwell Scientific Publications,
those in Table 1 for which transferable resistance Oxford.
has not been detected (5). The inability of some 10. Kasuya, M. 1964. Transfer of drug resistance be-
tween enteric bacteria induced in the mouse
coliforms to transfer resistance to S. typhi and intestine. J. Bacteriol. 88:322-328.
segregation of determinants during transfer 11. Manten, A., P. A. M. Guinee, E. H. Kampelmacher,
(Table 2; 19, 20, 27) may explain the low in- and C. E. Voogd. 1971. An eleven-year study of
cidence of R+ S. typhi cells resistant to chlor- drug resistance in Salmonella in the Netherlands.
amphenicol (11, 12). R+ bacteria in the hospital Bull. W. H. 0. 45:85-93.
12. Mare, I. J. 1967. Drug resistance in Salmonella
discharge were resistant to a broader spectrum typhosa. S. Afr. Med. J. 41:703-704.
of drugs than those in city sewage (Table 4). 13. Mar6, I. J. 1968. Incidence of R factors among
This shows that exposure to drugs not only se- gram negative bacteria in drug-free human and