Antibiotic Resistance Among Coliform and Fecal Coliform

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1976, p Copyright C 1976 Americn Society for Microbiology Vol. 9, No. 6 Printed in U.SA. Antibiotic Resistnce Among Coliform nd Fecl Coliform Bcteri Isolted from Sewge, Sewter, nd Mrine Shellfish MARYLYN D. COOKE Cwthron Institute, Nelson, New Zelnd Received for publiction 18 December 1975 Sewter nd shellfish smples collected in the vicinity of mrine sewge outfll were exmined for the incidence of ntibiotic resistnce mong coliform nd fecl coliform bcteri over 2-yer period. Seventy percent or more of these two groups of bcteri from both sources were resistnt to one or more ntibiotics. Forty-five percent of the isoltes resistnt to streptomycin or tetrcycline were cpble of trnsferring ll or prt of their resistnce pttern to n ntibiotic-susceptible strin of Escherichi coli K-12. Antibiotic resistnce, medited by extrchromosoml elements or R fctors, is widespred mong the Enterobctericee (22). R fctors my medite resistnce to s mny s eight ntibiotics simultneously nd confer resistnce to hevy metls such s nickel, mercury, nd coblt (17). They re trnsmissible mong grm-negtive bcteri such s Escherichi coli, Slmonell, nd Shigell nd to other unrelted bcteri such s Pseudomons eruginos (5). The widespred use of ntibiotics in griculture nd medicine is ccepted s mjor selective force in the incresing reports of high incidence of ntibiotic resistnce mong grm-negtive bcteri (2, 15). Coliform bcteri, generlly regrded s nonpthogenic indictors of pollution (9), re often used to study the bcteriologicl qulity of wter nd foods. It hs been demonstrted tht ntibiotic-resistnt coliform bcteri from effluents nd lnd runoff eventully my enter mrine receiving wters (7, 18-20). Filter-feeding shellfish such s scllops nd oysters tend to concentrte bcteri from overlying wters (16), nd experimentlly contminted oysters hve been shown to retin Slmonell typhimurium for up to 49 dys (11). As shellfish re often consumed rw or fter miniml cooking, they my serve s vehicle for trnsmission to humns of ntibiotic-resistnt bcteri crrying R fctors. In mny countries, the bcteriologicl quljity ofedible shellfish is bsed upon coliform nd fecl coliform stndrds, but little work hs been done to determine the incidence of ntibiotic resistnce mong coliform bcteri from such sources. This study ws undertken to investigte the incidence of ntibiotic resistnce mong coliform nd fecl coliform bcteri in smples of sewter nd shellfish collected ner city sewge mrine outfll. This outfll in Tsmn By, New Zelnd, ws commissioned in 1970 nd serves the city of Nelson (popultion, pproximtely 31,000), dischrging bout 5 x 106 liters of comminuted rw sewge per dy, minly from domestic sources. Smples were primrily collected for most probble number (MPN) coliform nd fecl coliform enumertion to monitor nd guge the extent of pollution of shellfish beds in the vicinity of the outfll. MATERIALS AND METHODS Smpling. Sewter nd shellfish smples were collected t pproximtely 8-week intervls over period of 2 yers from ech of eight loctions. Four loctions, ech pproximtely 3,000 feet (c. 900 m) prt, were on n rc of rdius 5,000 feet (c. 1,500 m) from the end of the outfll pipe; the other four loctions, similrly rrnged, were 10,000 feet (c. 3,000 m) from the outfll pipe. From ech loction, t ech smpling visit, two wter smples were tken, one just below the surfce, the other just bove the sebed, which vried between 55 feet (c. 16 m) nd 75 feet (c. 23 m) deep. Shellfish were gthered nd plced in plstic bgs for trnsport to the lbortory. Smples were processed within 2 h of collection. Isoltion of bcteri. Shellfish smples were prepred for MPN testing in ccordnce with methods recommended by the Americn Public Helth Assocition (1). McConkey broth (Oxoid CM5) ws used s the presumptive medium for both shellfish nd wter smples nd ws incubted t C for h. For some sewter smples, duplicte sets of minerls-modified glutmte medium (Oxoid) were lso inoculted for estimtion of presump- 879

2 880 COOKE tive coliforms. Tubes showing positive fermenttion were subcultured into brillint green bile (Oxoid; BGB) broth nd incubted t C for 48 ± 2 h to confirm the presence ofcoliform bcteri. Positive presumptive tubes were lso inoculted into further tube of BGB broth nd tube of tryptone wter (Difco), nd both medi were incubted t C for 24 ± 2 h. Growth nd gs production in BGB broth were considered positive for fecl coliform bcteri. The tryptone wter ws tested for production of indole with Kovcs regent, nd positive result in both BGB broth nd tryptone wter ws considered positive for E. coli (14). To isolte coliform bcteri in pure culture, gs-positive tubes of BGB broth were streked on Mc- Conkey gr (Oxoid CM7) nd incubted t 37 ± 0.5 C for h. One representtive lctose-positive colony of ech morphologicl type ws purified by restreking on the sme medium. Antibiotic susceptibility testing. Antibiotic resistnce ptterns of ll isoltes were determined by overlying mesured 10-ml ntibiotic-free Mueller- Hinton gr (Difco) pltes with 0.1 ml of 24-h nutrient broth culture (Oxoid CM67) in 2.5 ml of molten soft gr (0.6% Dvis gr). Antibiotic-impregnted pper disks were plced on the surfce of the inoculted pltes nd incubted t 37 ± 0.5 C for 18 ± 2 h. Isoltes were recorded s "resistnt" when there ws no inhibition of growth round the edge of the disk nd s "exhibiting prtil resistnce" when the zone of inhibition ws significntly reduced compred with control ntibiotic-susceptible E. coli strin. The following ntibiotics were used to determine ptterns of resistnce (concentrtion in microgrms per disk): mpicillin, 10; cephlothin, 20; chloromycetin, 10; gentmicin, 10; knmycin, 10; nlidixic cid, 5; prmomycin, 10; streptomycin, 10; sulffurzole, 250; tetrcycline, 10; nd trimethoprim, 2.5 (ll from BBL); nd rifmpin, 5 (from Difco). Representtive isoltes were subsequently identified by methods outlined by Edwrds nd Ewing (6). Trnsfer of ntibiotic resistnce. A rndom selection of the lctose-positive, ntibiotic-resistnt isoltes susceptible to nlidixic cid were used s prospective donors of resistnce to n ntibiotic-susceptible F- derivtive ofe. coli K-12, J6-2pror trp- hislcu, resistnt to 50,ug of nlidixic cid per ml. Mtings were done by mixing 0.1 ml of logrithmic donor culture with 1.0 ml of overnight recipient culture in 5.0 ml of nutrient broth, nd the mixed culture ws incubted for 2 h on rotting turntble t 37 C. This short time of mting, in comprison with overnight incubtion of the mixed donor nd recipient cultures used by others (12, 21), ws chosen in order to concentrte on isoltes likely to be derepressed (13) nd cpble of trnsferring their resistnce determinnts under less fvorble conditions in the nturl environment. The mixed culture ws then vigorously shken with Rotmixer to seprte mting pirs nd diluted in buffer, nd 0.2- ml volumes were plted in soft-gr overlys on miniml medium fully supplemented with mino cids required by the recipient strin nd contining 50,±g of nlidixic cid per ml nd single ppro- ANTIMICROB. AGENTS CHZMOTHER. prite ntibiotic, chloromycetin, streptomycin, tetrcycline, or knmycin, t concentrtion of 20,ug/ml. The medi used were selective for ntibioticresistnt recombinnts of J6-2, since growth of the prospective donor ws prevented by nlidixic cid nd growth of the recipient ws prevented by the presence of n pproprite ntibiotic. After incubtion t 37 ± 0.5 C for 72 h, recombinnts were purified by restreking on the sme selective medium. The ntibiotic resistnce pttern of t lest five recombinnts ws determined s described bove to scertin whether ll or prt of the ntibiotic resistnce pttern of the donor hd been trnsferred to the recipient. Controls with donor strins crrying wildtype R fctors, Rl, R64, nd R144 (13), resistnt to the pproprite ntibiotic, were included with ech set of mting experiments. RESULTS MPN vlues. Smples of sewter nd shellfish tken on two occsions, 4 months prt nd 6 months before the outfll ws commissioned, showed no contmintion with fecl coliform bcteri nd extremely low MPN vlues for presumptive coliforms (i.e., <20/100 ml or 100 g, respectively). After the outfll ws commissioned, considerble fluctution ws recorded in MPN vlues, prticulrly for sewter smples, between the vrious loctions nd between different smpling dtes. This ws in lrge prt dependent on wind nd current direction over the smpling re, cusing vritions in the dispersl of the sewge. MPN vlues for fecl coliforms from the shellfish smples, using McConkey broth, hve shown grdul increse over the period of study. A men MPN vlue of 60 fecl coliforms/100 g of shellfish ws obtined over the first yer of the study, with vlues rnging between 5 nd 160/100 g on different smpling dtes. During the second yer, the men MPN vlue incresed to 225 fecl coliforms/100 g of shellfish. The use of minerls-modified glutmte medium in prllel tests on sewter gve significntly higher MPN presumptive coliform counts, which were subsequently confirmed s fecl coliforms nd E. coli, by comprison with results in McConkey broth. However, Tble 1 presents men MPN vlues for sewter smples from McConkey broth lone. The MPN presumptive coliform index ws less thn 1/100 ml for 40.6% of the surfce-wter smples nd 52.8% of the deep-wter smples. In the shellfish smples, the decrese in MPN vlues for confirmed coliforms s compred with presumptive coliforms (flse-positive presumptives) cn be explined by substrte fermenttion of glucose in the shellfish tissue. Antibiotic resistnce in isoltes from se-

3 VOL. 9, 1976 wter. The incidence of ntibiotic resistnce mong the coliform nd fecl coliform bcteri from the shellfish nd sewter smples is presented in Tbles 2 to 5, where dt from ech of the eight sites hve been grouped for ech smpling dte. More thn 70% of ll coliform isoltes from sewter were resistnt to one or more of the ntibiotics used (Tble 2). A similrly high overll incidence of resistnce ws found mong fecl coliforms from the sme source, lthough here it ws found tht the percentge incidence of resistnce ws more vrible on different smpling dtes (Tble 3). Resistnce to mpicillin, sulffurzole, or rifmpin ws TABLE 1. FECAL COLIFORMS FROM SEAWATER AND SHELLFISH 881 exhibited by more thn 30% of the isoltes. Resistnce to cephlothin ws more prevlent mong coliform bcteri (23.6%) thn mong the fecl coliforms (11.1%). Resistnce to other ntibiotics showed similr overll frequency of occurrence mong the coliforms nd fecl coliforms but ws noticebly more vrible mong the fecl coliform isoltes on different smpling dtes. The frequency of multiple resistnce, tht is, isoltes exhibiting simultneous resistnce to more thn one ntibiotic, ws found to be 49.6% mong the coliform isoltes nd 41.8% mong the fecl coliforms. Antibiotic resistnce mong isoltes from shellfish. The frequency of ntibiotic resistnce Summry of men MPN vlues of coliform nd fecl coliform bcteri in sewter nd shellfish smples collected between June 1972 nd June 1974, inclusive MPNs per 100 ml or 100 g Source No. of smples Presumptive Confirmed Fecl colicoliforms coliforms forms E. coli Surfce wter Deep wter Shellfish 88 2, TABLE 2. Incidence of ntibiotic resistnce mong coliform bcteri from sewter Smple No. No. % Resist- % Resistnt isoltes exhibiting resistnce to: % Mulrun no. tested resistnt nt tiply mp SUI cep tet chl str nl kn tri pr rif resistnt NT b Totl j mp, Ampicillin; sul, sulffurzole; cep, cephlothin; tet, tetrcycline; chl, chloromycetin; str, streptomycin; nl, nlidixic cid; kn, knmycin; tri, trimethoprim; pr, prmomycin; rif, rifmpin. b NT, Not tested. TABLE 3. Incidence of ntibiotic resistnce mong fecl coliform bcteri from sewter Smple No. No. % Resist- % Resistnt isoltes exhibiting resistnce to: % Mulrun no. tested resistnt nt tiply mp Sul cep tet chl str nl kn tri pr rif resistnt NI NT NT NT NT NT 28.6 Totl For bbrevitions, see footnote, Tble 2. b NT, Not tested.

4 882 COOKE TABLE 4. Incidence of ntibiotic resistnce mong coliform bcteri from shellfish Smple No. No. % Resist- % Resistnt isoltes exhibiting resistnce to: % Mulrun no. tested resistnt nt mp psul ycepti MpSI cep tet str nl kn t rf resistnt NT b NT Totl For bbrevitions, see footnote, Tble 2. b NT, Not tested. TABLE 5. Incidence of ntibiotic resistnce mong fecl coliform bcteri from shellfish % Resistnt isoltes exhibiting resistnce to: % Mul- Smple No. No. % Resist- tiply run no. tested re3sistnt nt mp sul cep tet chl str nl tri rif resistnt NT b NT NT NT NT 59.1 Totl For bbrevitions, see footnote, Tble 2. b NT, Not tested. ANTIMICROB. AGENTS CHRMOTHER. mong coliform bcteri from shellfish smples ws 88.7% (Tble 4). Of the fecl coliforms, 72.8% were ntibiotic resistnt (Tble 5), frequency similr to tht found in both coliform nd fecl coliforms from the sewter smples. The incidence of sulffurzole-resistnt coliforms ws in generl lower (12.2%) thn tht found mong the isoltes from sewter (35.4%) or mong the fecl coliforms from the shellfish smples (29.7%). A higher frequency of multiple resistnce ws found in both coliforms nd fecl coliforms from shellfish (>60%) s compred with isoltes from sewter. No gentmicin-resistnt isoltes were obtined from ny source. No prmomycin-resistnt isoltes, chloromycetin-resistnt coliforms, or knmycin-resistnt fecl coliforms were isolted from shellfish. Prmomycin-resistnt isoltes were lwys simultneously resistnt to knmycin (3). Trnsferble ntibiotic resistnce. Selected isoltes, susceptible to nlidixic cid nd resistnt to streptomycin, tetrcycline, chloromycetin, or knmycin, were used s prospective donors of resistnce in mting experiments. A totl of 40.9% of the tetrcycline- nd 58.6% of the streptomycin-resistnt isoltes were cpble of trnsferring ll or prt of their resistnce pttern to the ntibiotic-susceptible recipient strin. Similr frequencies of trnsmissible R fctors were found mong coliform nd fecl coliform isoltes. Only two isoltes were chloromycetin resistnt nd nlidixic cid susceptible, nd neither trnsferred their chloromycetin resistnce determinnt. No explntion cn be offered for the fct tht no isoltes trnsferred resistnce to knmycin despite extensive retesting. This result ws unexpected s high incidence of trnsferbility (>60%) mong knmycin-resistnt coliform bcteri isolted from polluted river wter ws shown by Grbow et l. (8). Similr minimum inhibitory concentrtions to knmycin were exhibited by isoltes from sewter nd shellfish when compred with wildtype R fctors R144 nd R163 (M. Cooke, unpublished dt). Cephlothin resistnce did not trnsfer s n unselected mrker from ny donor strin resistnt to this ntibiotic. DISCUSSION The high incidence of ntibiotic resistnce mong both coliform nd fecl coliform bcteri from sewter nd shellfish smples tken

5 VOL. 9, 1976 ner the Nelson mrine sewge outfll emphsizes tht coliform bcteri cn no longer be regrded s hrmless indictors ofpollution (9). The results reported in this pper confirm tht survivl nd dispersl of ntibiotic-resistnt coliforms nd fecl coliforms occur in polluted sewter, s previously demonstrted (7, 19, 20). Fery et l. (7) found tht bout 20% of resistnt fecl coliforms from fresh wter nd slt wter contined R fctors, but unfortuntely no direct comprison with the results obtined here is possible, s these investigtors mde no distinction between the two wter sources. Smith et l. (20) demonstrted tht there ws no significnt chnge in the rtios of presumptive fecl coliforms nd presumptive fecl coliforms crrying R fctors determining mpicillin resistnce severl miles from mrine sewge outfll. Moreover, the results reported in the present pper show tht the filter-feeding shellfish tend to concentrte coliform bcteri, since MNP vlues for deep-wter smples were considerbly lower thn those for the shellfish. Over the period of study, only two deep-wter smples exceeded n MPN vlue of 70 presumptive coliforms per 100 ml, vlue currently used in the clssifiction of shellfishgrowing wters into the "pproved ctegory" (10). Since oysters nd scllops re often eten rw or fter miniml cooking, the results reported emphsize tht this is potentil public helth hzrd. Antibiotic-resistnt bcteri, including those infectiously resistnt by virtue of trnsmissible R fctor, usully outnumber pthogenic bcteri in wste wters nd sewge effluents. In this investigtion no Slmonell bcteri were isolted from shellfish smples (Cooke, unpublished dt). Ingestion of R- fctor-crrying coliforms by humns my led to trnsfer of resistnce to the norml intestinl flor or to susceptible pthogen. Although the miniml infective dose required to estblish R+ strins my be numericlly lrge (9, 15, 22), considerble numbers of coliform nd fecl coliform bcteri could be ingested from contminted shellfish nd be sufficient for trnsient coloniztion ofthe intestinl trct, even in the bsence of ntibiotic therpy. Limited studies of certin lctose-negtive isoltes nd of isoltes representtive of the norml bcteril flor of the shellfish which were cpble of growth t 11 C nd 15 C hve shown tht the mjority of these bcteri lso exhibit multiple ntibiotic resistnce, in some cses to s mny s eight of the ntibiotics used in the screening test (Cooke, unpublished dt). FECAL COLIFORMS FROM SEAWATER AND SHELLFISH 883 Sewge tretment plnts, lthough often reducing the number of coliform bcteri in effluents, re not selectively effective in removing ntibiotic-resistnt bcteri (21). Authorities responsible for estblishing nd mintining wter nd shellfish qulity criteri should tke into ccount the likely significnce to public helth of multiple ntibiotic resistnce mong coliform nd fecl coliform bcteri. However, it hs lso been demonstrted tht 79% of the coliform bcteri isolted from mussels collected from freshwter lkes known to be remote from importnt sources of humn nd niml pollution were resistnt to one or more ntibiotics (4). Furthermore, ntibioticresistnt fecl coliforms my hve selective dvntge once disseminted into nturl wters. (M. Cooke, N.Z. J. Mr. Freshwter Res., in press). ACKNOWLEDGMENTS I m indebted to the Medicl Reserch Council of New Zelnd for grnt to purchse equipment, nd to Joy M. Lmb for excellent technicl ssistnce. LITERATURE CITED 1. Americn Public Helth Assocition Recommended procedures for the exmintion of se wter nd shellfish, 4th ed. Americn Public Helth Assocition, Inc., New York. 2. Anderson, E. S The ecology of trnsferble drug resistnce in the Enterobcteri. Annu. Rev. Microbiol. 22: Chbbert, Y. A., J. G. Budens, nd D. H. Bounchud Medicl spects of trnsferble drug resistnce, p In G. E. W. Wolstenholme nd M. O'Connor (ed.), Bcteril episomes nd plsmids. Cib Symposium. Churchill, London. 4. Cooke, M. D Antibiotic resistnce mong coliform nd fecl coliform bcteri isolted from the freshwter mussel Hydridell menziesii. Antimicrob. Agents Chemother. 9: Dtt, N Epidemiology nd clssifiction of plsmids, p In D. Schlessinger (ed.), Microbiology Americn Society for Microbiology, Wshington, D.C. 6. Edwrds, P. R., nd W. H. Ewing Identifiction of Enterobctericee, 3rd ed. Burgess Publishing Co., Minnepolis. 7. Fery, T. W., A. B. Sturtevnt, nd J. Lnkford Antibiotic-resistnt coliforms in fresh nd slt wter. Arch. Environ. Helth 25: Grbow, W. 0. K., 0. W. Prozesky, nd J. S. Burger Behviour in river nd dm of coliform bcteri with trnsferble or non-trnsferble drug resistnce. Wter Res. 9: Grbow, W. 0. K., 0. W. Prozesky, nd L. S. Smith Drug resistnt coliforms cll for review of wter qulity stndrds. Wter Res. 8: Houser, L. S. (ed.) Ntionl Shellfish Snittion Progrm. Mnul of opertions. Prt 1. Snittion of shellfish growing wters. Public Helth Service Publiction no. 33. United Sttes Deprtment of Helth, Eduction, nd Welfre, Wshington, D.C. 11. Jnusen, W. A Oysters: retention nd excretion of three types of humn wterborne disese bcteri. Helth Lb. Sci. 11:20-24.

6 884 COOKE 12. Linton, K. B., P. A. Lee, M. H. Richmond, W. A. Gillespie, A. J. Rowlnd, nd V. N. Bker Antibiotic resistnce nd trnsmissible R-fctors in the intestinl coliform flor of helthy dults nd children in n urbn nd rurl community. J. Hyg. 70: Meynell, E., nd N. Dtt Mutnt drug resistnt fctors of high trnsmissibility. Nture (London) 214: Report The bcteriologicl exmintion of wter supplies, 4th ed. Her Mjesty's Sttioners' Office, London. 15. Richmond, M. H R fctors in mn nd his environment, p In D. Schlessinger (ed.), Microbiology Americn Society for Microbiology, Wshington, D.C. 16. Slnetz, L. W., C. H. Brtley, nd K. W. Stnley Coliforms, fecl streptococci nd Slmonell in se- ANTIMICROB. AGENTS CHEMOTHER. wter nd shellfish. Helth Lb. Sci. 5: Smith, D. H R fctors medite resistnce to mercury, nickel, nd coblt. Science 156: Smith, H. W Incidence in river wter of Escherichi coli contining R fctors. Nture (London) 228: Smith, H. W Incidence of R+ Escherichi coli in costl bthing wters of Britin. Nture (London) 234: Smith, P. R., E. Frrell, nd K. Dunicn Survivl of R+ Escherichi coli in se wter. Appl. Micro. biol. 27: Sturtevnt, A. B., nd T. W. Fery Incidence of infectious drug resistnce mong lctose-fermenting bcteri isolted from rw nd treted sewge. Appl. Microbiol. 18: Wtnbe, T Infectious drug resistnce in bcteri. Curr. Top. Microbiol. Immun. 56:43-98.