Isolation of Salmonellae and Other Potential Pathogens from

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, My 1976, p Copyright 1976 Americn Society for Microbiology Vol. 31, No. 5 Printed in U.SA. Isoltion of Slmonelle nd Other Potentil Pthogens from the Freshwter Aqurium Snil Ampullri KAREN H. BARTLETT AND T. J. TRUST* Deprtment of Bcteriology nd Biochemistry, University of Victori, Victori, British Columbi, Cnd V8W 2Y2 Received for publiction 6 Jnury 1976 The freshwter qurium snil (Ampullri spp.) ws demonstrted to crry s mny s 108 vible mesophilic bcteri per g of met plus shell. Some 16 gener of bcteri were identified, with grm negtives predominting. Enrichment culture techniques enbled the isoltion of slmonelle from 24 of 42 lots of 200 g ech. The slmonelle comprised eight different serotypes, including Slmonell newport, Slmonell sint-pul, nd Slmonell infntis. This ssocition of slmonelle with snils my contribute to cses of humn slmonellosis, since other qurium species hve lredy been shown to contribute to mny such cses. The snils were lso found to commonly hrbor Pseudomons eruginos nd, occsionlly, Edwrdsiell trd. Orgnisms of the genus Slmonell re mong the most widely distributed pthogens in nture, nd Slmonell infection is widespred in humns nd other nimls throughout the world (4, 18). Becuse of its universl incidence, its diverse mnifesttions, nd its complex epidemiology, slmonellosis now constitutes serious thret to the helth of humns nd other nimls (18, 25). The problem is of such mgnitude tht, in spite of numerous publictions on the slmonelle nd their effects on vrious hosts, few countries cn ssess ccurtely the nnul toll of humn illness or economic loss of livestock s result of these infections (18). To erdicte such n infection, it is necessry to identify the min reservoirs nd vehicles of Slmonell nd to brek the links in the chin of trnsmission of the orgnism to humns. Recently, there hs been considerble publicity bout the public helth risks ssocited with the sle of smll pet qurium turtles nd tortoises. One estimte puts the number of cses of humn slmonellosis ssocited with these pets t 300,000 per nnum in the United Sttes (27). In Cnd, the ssocition of slmonelle with these species hs resulted in lws bnning the importtion of qurium turtles nd tortoises. In the United Sttes, the Food nd Drug Administrtion hs lso bnned the interstte sle of these nimls. For the pst 2 yers, this lbortory hs been exmining the bcteril popultion of other qurium species (23) nd qurium diets (24). Recently, we turned our ttention to the freshwter qurium snil (Ampullri spp.), since it hs been observed tht edible snils obtined from swmps in Morocco for consumption in North Americ crry with them Slmonell species (2). Significnt numbers of qurium snils re sold to the public, nd, if crrying slmonelle, these snils my present public helth risk similr to tht presented by the qurium turtles. The present study therefore ws designed to determine the microflor ssocited with the common qurium snil, to quntitte their bcteril lod, nd to demonstrte the presence or bsence of slmonelle in this microflor. 635 MATERIALS AND METHODS Snil smples. A totl of 49 smples of snils were purchsed from 33 retil outlets in Duncn, Nnimo, Port Alberni, Vncouver, nd Victori, British Columbi. Snils were supplied in plstic continers contining wter tken from the qurium in which the snils hd been housed. Smples were trnsported to the lbortory nd ssyed immeditely. Snil smples weighed from 3.2 to 50.6 g, with men weight of 8.2 g. An dditionl 42 lots of 200 g ech were tested. These snils were obtined directly from supplier in Florid. Quntittive bcteriologicl exmintion. Smples were diluted with iced 0.1% (wt/vol) peptone wter (ph 7.2) nd homogenized in sterile blending jr t high speed six times, for 30 s ech time. To ensure tht overheting did not occur, the diluted smple ws cooled in n ice bth between homogeniztions. Duplicte dilutions were prepred in 0.1% peptone wter, nd the vible mesophilic bcteri present were enumerted on Trypticse soy gr (Bltimore Biologicl Lbortories [BBL]), Mc- Conkey gr (BBL), nd pseudocel gr (BBL) by the drop-plte method of Miles nd Misr (16).

2 636 BARTLETT AND TRUST Inoculted Trypticse soy gr ws incubted erobiclly nd nerobiclly t 20, 30, or 37 C for 48 h, wheres inoculted McConkey gr nd pseudocel gr were incubted erobiclly t 37 C for 48 h. Anerobiosis ws obtined by using nerobic jrs with disposble hydrogen-crbon dioxide genertor envelopes (BBL). The procedures of the Americn Public Helth Assocition, Inc. (1) were followed for most-probble-number estimtes. Presumptive coliforms were estimted by the 10:1:0.1 multitube method by using McConkey broth (BBL) (8, 27) nd were confirmed by seprte loop trnsfer from positive presumptive coliform tubes to McConkey broth nd incubted t C. The most-probble-number estimtion of enterococci ws crried out in ethyl violet zide broth (BBL) (14) nd ws confirmed by seprte loop trnsfers to fresh ethyl violet zide broth. Qulittive bcteriologicl enumertion. A representtive of ll the colony types ppering on pltes contining 30 to 50 colonies ws selected. Three different persons were used to pick colonies to further reduce selection bis. The isoltes were then purified nd mintined by weekly trnsfer on Trypticse soy gr nd storge t 4 C. Selenite-F broth (BBL) nd tetrthionte-brillint green broth (27) were lso inoculted with smples nd incubted t 37 nd 41.5 C. After 48 h the Selenite-F nd tetrthionte-brillint green broths were streked onto Slmonell-Shigell gr (BBL), brillint green gr (BBL), nd bismuth sulfite gr (BBL). Similrly, cetrimide broth ws inoculted nd incubted t 42.5 C nd then streked onto pseudocel gr (BBL). The tests used to chrcterize the isoltes were those previously described (23) nd were obtined from Edwrds nd Ewing (9), Skermn (21), nd Smith et l. (22). Finl identifiction of the isoltes ws bsed on the schemes of Buchnn nd Gibbons (5), Shewn et l. (20), Bin nd Shewn (3), Cown nd Steele (7), Hugh (11), Edwrds nd Ewing (9), Wever et l. (26), nd Lewis (13). Seprtion of Vibrio species from the otherwise Vibrio-like nerogenic eromonds nd plesiomonds ws fcilitted by testing for cetein production, sensitivity to compound 0/129, fermenttion of inositol, liquefction of geltin, nd decrboxyltion of lysine (10). Identifiction within the Enterobctericee ws confirmed by using the API TABLE 1. APPL. ENVIRON. MICROBIOL. system (Anlytb Products, Inc., New York). The serotypes of isoltes biochemiclly identicl to slmonelle were determined with "O" nd "H" ntiser (BBL) nd confirmed serologiclly by the Ntionl Enteric Reference Lbortory. RESULTS Quntittive bcteriologicl exmintion. The results in Tble 1 show tht snils re populted by s mny s 108 vible mesophilic bcteri per g of met plus shell. The men counts obtined when plted dilutions were incubted t 20, 30, or 37 C were similr, s were the numbers of bcteri recovered when pltes were incubted nerobiclly. As mny s 106 of these orgnisms were cpble of growth in the presence of cetrimide t 37 C (on pseudocel gr), lthough the mjority of these orgnisms did not produce pyocynin, suggesting tht they were not Pseudomons eruginos. Typicl pigment-producing strins of this orgnism were, however, isolted on occsion from dilutions s high s Some 107 of the orgnisms present were cpble of growth in the presence of bile slts (on McConkey gr), suggesting likelihood of fecl origin for mny of the orgnisms. Of these, s mny s 106 were lctose fermenters. The results of the most-probble-number ssys performed on the snils re shown in Tble 2. Using this technique, s mny s 10" completed coliforms per 100 ml were demonstrted, nd countble numbers of coliforms were present in ll smples tested. As mny s 105 of these orgnisms were fecl coliforms, which were recovered from 6 of the 23 smples tested. All smples tested contined countble enterococci with numbers s high s 105/100 ml. Qulittive bcteriologicl exmintion. Some 727 of the most numerous species were chrcterized, nd the results re shown in Tble 3. One of the most common gener isolted ws Pseudomons. The mjority of the Totl bcteril numbers present on freshwter qurium snils No. of smples Estimted no. of vible orgnisms Incubtion conditions" tested Men Rnge Aerobic incubtion 20 C, TSA x x x C, TSA x x x C, TSA x x x C, PA x 105 TFC(1)-5.3 x C, Mc (totl) x x x C, Mc (lctose +) x x x 107 Anerobic incubtion 37 C, TSA x X x 108 TSA, Trypticse soy gr; PA, pseudocel gr; Mc, McConkey gr. TFC(n), Too few orgnisms to count in (n) smples.

3 VOL. 31, 1976 TABLE 2. SALMONELLAE IN AQUARIUM SNAILS Most probble numbers of coliforms nd enterococci populting freshwter qurium snils No. of No. of posi- MPNI/100 ml in positive smples Assy tionci smples tive smtions" tested ples Men Rnge Completed coliforms 02, 37 C, Mc x x x 109 Fecl coliforms 02, 44.5 C, Mc x x x 105 Confirmed enterococci 02, 37 C, EVA x x x 105 Mc, McConkey broth; EVA, ethyl violet zide broth. bmpn, Most probble numbers. 637 TABLE 3. Frequency of isoltion of bcteril species from freshwter qurium snils No. of sm- No. of Species isolted isoltes No. of which ples from iso- smples ses lted tested Acinetobcter Aeromons Alcligenes Bcillus Chromobcter Citrobcter lb Clostridium Edwrdsiell Enterobcter Escherichi Flvobcterium Proteus Pseudomonsc Slmonell Streptococcus Vibrio Weights of 49 smples (3.2 to 50.6 g). b Weights of dditionl 42 smples (200 g). c P. eruginos isolted on 28 occsions from 28 smples. pseudomonds were nonpigmented species; however, typicl pyocynin-producing P. eruginos ws isolted from 28 smples, wheres fluorescent strins were isolted on 14 occsions. The most common eromond isolted ws Aeromons hydrophil, recognized in 19 smples. The Enterobctericee were well represented nd comprised 69% of the isoltes. A lrge number of Citrobcter were chrcterized, C. freundii on 244 occsions nd C. diversus on 76 occsions, in prt due to the fct tht H2S-producing colonies on Slmonell-Shigell gr pltes were closely exmined to determine the presence of Slmonell. Proteus species were identified in 62 smples nd included P. mirbilis, P. morgnii, P. rettgeri, nd P. vulgris. Escherichi coli ws observed in 20 smples, nd in 21 smples contining Enterobcter 68% were E. cloce nd 32% were E. hfnie. No Slmonell were isolted from the first 49 smples of snils (verge weight, 8.2 g). However, when the smple size ws incresed Slmonell ws redily isolted. When n dditionl 42 lots of 200 g ech were processed by the regulr procedures, slmonelle were isolted from 24 lots. These slmonelle were shown to belong to eight different serotypes (Tble 4). It should be noted tht more thn one serotype per smple ws encountered in 10 of the smples tested. In ddition to these slmonelle, Edwrdsiell trd ws identified on 10 occsions. Grm-positive identifictions include 30 isoltions of enterococci nd 29 isoltions of clostridi, which included Clostridium bifermentns, C. botulinum, C. butyricum, C. fllx, C. glycolicum, C. perfringens, C. sordelii, nd C. subterminle. DISCUSSION This study hs reveled previously unreported zoonotic reservoir of slmonelle. This disquieting finding hs obvious public helth implictions since it seems likely tht the snils contminte other qurium species, s well s the wter nd hrdwre of quri, with these pthogenic bcteri. There is reson to believe tht this ssocition of slmonelle with snils will contribute to cses of humn slmonellosis, since other qurium species, nmely, the smll terrpins, hve lredy been shown to contribute to mny cses of humn slmonellosis. It would seem desirble tht regultory ction be tken to control the interntionl nd intrntionl shipment nd subsequent sle of qurium snils unless they cn be certified Slmonell-free. At the very lest, those persons hndling lrge shipments of these invertebrtes must be mde wre of the possible risk of infection. Vrious gencies, including the World Helth Orgniztion, re becoming concerned with the incresing incidence of Slmonell infections worldwide. Of the slmonelle detected in the current investigtion, Slmonell newport, S. sint-pul, nd S. infntis re mong the 10 most frequently isolted serotypes from humn sources (6); however, incresing numbers of new serotypes re lso being ssocited with humn infections. It is

4 638 BARTLETT AND TRUST TABLE 4. Frequency ofisoltion ofslmonelle from 200-g smples of freshwter qurium snils No. of sm- Species No. of iso- ples from ltes which isolted" Slmonell florid S. tennessee 9 7 S. hvn 6 4 S. infntis 4 4 S. sint-pul 4 1 S. newport 3 3 S. jv 2 2 S. mikwsim 1 1 Totl of 42 smples tested. worth noting tht the biochemicl chrcteristics nd serologicl properties of number of these isoltes were demonstrted to be unique, nd complete description of these nd other unique slmonelle isolted from qurium sources will pper in subsequent report. The present investigtion hs reveled tht qurium snils re lso crriers of E. trd. This species is cpble of producing disese ptterns similr to those cused by Slmonell (12) s well s other diseses of humns nd other nimls (19). The ssocition of P. eruginos with qurium snils my lso hve public helth significnce. This is the first report of such n ssocition, nd the findings point to the qurium s being nother source of nosocomil infections with this species. Although it is hrd to quntitte the numbers of quri present in hospitls nd rest homes, it is certinly not uncommon to find quri in peditric wrds, nd in less creful hospitls quri re even found in burns wrds. These quri re mintined by hospitl stff, presenting the opportunity for these workers to come into direct contct with P. eruginos. If dequte disinfection procedures re not followed fter contct with quri, chnce certinly exists for the spred of this potentil pthogen to ptients. The sme cn be sid for doctors' nd dentists' offices. It is importnt to note tht since the snils will contminte the qurium. wter direct contct with the snils is not necessry, nd simple contct with the wter or qurium hrdwre will suffice. In fct, the potentil often exists for more widespred distribution of the orgnism from the qurium by erosol formed by the ction ofthe qurium ertor. Becuse of this potentil public helth risk posed by P. eruginos, studies hve been initited to type isoltes of this orgnism from qurium sources to determine whether they include types known to be ssocited with humn infections. APPL. ENVIRON. MICROBIOL. The other bcteril species found to be ssocited with the snils were similr to those found in wter contining ornmentl fish nd smll pet green turtles, lthough the totl numbers of these bcteri on the snils were generlly higher thn those present in the wter in these other studies. In terms of humn infection, most of these orgnisms cn be regrded s nonpthogens. However McCoy nd Seidler (15) hve suggested tht the possibility of humn infections from some of these species should not be overlooked. For exmple, there re incresing numbers of reports of humn infections in which fluorescent pseudomonds, eromonds, nd Citrobcter nd Enterobcter species re implicted. These infections include genito-urinry trct infections, enterocolitis, nd dirrhe in children nd bcteremi nd septicemi in debilitted persons. All these species re present in significnt numbers on the qurium snils exmined. The methods used for commercil production of qurium snils fcilitte their ccumultion of lrge numbers of microorgnisms, since these invertebrtes breed nd feed on mud flts. If these snils re to continue to be offered for sle to the public, studies must be initited to determine the procedures necessry to rid them of pthogenic microorgnisms. ACKNOWLEDGMENTS We re indebted to H. Lior, Chief, Ntionl Enteric Reference Centre, Bureu of Bcteriology, Lbortory Centre for Disese Control, Ottw, for the Slmonell serotype determintions. LITERATURE CITED 1. Americn Public Helth Assocition Stndrd methods for the exmintion of wter nd wstewter, p Americn Public Helth Assocition Inc., New York. 2. Andrews, W. H., C. R. Wilson, A. Romero, nd P. L. Poelm The moroccn food snil, Helex spers, s source of Slmonell. Appl. Microbiol. 29: Bin, N., nd J. M. Shewn Identifiction of Aeromons, Vibrio nd relted orgnisms, p In B. M. Gibbs nd D. A. Shpton (ed.), Identifiction methods for microbiologists, prt B. The Society for Applied Bcteriology Technicl Series no. 2. Acdemic Press Inc., New York. 4. Bowmer, E. J The chllenge of slmonellosis mjor public helth problem. Am. J. Med. Sci. 247: Buchnn, R. E., nd N. E. Gibbons (ed.) Bergey's mnul of determintive bcteriology, 8th ed. The Willims & Wilkins Co., Bltimore. 6. Center for Disese Control Slmonell surveillnce report. Center for Disese Control, Atlnt. 7. Cown, S. T., nd K. J. Steele Mnul for the identifiction of medicl bcteri. Cmbridge University Press, Cmbridge. 8. Deprtment of Helth nd Socil Security The bcteriologicl exmintion ofwter supplies, 4th ed. Her Mjesty's Sttionery Ofrice, London.

5 VOL. 31, Edwrds, P. R., nd W. H. Ewing Identifiction of Enterobctericee, 3rd ed. Burgess Publishing Co., Minnepolis. 10. Evelyn, T. P. T First records of vibriosis in Pcific slmon cultured in Cnd, nd txonomic sttus of the responsible bcterium, Vibrio nguillrum. J. Fish. Res. Bord Cn. 28: Hugh, R Pseudomons nd Aeromons, p In J. E. Blir, E. H. Lennette, nd J. P. Trunt (ed.), Mnul of clinicl microbiology. Americn Society for Microbiology, Bethesd, Md. 12. Jordon, G. W., nd W. K. Hdley Humn infection with Edwrdsiell trd. Ann. Intern. Med. 70: Lewis, D. H Predominnt erobic bcteri of fish nd shellfish. Se-Grnt College publiction TAMU- SG , Texs A & M University, College Sttion. 14. Litsky, W., W. L. Mllmnn, nd C. W. Fifield Comprison of the most probble numbers of Escherichi coli nd enterococci in river wters. Am. J. Public Helth 45: McCoy, R. H., nd R. J. Seidler Potentil pthogens in the environment: isoltion, enumertion, nd identifiction of seven gener of intestinl bcteri ssocited with smll green pet turtles. Appl. Microbiol. 25: Miles, A. A., nd S. S. Misr The estimtion of the bcteril power of the blood. J. Hyg. 38: Morse, E. V., nd M. A. Duncn Slmonellosisn environmentl helth problem. J. Am. Vet. Med. Assoc. 165: Robinson, R. A Slmonell infection: dignosis nd control. N. Z. Vet. J. 18: Shrm, V. K., Y. K. Kur, nd I. P. Singh SALMONELLAE IN AQUARIUM SNAILS 639 Frogs s crriers of Slmonell nd Edwrdsiell. Antonie vn Leeuwenhoek J. Microbiol. Serol. 40: Shewn, J. M., G. Hobbs, nd W. Hodgkiss A determintive scheme for the identifiction of certin gener of Grm-negtive bcteri, with specil reference to the Pseudomondcee. J. Appl. Bcteriol. 23: Skermn, V. B. D A guide to the identifiction of the gener of bcteri, 2nd ed. The Willims & Wilkins Co., Bltimore. 22. Smith, D. B., K. M. Tomfohrde, D. L. Rhoden, nd A. Blows API system: multitube micromethod for identifiction of Enterobctericee. Appl. Microbiol. 24: Trust, T. J., nd K. H. Brtlett Occurrence of potentil pthogens in wter contining ornmentl fishes. Appl. Microbiol. 28: Trust, T. J., nd V. G. Money Bcteril popultion of diets for qurium fishes. J. Fish. Res. Bord Cn. 29: Veterinry Record Slmonellosis: bne not yet beten. Vet. Rec. 96: Wever, R. E., H. W. Ttum, nd D. G. Hollis The identifiction of unusul pthogenic Grm-negtive bcteri. Center for Disese Control, Atlnt. 27. Wells, J. G., G. M. Clrk, nd G. K. Morris Evlution of methods for isolting Slmonell nd Arizon orgnisms from pet turtles. Appl. Microbiol. 27: World Helth Orgniztion Interntionl stndrds for drinking wter, 2nd ed. World Helth Orgniztion, Genev. Downloded from on November 20, 2018 by guest