Coliforms Isolated from Water Samples

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1983, p /83/ $02.00/0 Copyright C 1983, American Society for Microbiology Vol. 45, No. 1 Antibiotic Resistance Among Different Species of Fecal Coliforms Isolated from Water Samples MAARIT NIEMI,1 MERVI SIBAKOV,2 AND SEPPO NIEMELA3* National Board of Waters, SF Helsinki, 10,1 National Public Health Institute, SF Helsinki 28,2 and Helsinki University Department of Microbiology, SF Helsinki, 71,3 Finland Received 27 January 1982/Accepted 8 September 1982 The distribution of resistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin among fecal coliforms in sewage, surface waters, and sea water was investigated. The incidence of resistant strains among isolates varied significantly among the water samples, without obvious connection with the water source or the level of pollution. The average frequency of multiple resistance was not always high in the same samples in which the overall resistance was high. The species composition varied considerably in different water samples. A significant correlation was observed between the relative frequency of Klebsiella species and the incidence of ampicillin resistance in water samples. The importance of species composition of fecal coliforms, affected by their source and by the aquatic environment, on the resistance pattern is noted. During recent years, the distribution of antibiotic-resistant strains of Enterobacteriaceae in the aquatic environment has been studied in different parts of the world. For example, river and sewage waters in South Africa (9, 10), surface waters, sea water, and shellfish in New Zealand (3, 4), sewage in Canada (1), and surface waters, sea water, and sediments in the United States (8, 12, 13) have been investigated for the presence of antibiotic-resistant Enterobacteriaceae. The majority of the studies focused on transferable drug resistance because of its practical importance. Species are identified, if at all, only after strains have been found drug resistant. In one study (2), coliforms were identified, but the effect of species composition was not fully discussed. In this study, the distribution of resistance to antimicrobial drugs among fecal coliforms in sewage, surface waters, and sea water was investigated without differentiating transferable and nontransferable resistance, but paying attention to the effect of the species composition of the sample on the incidence of resistance and resistance patterns. MATERIALS AND METHODS Water samples. Duplicate water samples were taken in sterile glass bottles. Samples were transported to the laboratory within a few hours after sampling. Samples were taken from sewage, rivers, lakes, and the Baltic Sea (Table 1). Determination of fecal coliforms. The colony counts shown in Table 2 were made by the membrane filtration technique, counting the typical blue colonies on mfc medium incubated for 1 day at 44 C. The original composition of the medium (7) was modified by adding agar (13 g liter- 1) and substituting water blue (Gurr) at a concentration 0.25 g liter-' for aniline blue. Identification of bacteria. Our intention was to isolate equal numbers (15 to 20 colonies) of bacteria from each of four parallel plates. Starting from a selected point, all colonies were isolated until the desired number was counted. For some samples, this was not achieved due to small colony numbers in some plates. All colonies were isolated from plates with fewer than 15 colonies. On isolation, the colonies were characterized as typical (totally blue), intermediate (partly blue or blue mixed with other hues), or atypical (nonblue). The strains were purified by streaking on standard plate count agar and identified by using API 20E kits (Analytab Products, Inc.). Antibiotic sensitivity tests. To test antibiotic sensitivity, the routine method of the Finnish Central Public Health Laboratory (a modification of the ICS agar diffusion method [5]) was used. The isolates were purified by spreading on Mueller-Hinton agar. Ten 1- day-old colonies were picked up into saline solution. One drop (0.03 ml) of this suspension was transferred into 10 ml of saline with a Pasteur pipette. This dilution was spread over Mueller-Hinton agar plates (ca. 14 cm), excess liquid was removed, and after 30 min of absorption time at 37 C, the disks (Neo-Sensitabs; Rosco) were transferred onto the plates. The plates were incubated at 37 C overnight. The inhibition zones were measured, and the average of duplicate plates was used to estimate the sensitivity according to the manufacturer's instructions. The strains were coded as resistant or sensitive, with intermediate strains being included in the resistant class. RESULTS A total of 812 strains were identified from 14 water samples with different concentrations of 79

2 80 NIEMI, SIBAKOV, AND NIEMELA TABLE 1. Sources of water samples Sample Site Date of Type of the sample no. sampling (1979) 1 Suomenoja treatment plant 7 June Untreated domestic sewage 2 Viikki treatment plant 19 June Untreated domestic sewage 3 Viikki treatment plant 25 April Treated domestic sewage 4 Viikki treatment plant 8 August Treated domestic sewage 5 Rinnekoti asylum 5 September Chlorinated domestic sewage 6 Saarioinen Ltd. 18 April from food industry 7 Viikki experimental farm 27 June from cow stables 8 River Vantaanjoki 2 May Polluted with domestic, industrial, and 9 River Vantaanjoki 25 July agricultural wastes 10 River Risubacka a 12 June Polluted with domestic and industrial wastes 11 Lake Enajarvi 17 July Polluted with domestic sewage 12 Lake Hiidenvesi 17 July Contaminated from non-point sources 13 Katajaluoto Sea area 3 April Polluted brackish water in the vicinity of 14 Katajaluoto Sea area 22 May Helsinki City APPL. ENVIRON. MICROBIOL. fecal coliforms (Table 2). A total of 645 strains of the isolates (80%) produced typical colonies on mfc agar, 76 strains (9%) were intermediate, and 90 strains (11%) were atypical. Resistance to one or more of the four antibiotics or to sulfonamides was detected in 29% of the strains that produced typical colonies, in 28% of the strains that produced intermediate colonies, and in 48% of the strains that produced atypical colonies. The atypical strains would normally not be counted in the standard water analysis. Since, however, most of them (71%) proved to be Enterobacteriaceae and their fractions in different samples were not significantly different, we decided to include them in the analysis of antibiotic resistance. Furthermore, there is unpublished evidence that lactose-positive coliforms may produce atypical colonies appearing lactose negative under the harsh conditions of the mfc procedure. The high proportion of resistant strains (48%) in this group is chiefly due to Klebsiella pneumoniae and Enterobacter cloacae strains with a high frequency of resistance (over 80%). The incidence of resistant strains varied significantly (X2 = 59.8; df = 13; P < 0.1%) in the TABLE 2. Incidence of resistance and multiple resistance among bacteria isolated from different water samples Source and Fecal coliforms No. No. resistant No. multiply sample no. per 100 ml isolated (%)a resistant (%)b x (49) 14 (20) x (51) 10 (15) x (30) 2 (3) x (24) 3 (5) x (34) 13 (17) X (21) 2 (3) x (12) 5 (10) x (36) 6 (12) x (15) 1 (2) 10 ca (29) x (44) 10 (26) x (18) 11 (18) x (24) 6 (10) x (45) 10 (14) a Total, 31%. b Total, 11%.

3 VOL. 45, 1983 Source and TABLE 3. ANTIBIOTIC RESISTANCE OF FECAL COLIFORMS 81 Resistance of bacteria to different antibiotics in water samples No. (%)a of isolates resistant to: sample no. Ampicillin Chloramphenicol Sulfonamides Tetracycline Streptomycin 1 29 (42) 5 (7) 11 (16) 7 (10) 4 (6) 2 23 (35) 1 (2) 13 (20) 11 (17) 4 (6) 3 16 (27) 1 (2) 0 3 (5) (12) 2 (3) 4 (7) 8 (14) 2 (3) 5 10 (13) 3 (4) 16 (21) 12 (16) 7 (9) 6 12 (15) 1 (1) 2 (3) 4 (5) (10) 5 (10) 2 (4) 8 15 (30) 5 (10) 4 (8) 6(12) 0 9 6(10) 1 (2) 2 (3) 3 (5) 1 (2) 10 3 (21) 0 1 (7) (8) 2 (5) 10 (26) 17 (44) 2 (5) 12 9 (15) 0 9 (15) 7 (12) 4 (7) 13 9(15) 3 (5) 6(10) 6(10) (31) 7 (10) 12 (17) 9 (13) 1 (1) a Totals: ampicillin, 20%; chloramphenicol, 4%; sulfonamides, 12%; tetracycline, 12%; streptomycin, 3%. water samples (Table 2). There was no obvious connection with the water source or the level of contamination. The incidence of resistance was high in untreated domestic sewage (samples 1 and 2) and in some brakish water and lake water samples (samples 11 and 14). The average frequency of multiple resistance was 11%. It was not high in the same samples in which the overall resistance was high (Table 2). The resistance of bacteria to different antibiot- TABLE 4. Species composition of water samples No. (%)' of isolates Source and sample no. E. coli K. pneumoniae E. cloacae Other Entero- Unidentified and non-enterobacteriaceae bacteriaceae 1 33 (48) 17 (25) 10 (14) 2 (3) 7 (10) 2 37 (55) 14 (23) 10 (15) 5 (8) 1 (2) 3 29 (48) 10 (17) 10 (17) 4 (7) 7 (12) 4 29 (49) 10 (17) 12 (20) 4 (7) 4 (7) 5 56 (73) 13 (17) 1 (1) 1 (1) 6 (8) 6 26 (33) 7 (9) 31 (40) 3 (4) 11 (14) 7 36 (73) (12) 7 (14) 8 30 (60) 4 (8) 5 (10) 5 (10) 6 (12) 9 44 (73) 4 (7) 3 (5) 2 (3) 7 (12) 10 9 (64) 3 (21) 1 (7) 0 1 (7) (85) (15) (87) 2 (3) 0 0 6(10) (59) 7 (12) 8 (14) 7 (12) 2 (3) (54) 18 (25) 0 4 (6) 11 (15) a Totals: E. coli, 60%o; K. pneumoniae, 13%; E. cloacae, 11%; other Enterobacteriaceae, 6%; unidentified and non-enterobacteriaceae, 10%.

4 82 NIEMI, SIBAKOV, AND NIEMELA APPL. ENVIRON. MICROBIOL. TABLE 5. Incidence of resistance to different antibiotics among bacterial strains No. (%) of isolates resistant to: No. (%) of Species Ami Ampi Chlor- Sulfo Sloa T Strep- isolates amphe- Tetracycillin tomy- Total multiply nicol mide cline cin resistant E. coli 30 (6) 15 (3) 67 (14) 76 (16) 21 (4) 103 (21) 59 (12) K. pneumoniae 66 (61) 1 (1) 10 (9) 4 (4) 2 (2) 70 (64) 10 (9) E. cloacae 34 (38) 2 (2) 7 (8) 5 (6) 3 (3) 38 (42) 9 (10) Other Enterobacteriaceae 9 (21) 1 (2) 1 (2) 3 (7) 0 12 (28) 2 (5) Unidentified and non- 25 (30) 12 (15) 10 (12) 10 (12) 1 (1) 30 (37) 14 (17) Enterobacteriaceae Total 164 (20) 31 (4) 95 (12) 98 (12) 27 (3) 253 (31) 94 (12) ics varied from one water sample to another (Table 3). Again, no obvious connections between resistance and water source can be seen. Table 4 summarizes the results of the identifications. The species composition is found to vary considerably from Escherichia coli-dominated lake water samples (11 and 12) to the much more diverse populations of most waste waters. To connect the resistance data with the species composition, the relative proportion of each species (Table 4) in turn was paired with relative incidence (p) of drug resistance (Table 3) in each sample. Their correlation coefficients were computed after arc sin transformation (x = arc sin VT) Another set of correlations was computed between similarly transformed paired resistance values of different drugs to examine the sources of multiple resistance. The analyses showed that high overall incidence of resistance in the water samples seemed to be connected with a high proportion of Klebsiella strains. In fact, the only statistically significant positive drug versus species correlation was that of relative Klebsiella frequency and incidence of ampicillin resistance (0.789; significant at the 99.9% confidence level). The highest incidence of multiple resistance was found in the unidentified group (Table 5). However, the group was so small and scattered that the correlation coefficient was not statistically significant. The incidence of multiple resistance actually seemed to depend more on the dominance of E. coli. These findings are supported by the results in Table 5, which show, furthermore, that the high resistance incidence of especially Klebsiella, but also of Enterobacter, is chiefly due to ampicillin resistance. The correlations of sulfonamide, tetracycline, and streptomycin resistance were statistically significant. DISCUSSION Nearly one-third (31%) of the isolates were found resistant to one or more of five antimicrobial drugs (Table 2). The proportion of resistant strains was almost the same, 26%, in another set of 186 strains isolated 3 years earlier from the same kinds of water samples and tested against the same antimicrobial drugs (unpublished data). The comparison of the percentage of resistant strains with published work from other times and places is complicated because researchers have used different numbers and kinds of antibiotics in their studies. The percentages calculated in this study are, however, quite close to those reported for untreated (33%) and treated (28%) sewage for bacteria tested against six antimicrobial drugs (1). The much higher incidence of resistance (46 to 72%) reported by Cooke (3, 4) and Goyal et al. (8) for bacteria from aquatic environments may have a natural explanation in the fact that 11 antimicrobial drugs were tested. The fecal coliform group consists of different genera. The variation of the relative incidence of each genus between water samples can affect the percentage of resistant fecal coliform bacteria. For example, a high proportion of K. pneumoniae may increase the total amount of ampicillinresistant bacteria, because Klebsiella strains tend to be naturally resistant to this antibiotic. Bell et al. (2) found the high proportion of Aeromonas species to decrease the incidence of bacteria carrying R factors in a set of bacteria isolated as total coliforms. The effect of species composition is clearly evident in some examples. In Lake Enajarvi, the high incidence of sulfonamide, tetracycline, and multiple resistance and the low incidence of ampicillin resistance (Table 3, sample 11) was caused by the high incidence of E. coli strains (85%). Resistance to ampicillin is common in the samples from untreated sewage (Table 3, samples 1 and 2), in which only half of the strains were E. coli and the other half mainly Klebsiella and Enterobacter strains (Table 4). Resistance to ampicillin among fecal coliform bacteria reported in the literature varies from 5.6% in spring water (3) to 54% observed among bacteria isolated from sea, sewage, and shellfish (4). One explanation for this wide variation may be the difference in species composition of the samples. The species composition of the sample

5 VOL. 45, 1983 depends on the time elapsed after excretion, the proportion of stormwater (6) or kitchen water in the sewage (11), and the nature of the recipient; it probably does not depend much on the resistance to antibiotics. If the incidence of resistance to antibiotics and synthetic antimicrobial drugs is to be compared in different areas and at different times, it is necessary to identify the bacteria and to use the same set of antimicrobial drugs in the tests. It may be very misleading to record only the resistance incidence of "fecal coliforms" counted as typical colonies. The proportion of Klebsiella species is decisively important. ACKNOWLEDGMENTS This research was supported by a grant from the Maj and Tor Nessling Foundation. We thank Eva Nyreen and Rita Haaparanta for technical assistance and Kari Aalto for computing the correlation data. We thank Piro Lehtovaara for typing the manuscript and Aino-Maija Niemela for correcting the English. LITERATURE CITED 1. Bel, J. B Antibiotic resistance patterns of fecal coliforms isolated from domestic sewage before and after treatment in an aerobic lagoon. Can. J. Microbiol. 24: Bell, J. B., W. R. MaCrae, and G. E. Elliott Incidence of R factors in coliform, fecal coliform, and Salmonella populations of the Red River in Canada. Appi. Environ. Microbiol. 40: Cooke, M. D Antibiotic resistance in coliform and ANTIBIOTIC RESISTANCE OF FECAL COLIFORMS 83 fecal coliform bacteria from natural waters and effluents. N. Z. J. Mar. Freshwater Res. 10: Cooke, M. D Antibiotic resistance among coliform and fecal coliform bacteria isolated from sewage, seawater, and marine shell-fish. Antimicrob. Agents Chemother. 9: Ericsson, H. M., and J. C. Sherris Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathol. Microbiol. Scand. Sect. A 217(Suppl.): Geldreich, E. E., L. C. Best, B. A. Kenner, and D. J. Van Donsel The bacteriological aspects of stormwater pollution. J. Water Pollut. Control Fed. 40: Geldreich, E. E., H. F. Clark, C. B. Huff, and L. C. Best Fecal-coliform-organism medium for the membrane filter technique. Am. Water Works Assoc. J. 57: Goyal, S. M., C. P. Gerba, and J. L. Melnick Transferable drug resistance in bacteria of coastal canal water and sediment. Water Res. 13: Grabow, W. 0. K South African experience on indicator bacteria, Pseudomonas aeruginosa, and R+ coliforms in water quality control, p In A. W. Hoadley and B. J. Dutka (ed.), Bacterial indicators/health hazards associated with water. ASTM STP 635. American Society for Testing and Materials, Philadelphia. 10. Grabow, W. 0. K., and 0. W. Prozesky Drug resistance of coliform bacteria in hospital and city sewage. Antimicrob. Agents Chemother. 3: Karlgren, L., K. Ljungstrom, E. Olsson, and V. Tullander Household wastewater. Composition and properties. The National Swedish Institute for Building Research. Meddelande/Bulletin M :1-44. (In Swedish.) 12. Kelch, W. J., and J. S. Lee Antibiotic resistance patterns of gram-negative bacteria isolated from environmental sources. Appl. Environ. Microbiol. 36: Koditschek, L. K., and P. Guyre Resistance transfer fecal coliforms isolated from the Whipping river. Water Res. 8: