Resistance transfer fecal coliforms isolated from the whippany river

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RESISTANCE TRANSFER FECAL COLIFORMS ISOLATED FROM THE WHIPPANY RIVER LEAH K. KODITSCHEKand PAUL GUYRE* Biology Department. Montclair State College. Upper Montclair, New Jersey 07043. U.S.A. (Receiccd 20 3,1arch 1973)

Abstract--Three sites on the Whippany River. a tributary of the Passaic Watershed in Morris County. N.J.. were monitored for resistance transfer coliforms. A high percentage of fecal coliform isolates shox',cd multiple antibiotic resistance. A significant number oflac- and lac~ isolates proved to be donors o[ mmsmissible antibiotic resistance plasmids {R donorsl, by conjugation with a recipient strain of Salmo*zelhz Oallimlrum. Re-examination of testing methods used for recreational and potable water sources is suggested b? these findings.

INTRODUCTION

The recent increase in multiple drug resistance among strains of Enterobacteriaceae has been widely documented (Anderson, 1968; Davies and Rownd, 1972; Meynell et al., 1968), and the medical significance of resistance transfer i, riro, associated with selection of multiple drug resistant pathogens has been emphasized (Aandahl, 1968 : [senberg et al., 1971 ; Kr~mdry et al.. 1972; Watanabe, 1972). R plasmids may code for resistance to heavy metals and other antimicrobials as well as antibiotics (Novick, 1969; Smith, 1967) and this may enhance survival of bacteria containing such plasmids. It has been suggested that R factor bacteria may be widely disseminated through food (KrOmdry et al., 1971) and water (Smith, 1970). The routine bacteriological examination of water is confined to confirmed tests for coliforms (Standard Methods, 1971). Little effort has been made in this country to determine the antibiotic spectrum of coliform populations in water environments, the sensitivity to other antimicrobials (with the exception of chlorine), or the incidence of R factor bacteria, even in areas where sewage effluents are discharged into recreational or potable water sources. Sturtevant et al., (1969, 19701, reported the presence of R factor coliforms in both raw and treated sewage, and the possibility exists that sewage effluents, storm run-off, as well as other surface contaminants, may contribute to the dissemination of antibiotic resistant bacteria through water. A recent study by Feary et al. (1972) shows a high incidence of antibiotic resistant coliforms in water of the Black Warrior-Tombigbu River System and the bay in which it empties.

The aim of this study was to examine selected sites within the Upper Passic Watershed, a potable water source in Morris County, where recent data showed 9 per cent of industrially zoned land is utilized (Lesser et al., 1970). It was felt that examination of water from an industrially underdeveloped area would be more representative of the overall incidence of antibiotic resistant and R factor bacteria at suburban river sites. and may be more pertinent than the examination of water at heavily polluted urban sites. Accordingly, three sites on the Whippany River in proximity to the Morristown Sewage Treatment Plant were selected, as shown in Fig. 1 : these sites are designated for routine

~iver

" P. Guyre is currently a graduate student at the Univer- Fig. l. Map of section of the Whippany River showing samsitx of Nev, Hampshire. Durham. N.H. pling sites 280. 260 and 240. 74.7

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LLaH K. KODIrSCHEKand P~L L Gt. ~Rk

sampling by the Passaic Valley Water Commission as follows: site 280. immediately above the sewage outfall: site 260, shortly after the outfall: and site 240. approximately l mile down-stream tLesser et al.. t9701. It was hoped that these sites might show the effect of sewage treatment discharge and its subsequent dilution on the fecal coliform population in a section of this river. Samples were screened for fecal coliforms, multiple antibiotic resistant fecal coliforms, and R factor donors. The results of this preliminary study suggest the need for further examination of water sources as a possible vehicle for R factor dissemination.

MATERIAL AND METHODS

1. Sampling procedure Standard procedure was used to obtain surface water samples, which were refrigerated and processed in the laboratory by membrane filtration within 2 h after collection. A modification of F'WPCA methods (1968), and the method of Geldreich (1970), which was found to be superior to other standard selective techniques For fecal coliform enumeration, was used exclusively. After filtration of appropriate sample aliquots, millipore filters were placed on m F C medium (Difco mFC broth plus 1.5% agar) alone, or supplemented with 40 ILg per ml chlortetracycline (Chtet) and incubated in closed bags, submerged in a constant temperature water bath at 44.5 +_ 0.5 :C for 20 4-_ 2 h. All bluecolonies were counted as fecal coliforms; colorless or pink colonies were scored as lac-.

Table

2. [sol,.~ticm of potenti,.¢.l c[onor'; Fecal colitbrm colonies isolated on mFC + Chtet were picked and spotted on tr)pticase so5 agar ITS-k) supplemented ~ith the appropriate antibiotic. Onl) isolates which grew on TSA - Chtet, but failed to gro~ on TSA - Chtet - nalidixic acid {40,~g NA per ml) were screened for R factor donor activit) in conjugation with Salmo~wlla ~j~dlimlrmn 244. a recipient which is Chtet'. N.U.

3. 3httitlg proccdure {ttatcz~zahe and Fukasawc~ {1961) (a) Donors and recipients were inoculated into trypticase soy broth (TSB} at 37:C. {b) After 10-fold dilution or" overnight cultures with TSB, donor and recipient cultures were incubated with aeration (~vater bath shaker) at 37=C to exponential phase {o.d. 0.2-0.6 on Bausch & Lomb Spectronic 20). (c) Conjugation mixes were adjusted on the basis of o.d. readings to a ratio of approximately 1 x l0 s donors to 5 × 10 s recipients in a total vol of 1.5 ml, and incubated stationary at 37:C for 1 h. (d) Recombinants were scored by growth on TSA + Chtet + NA in comparison with controls, (potential donors and recipients spotted on the same medium).

4. Antibiotic spectrum of isohltes Isolates were screened for antibiotic resistance on plates of TSA pills seeded semi-solid overlay. Sensi discs (BBD of the following antibiotics were placed on the seeded plates: chlortetracvcline (A-30l~g). tetracycline (Te-30/~g), streptomycin IS-10 iLg), colymycin {C1-2 ~zg), Kanamycin (K-30 !tg}. penicillin (P-10 units), chtoram-

Incidence of fecal coliforms showing Chtet resistance and donor activit) in samples from site 280 Per cent

Date of sample 8 June 1971 i5 Nov. 197l 29 No,,'. I971 27 Dec. 1971 28 Dec. 1971 10 Jan. 1972 19 Jan. 1972 7 June 1972 12 June 1972 26 June 1972 30 June 1972 Total

Temperature CC) 22 7 6.8 8 8 4 4 17 15.5 16 17.5

Fecal coliforms*

Fecal coliforms resistant to Chtet*

Total resistant to Chtet (?,]}

No. of isolates tested for donor activit~

No. of isolates showing donor activity

2500 9500 13.200 84 96 4400 455 83.000 188,000 10.000 6500 28.885

300 450 345 22 44 670 31 3700 8100 350 375 1308

i2.0 4.7 2.6 26.2/ 45.8 J 15.2 6.8 4.5 4.3 3.5 5.8 4.5

3 9 17

1 1 7

2s 24 13 31 20

3

12.0

12 6 22 3

50.0 46.2 71.0 15.0

-142

-55

38.7

*Average counts per 100 ml of sample, usually based on five replicate filtrations per sample.

tested showing donor activity 33.3 li.1 41.2

Resi>tance transfer t)cal coliforms

729

Table 2. Incidence of fecal colilorms sho~ing Chtet resistance and donor activity at three sites on the \\hippan.~ Rixer

Site no.

A~erage t)cal colit'orms*

A~erage fecal coliforms resistant to Chtet*

Fecal coliform resistant to Chtet 1",,1

No. of isolates tested for donor acti~it~

No. sho~ ing donor activit~

sho~ ing donor actix it> C,,I

280 260 240

28.8S5 14,161 2215

1308 480 55

4.5 3.4 2.5

142 129 82

55 2l 30

3~.7 16.3 36.6

* A~erage total counts: total of counts per I00 ml. Number of samples represented for each site varies.

phenicol (C-30 llg). triple sulfa {SSS-0.25 rag), sulfacetamide (SA-0.25 mg). nalidixic acid INA-30 #g). After incubation at 3 7 C for 18-20h, plates were scored visually. A zone of inhibition of 10 m m dia or greater was considered to indicate antibiotic sensitivity. G r o w t h of the isolate culture within I mm of the sensi disc, or closer was scored resistant.

RES.L LTS Considerable fluctuation in total counts and in the antibiotic resistance of isokttes was observed in diftbarent samples for the same site. Table 1, which shows detailed data for samples at site 280, demonstrates this variability. It will be noted that two samplings on different days, when the water temperature was 4~C, gave 10-fotd difference in fecal coliforms, and the Chtet ~ cot, hiS varied by a factor of 20. It is apparent, therefore, that temperature is only one of a n u m b e r of complex variables involved in coliform survival in this aqueous environment. The two samples which show the highest fecal coliform counts, on 7 June and 12 June 1972, were taken during a week of heavy rain and flooding. It is noteworthy that the sample taken at the beginning of this flood period (7 June) shows the highest percentage of R donor bacteria.

A summary of data at the three sites in the vicinity of the sew;tge disposal plant is sho~vn in Table 2. This data shows that overall, the average fecal coliform count is highest at site 280, prior to the discharge of chlorinated outfall from the sewage disposal plant, and lower after this outfall (site 260 and 240). The widest variations in individual sample counts (not shown) were found at site 260, at the site of the outfall. This may reflect variations in chlorine content of the sewage plant effluent. Closer examination of the data in Table 2 indicates that in spite of the reduction in average fecal coliform counts, and average Chtet ~fecal coliform counts by sewage plant discharges, the percentage of donors a m o n g these isolates increases sharply at site 240, a short distance from the selvage outfall at site 260. It is obvious from the data in Table 2 that a relatively high percentage of Chtet ~isolates tested from the three sites proved to be donors. Attention should also be drawn to the significant n u m b e r of lac- Chtet ~ organisms found at each site, (Table 3). Although we examined lac~ Chtet ~ isolates for d o n o r activity from only one sampling at each site, there is supporting evidence in the literature (Hoar, 1970; Meynell et al., 1968) that a n u m b e r of lac- pathogens and non-pathogens are high frequency donors of the R plasmid. In fact, a lac- derepressed F ~ d o n o r

Table 3. Incidence of lac- organisms showing Chtet resistance and donor activity at three sites on the Whippany River

Site no.

Average laccolonies counted*

Average lacchlortetracycline resistant*

280 260 240

1952 2783 1835

69 63 5

Lacchlortetracycline resistant ("o)

Lac- Chtet ~ isolates tested for donor activity+

LacChtet ~ donors~

Chtet r lacisolates showing donor activity{°~)

3.5 2.3 0.3

9 7 3

8 ~ 1

88.9 28,6 33,3

* Average counts per 100 ml of sample, usually based on five replicate filtrations per sample. ~ Lac-. Chtet r isolates from one sample for each site were tested for donor activity.

LF:,',H K. KODITSCHEK and PAL L GL "~RE

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Tabie 4. [ncidence of resistance patterns and chlortctrac~cline resistance transferability Sampling site

Classification*

Patterns +

Transferred resistance

No.

280

a b c d e f

Te{. SA, P. SSS, C1, A, S Te. SA. K, P, SSS. CI. A. S Te. SA, P. SSS. A. S Te. SA, K. P. SSS. A. S Te. SA. P, SSS, A Te, SA, P, SSS, CI, A Other patterns Total

I8 I3 1[ 9 9 6 32 98,~

8 6 7 6 6 5 14 52

260

a c ,, b h e f i

Te, SA, P, SSS, C1, A, S Te, Sa, P, SSS, A, S Te, P . A Te. SA. K, P. SSS, C1, A, S Te. P, CI, A, S Te, SA. P. SSS. A Te, SA, P. SSS. CI, A Te, P, A, S Other patterns Total

22 22 I0 9 6 6 6 6 33 12~

4 2 1 2 0 [ 0 4 6 20

240

a c b f i

Te. SA, P, SSS, C1, A, S Te, SA, P, SSS, A. S To, SA, K, P, SSS, CI, A, S Te, SA, P, SSS, C1, A Te, P, A. S Other patterns Total

18 15 9 6 6 30 84§

7 2 2 i 2 15 29

* Classification of resistance patterns to Pacilitate comparison. t All isolates tested were nalidixic acid sensitive (see Methods). ~. Abbreviations: Te, tetracycline; SA, sulfanilamide; K, kanamycin; P, penicillin; SSS, triple-sulfa; CI, coly-mycin; A, aureomycin; S, streptomycin (BBL sensi-discs). § Totals deviate from those in Table 2 since not all isolates were screened for antibiotic sensititivity.

40 [--

strain o f E. coil D H 4, o b t a i n e d from Dr. H o a r , was used in this study as a c o n t r o l for c o n j u g a t i o n procedures. This p o i n t bears emphasis, because routine water analysis specifically excludes e x a m i n a t i o n o f lac- o r g a n i s m s , a n d our limited data indicates a high incidence o f d o n o r s a m o n g l a c - C h t e t r isolates. The antibiotic resistance p a t t e r n o f lac* isolates is s h o w n in Table 4 a n d Fig. 2. It m u s t be n o t e d that a very high p r o p o r t i o n o f all lac + isolates at each site s h o w multiple antibiotic resistance, with a peak o f

[ ] Site 280 [ ] Site 2 6 0

| ~

•Site 240

30

ul

~

2o

Table 5. Multiplicity of drug resistance oflac" isolates from simultaneous samplings at three sites on the Whippany River

i0

2-3

4

5

6

7

Site no.

2-3

280 260 240

0* 2.4 0

Number of Antibiotics 4 5 6 7

8

8

Of antibiotics Fig. 2. Percentage of multiple drug resistant lac- isolates from three sampling sites on the Whippany River. ~ Antibacterials used: nalidixic acid and chloramphenicol plus those listed in footnote ++,Table 4.

1.9 4.7 3

3.9 16.7 0

27.0 14.3 23.5

44.2 38.0 44. l

23.0 23.8 29.4

* Figures represent percentage of resistant isolates for the samples obtained simultaneously at the three sites. For antibiotics used see footnote Fig. 2.

Resistance transfer fecal coliforms resistance to six and seven antibiotics. Time limitations prevented the simultaneous sampling and processing of samples for all three sites, but such data is available for txvo samplings. ITable 51. It is again apparent that the outfall at the sewage disposal plant did not affect the high percentage of fecal coliforms resistant to six, seven and eight antibiotics at sites 260 and 240. DISCL SSION

Our results indicate that selected sites on a potable water source in a suburban area show significant numbers of antibiotic resistant fecal coliforms which are capable of transmitting this resistance to a recipient Salmonella strain during combined culture {coniugation mix) for 1 h. It is obvious that the system used in this study revealed only those donors which are Chte¢, NA ~ and that bv the use of other recipient strains, the incidence of multiple antibiotic resistant donors in this potable water source could be shown to be much higher than that reported here. Similar results have been reported in a much more extensive sampling of British rivers by Smith {1970). His data shows the incidence of tetracycline resistant coliform donors to bc greater than 50 per cent, and an incidence of chloramphenicol resistant donors of over 80 per cent. His observation that "'the concentration of antibiotic resistant coliform organisms was always much lower in the effluent than in the inflt.ent sewage" seems to agree ,aith our results, in that the counts for site 260 are usually lower than those for site 280, before the sewage treatment outfaU, (Fig. 1). Our data also supports his conclusion that the chlorine in effluents which are discharged into streams does not have any prolonged effect on the antibiotic resistant population of such streams. In contrast, Sturtevant et al, (1969, 1970) concluded from their study at five sewage plants in Jefferson County, Alabama, that the incidence of antibiotic resistant bacteria was not significantly lower in the effluents compared with the raw sewage. A draft report of a County and Municipal Government Study Committee in New Jersey (1973) has documented that during periods of high runoff, the outflow of untreated sewage may' be discharged into the Passaic River. Even under routine conditions, sewage treatment facilities in this area are overtaxed and understaffed, and residual chlorine in effluents may be below standard, This dilemma is so serious, that the Passaic Valley Water Commission is seeking court in.iunctions to block further development along the Passaic River Basin. Thus, it is not surprising to find high fecal coliform counts and antibiotic resistant coliforms in the Whippany River. What may be more significant, is our finding that low fecal coliform counts in this water may not insure

-51

freedom from health hazard. Our results sho~ that lo,a temperature. ~hich may reduce the fecal coliform population, does not pre~ent the survival of antibiotic resistant bacteria, and R factor donors. Thus. e~en in winter, when the fecal coliform counts tended to be lower, the percentage of R donor fecal coliforms was quite high: data in Table l indicate that water samples showing less than 500 fecal coliforms per 100 ml, which is within the suggested tolerance for recreational water tGeldreich, 1970) could disseminate multiple resistant bacteria, including R donors. Overall. the lack of direct correlation between fecal coliform count and percentage of R donors in these samples, raises the possibility that this type of water contamination may' originate from sources other than fecal pollution. Recent reports by Krem~,ry et al. (1972) show that a variety of foods tested in Czechoslovakia contained R factor donor strains of bacteria. Watanabe and coworkers (1971) have demonstrated the presence of R factor donors (non-coliform) isolated from fresh water fish in Japan. Additionally, Hendricks (1970) has demonstrated that intestinal pathogens may be sequestered in stream sediments. Some of our studies on marine sediments at ocean dump sites near New Jersey, (unpublished data), revealed high concentrations of R factor donor bacteria in sediments as compared with deep or surface water samples. Thus, the routine sampling of water alone may not be adequate for complete surveillance of water safety. The sanitary significance of fecal coliforms has been emphasized in standard methods, and re-evaluated by' Geldreich (1965) as a reliable indicator of pollution in recreational and potable water, However, fecal coliform analysis excludes consideration of lac- bacteria, and there is sufficient data to indicate that R plasmids are associated with a wide variety of non-lactose fermenting bacteria which might be established in a water environment (Watanabe et al., 1971). If projected plans of the County and Municipal Government Study Committee (1973), to improve water quality in the Passaic River Basin for recreational and continued potable use are to be implemented, it may be desirable to supplement routine coliform estimation of water samples with additional monitoring of water and sediments, by using other indicators. Our findings suggest that the problem of possible water dissemination of R factor donor bacteria should receive some consideration in future monitoring programs in this area, as well as in other technologically developed countries. Acknowledgements--This investigation was supported m part by funds from a Montclair State College Faculty Improvement Grant and from the Edwin E. Aldrin Fund. We wish to thank Dr. J. Unowsky of Hoffman-LaRoche, Inc.

".~

LZ-AH K. KODITSCHEKand P~,LL Gt.'~R~

for providing a culture of Saimoaellu cjalli~kzrtlm 2...t-l.. Dr. E. L. Dulane.v. Merck and Co.. Inc. and Mr. F. Brezenski. Region II. Environmental Protection Agency. for heip(ul suggestions.

REFERENCES

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Kr~mer? V., Rosival L.. V~mota F. and Hegzlar M. (1972) Problems of transferable antibiotic resistance in Czechoslovakia. In, First [at. S~pnp. In]7 .4~ztibiotic Resistance cEdited by Kr~mer.v V.. Rosival L. and Watanabe T.) pp. 13-10. Springer-Verlag. New York. Lesser A.. Spinner A. and Tirabassi M. 11970) An engineering economic study of industrial growth potential of the upper Passaic River Basins. Ofiqce of Water Resources Research. Me~ nell E.. Meynell G. G. and Dam, H. ~~968) Phylogenetic relationships of drug-resistant factors and other transmissible bacterial plasmids. Butt. Ret'. 32, 55-83. Novick R. P. {1969) Extrachromosomal inheritance in bacteria. Butt. Ret'. 33, 210-263. Smith D. H. (1967) R factors mediate resistance to mercury nickel and cobalt. Science N.E 156, 1114-i 116. Smith H. N. (1970) Incidence in river water of E. coil containing R Factors. ,Vuttzre. Lo~ld. 228, 128&-1288. Standard Methods for the Examination of Water and Wastewater (1971) I3th edn. APHA. New York. Sturtevant A. B. Jr. and Feary T. W.(I969) Incidence of infectious drug resistance among lactose fermenting bacteria isolated from raw and treated sewage. Appl. Microbiol. 18, 918 924. Sturtevant A. B. Jr., Cassell G. H. and Feary T. W. (1970) Incidence of infectious drug resistance among fecal coliforms isolated from raw sewage. AppI. Microhiol. 21, 4-87-49 I. Watanube T. (1972) Further outlooks of antibiotics in the shadov~s of resistance factors. In First Int. Syrup. hgi Antiohiotic ResisraJzce (Edited by' Kr6m~:rv V.. Rosival L. and Watanabe T.) pp. 9-12. Springer-Verlag, New York. Watanabe T.. Aoki T., Ogata Y. and Egusa S. (1971) R factors related to fish culturing. Ann. N.E ,4cad. Sci. 182, 383-410. Watunabe T. and Fukasawa T. (1961) Episome-mediated transfer of drug resistance in Enterobaeteriaceae--I: Transfer of resistance factors by conjugation. J. Bact. 81, 669-678,