Certain bacteria, some of medical interest, associated with the slug Limax maximus

JOUBiSAL

OF

INVERTEBHATE

Certain

PATHOLOGY

Bacteria,

15,

306-312

(1969)

Some of Medical Interest, the Slug Limax maximus

Associated

with

L. P. ELLIOTT Department

of Biology,

Western

Kentucky Received

University, Bowling July1 4, 1969

The isolation and identification of microorganisms from testinal tract of the slug Limux maximus are presented. Of the surface of the slug, coliforms predominated. Identified human beings were Escherichia coli, serogroups 3, 25, aeruginosa. From the intestinal tract, the coliforms were likely pathogens identified from the tract were Escherichia 83, 139, 0X36, 144/0X44, ClostTidium perfringens, and appears that this slug serves as a carrier and possible microorganisms.

Slugs are mollusks belonging to the class Gastropoda. Limax maximus is an European species that is found commonly in the Bowling Green, Kentucky, area. It is easily identified by its gray color with alternate longitudinal rows of spots and black stripes, replaced by irregular blotches on the mantle. This slug is dirty white below, and the body is covered with coarse elongated tubercles (Pratt, 1916). Dissection is not difficult because of the slug’s posterior bilateral symmetry, its soft outer covering, and the lack of excessive body fluids. Limax maximus can be found on or beneath rocks, logs, or garbage pails; on dishes where food has been set out for animals; or crawling across sidewalks at night. It apparently feeds on plant material (unfortunately, in gardens) and on decaying matter. It requires a moist habitat at all times and is not often found in woods or in places far from man’s habitation. Because these invertebrates are common in man’s environment, it is likely that people make contact with their exterior and intestinal flora, and thus these flora should

be analyzed pathogens.

Green,

Kentucky

42101

the surface and the inthe isolates identified from as possible pathogens for 0X36, and Pseudomonas most prevalent. The most co& serogroups 3, 26, 73, Streptococcus faecalis. It transmitter of pathogenic

for their

potential

as human

MATERIALS AND METHODS Slug Specimens. Fifteen slugs (Limax maximus) were collected from yards in the Bowling Green area in December 1967 and during the period from March to July in 1968 (Table 1). Because of cold weather, none was found in January and February. The slugs were picked up with a sterile paper towel, placed in a tared sterile container, and brought to the laboratory and weighed. The average weight of the slugs was 6.6 g; the heaviest weighed 9.6 g and the lightest weighed 3.8 g. Analysis of Exterior Flora. A captured slug was immediately immersed in 63 ml of 0.1% peptone contained in an Erlenmeyer flask, and was agitated for about 2 min to wash the microbial flora from its surface. Decimal dilutions of the surface washing were made in g-ml peptone-broth blanks. Peptone-broth dilution blanks were used in all experiments because the broth was found by Hauschild et al. (1967) to en-

BACTERIA

TABLE

1

ASSOCIATED

WITH

SLUG

307

Decimal dilutions were made in peptone broth from which aliquots were removed and plated to obtain aerobic and anerobic Weight plate counts. The coliform analysis was of slug made as previously described. Slug no. Date captured (8) From the original peptone broth, 0.1 ml 1 Dec. 4,1967 5.1 was transferred to duplicate enterococci pre2 Dec. 19,1967 5.6 sumptive broth (Difco) which was in3 March 12,1968 3.8 cubated at 45°C for 24 to 48 hr. Positive 4 March 29,1968 6.3 5 April 5,1968 5.5 tubes were streaked on human blood-agar 6 April 12, 1968 5.4 plates (“Manual of Microbiological Meth7 April 19, 1968 5.3 ods,” 1957) to determine their hemolytic 8 April 26, 1968 6.7 activity and to provide stock cultures for 9 17,1968 7.8 May 10 25,1968 9.6 later identification, May 11 June 11,1968 7.2 Sulfite, polymyxin, sulfadiazine (SPS) 12 17,1968 8.5 agar plates (Difco) were inoculated and in13 ;z:: 26,1968 7.4 cubated in an Anaero-Jar (Case Labora14 July lo,1968 6.6 tories, Chicago, Illinois) at 37°C for 24 hr, 15 July 17,1968 8.0 and then the black colonies were counted. Anaerobiosis was established in the Anaerohance quantitative recovery of Clostridium Jar by three consecutive flushings with a perfringens. One ml of each dilution was mixture of 90% N2 and 10% C02. This then transferred to duplicate petri dishes to same procedure was followed for the total which plate count agar (Difco) was added. anaerobic count. The plates were incubated at 37°C for 24 A calibrated loop was used to remove hr and then the resultant colonies were 0.01 ml from the initial peptone broth, counted. which was streaked onto triplicate Littman Tubes of brilliant green lactose bile broth, oxgall agar (Difco) plates and incubated 2% (Difco ), were inoculated with proper at room temperature (approx. 23°C) for 5 portions and incubated at 35°C for 24 hr. days. Then the fungi were counted and Stock cultures were made of transfers from identified or transferred to a slide-chamber eosin methylene blue (EMB) agar plates, culture for identification. to be used later for further testing. Further indentifkation procedures. ColiAnalysis of Intestinal Flora. The slug forms were recognized by their typical was transferred from the flask to a beaker appearance on EMB agar and classified by and immersed in 70% alcohol for 1 min to the standard IMViC (indole, methyl red, kill the exterior flora, and then it was Voges-Proskauer, citrate) reactions (Edaseptically dissected with the aid of a wards and Ewing, 1962). The cultures binocular dissecting microscope. The ap- identified as Escherichia coli were sent to proximately 3-inch-long digestive tract was Dr. Paul J. Glantz (Escherichia coli Typing removed, opened, and its contents trans- Center, University of Saskatchewan, Saskaferred to 9 ml of peptone broth. Gram toon, Canada), who examined them serologically with all available standard E. co& strains were made of microorganisms from these initial tubes to gain a general indica- serums and with 45 unclassified E. coli 0 tion of the possible flora. (No search was serums even after heating the antigens at made for protozoa such as the ciliates 121°C. Cultures reacting with unclassified antiserums were labeled OX. Strains unstudied by Brooks, 1968.) DATEOF

SLUGANALYSIS,ANDWEIGHTOFTHE SLUG (Limax maximus)

308

ELLIOTT

groupable with any of the E. coli 0 or OX antiserums were designated negative (N). Morphologically, those organisms that were pleomorphic, nonsporeforming rods, fragmenting and becoming coccoid with age, were classified as to genus and species by published descriptions ( Bergey’s “Manual of Determinative Bacteriology,” Breed et al., 1957), and according to characteristics determined by accepted methods (Sguros, 1955; and the Society of American Bacteriologists’ “Manual of Microbiological Methods,” 1957). The enterococci were tested further for growth in broth medium containing 0.1% tellurite, growth on methylene blue azide (MBA) medium, and for acid production from glucose in Snyder’s test agar (Difco) as described by Smith and Bodily ( 1967). The nonlactose fermenters that were isolated on EMB agar were tested for cytochrome-oxidase activity with Patho Tee-Co differential test paper ( General Diagnostics Division, Warner-Chilcott Laboratories, Morris Plains, New Jersey). The production of pyocyanine, pyorubrin, and fluorescence pigments and growth at 4°C and 41°C were determined as described by Sutter (1968). Growth on centrimide (Pseudosel agar, BBL) and SS agar (Difco) was also tested. Suspected Clostridium perfringens colonies were tested for nitrate reduction and motility ( Angelotti et al., 1962). These organisms were also tested for the production of opalescence on egg-yolk plates and on human-blood-agar plates hemolysis (“Manual of Microbiological Methods,” 1957). RESULTS

Exterior Flora. The average number of fungi yielded by the slug was 1306. Molds were identified according to Bamett ( 1960). Those isolated in the order of predominance, were Penicillium, Aspergillus, MUCOT, Fus-

arium, and Botrytk Yeasts were found in all samples but not in as great numbers as were molds. The only yeast identified as to genus was Rhodotorula. The average bacterial count was 14 X 104/slug. No correlation was found between counts and season of the year. In four analyses, Bacillus cereus var. mycoides was noticed because of its characteristic growth. Two slugs yielded Arthrobacter aurescens as determined by established criteria (Bergey’s Manual of Determinative Bacteriology,” 1957; Sguros, 1955). The average MPN of coliforms on the surface of the slug was 55/slug. All slugs but four yielded coliforms from their body surface. One colony of each predominant type was picked from the EMB plates and purified. The bacteria were classified into genera and, when feasible, into species from descriptive tests outlined in the section on

IDENTITY OF SLUG

OF AND

TABLE 2 BACTERIA ISOLATES SEROCEWUP OF THE

FROM

SURFACE co&

Escherichia

Slug no.

Culture no.

Generic name

Sero-

2

2A

Enterobacter aerogenes

3

3c 3D

6

6B

7

8

6C 7A 8B

Escherichia coli Escherichiu coli Enterobacter aerogenes Escherichiu coli Escherichiu coli Pseudomonas

9 10

9A 10A

11

11c

12

12A

13

13A

14

15A

group

3 Na

OX36

25

SP.

a N denotes

Escherichia coli Pseudomonas aeruginosa Enterobacter aerogenes Pseudomonas aeruginosa Escherichia coli freundii var. I Enterobacter aerogenes E. coli negative

to serologic

N

tests.

BACTEXUA ASSOCIATED WITH

SLUG

309

Material and Methods. Of these, 13 differWastewater, Am. Public Health Assoc., ent isolates were identified as shown in 1965), 2 Pseudomonas aeruginosa, and 1 Table 2. As can be seen, 6 isolates of Pseudomonas sp. were identified. E. co& Escherichia coli, 4 Enterobacter aerogenes, cultures 6C and 7A had yellowish pigments. 1 Escherichia freundii var. I (“Standard The two P. aeruginosa cultures synthesized Methods for the Examination of Water and pyocyanine and pyorubrin pigments and were fluorescent. They were also hemolytic, grew at 41°C but not at 4°C grew on SS TABLE 3 and centrimide agars, and were oxidase COLIFORMS INSIDE THE ALIMENTARY TRACT positive. The Pseudomonas sp. was fluoresOF SLUC cent, oxidase positive, grew at 4°C but not Slug Culture Seroat 41°C and did not grow on SS or acetno. Identitv no. ETOUD amide agar, and was nonhemolytic. 1 Enterobacter 1A Intestinal Flora. The flora was domaerogenes inantly composed of gram-negative bacilli, 1B Escherichia coli f reundii although some slides had a dominance of var. I gram-positive cocci in chains. Some gram2 2B Escherichia coli OX36 positive rods were observed. Two sIugs 2c Escherichia coli 139 yielded Bacillus cereus var. mycoides, and 3 3B Escherichiu coli 73 seven slugs yielded Arthrobacter aurescens. 3E Enterobacter aerogenes When duplicate plates of gut contents were 4 4A Escherichiu coli N incubated aerobically and anaerobically the N 4B Escherichiu coli average counts were, respectively, 56 X lo6 4W Colifom and 32 X 106. The MPN for coliforms was intermediate found to be 50 X 105. Twenty-four coliform Escher&h&z cold N 5 5A 6 6D Escherichiu coli 26 isolates from the gut were identified and, 7 7c Escherichia coli 83 as Table 3 demonstrates, 14 of these isolates 7E” Colifomn were E. coli, 5 were E. aerogenes, 4 were intermediate intermediate type coliforms, and 1 was Colifonn 8 80 E. freundii var. I. E. coli culture No. 2B, intermediate N 9 9B Escherichia coli 2C, and 4A had yellow pigmentation. A 10 Enterobacter 10 cross-absorbed serum was not available for aerogenes complete confirmation of the 0 group of 10B Escherichia coli N culture 11D. 11 11B Escherichia coli 13 1lDd Escherichiu coli 144/0x44 Table 4 summarizes the other bacteria 12 12c Enterobacter isolated from the intestinal tract. Ten slugs aerogenes harbored Clostridium perfringens, and the 13 13B Escherichiu coli N average count was 17 per total gut content. Escherichiu coli N 14 14B This number was quite low, and since every 14c Enterobacter colony was not tested for nitrate reduction aerogenes Coliform 15 15C~ and motility, it is realized that part of this intermediate number might be other sulfite reducing 0 IMViC reaction was - -t- + -. clostridia such as Clostridium bifermentans. a IMViC reaction was - + + -I-. Ten slugs harbored enterococci. From the c IMViC reaction was - + -I- -. presumptive enterococcus medium, positive * Crossreaction. tubes were streaked on blood agar plates, e IMViC reaction was - - + -.

310

ELLIOTT

mens in Yamada’s work, the lower bacterial counts on the body surfaces of L. maximus Streptococcus TRACT might thus be explained. In our studies, Count of 73% of the slugs carried colifonns on the C. perfringens/ surface of their bodies. Yamada et al. total contents Presence of ( 1960) also found a higher number of bacof tract S. faecalis Slug no. teria in the alimentary canal, with about 1 6 80% of the slugs having coliforms present 14 + 2 in the tract, while about 73% of the slugs 3 4 57 in this work carried coliforms. After feeding 34 5 the slugs on a diet containing an initial 10 + 6 inoculation of E. coli, they found that viable + 7 15 E. coli were excreted with some being re+ 8 tained in the tract for 8 days. They sug9 14 + 10 + 10 gested that slugs are potential carriers of 11 + pathogenic microbes. 12 In analyzing Tables 2, 3, and 4, some in13 4 + teresting observations can be made. Slug + 14 15 5 + 7 was interesting because different E. coli serogroups were carried on the surface than 10 No. positive 10 were present in the intestinal tract. Slug 6 carried an enteropathogenic E. coli strain and no hemolysis was noted. All isolates which is known to cause epidemic diarrhea grew on the MBA agar. The organisms of the newborn ( Bailey and Scott, 1966). were positive for the tests described in the A number of E. coli serogroups was isosection on Materials and Methods and were lated. Of these Glantz and Jacks (1968) identified as Streptococcus faecalis. reported that E. coli serogroup 3, 25, 26, 73, 83, and 139 were found in bodies of water. They discussed many ways in which DISCXJSSION such waters might be contaminated with Account was taken of the bacteria and E. coli and factors that influence their fungi carried on the slug’sskin. It is difficult survival. Whether or not L. maximus conto distinguish normal flora from transient tacted E. coli from polluted water is specuflora since the slug is in constant contact lative. E. coli 0139 is common in pigs and with the soil. pathogenic for them (P. Glantz, personal Yamada et al. (1960) found that the bac- communication). Slug 6 carried E. coli terial population ranged from 12 X lo4 to OX36 on its surface, while slug 2 harbored 37 X lo8 on the body surface of the slug this type in its intestinal contents. This Limux fiavus, and more than half of them serogroup has been isolated from the feces carried coliforms. They found the numbers of a healthy pig (Glantz, 1968). E. coli in the alimentary canal of the slug to be 0144/0X44 has been isolated from pigs by between 29 X lo5 to 26 X lo7 per individGlantz (P. Glantz, personal communicaual, with about 80% carrying coliforms. To tion). The slugs’ gluttonous eating of a a degree, these results agree with what was variety of foods might explain why they found in our work. Since the slugs studied harbor these bacteria, particularly E. coli. in this work were not specifically collected The distance a slug roams in search for food from polluted habitats as were the speci- is not known, although probably it is not SLUGS

TABLE 4 Clostridium perfringens faecalis IN THEIR INTESTINAL

HARBORING

AND

BACTEXIA

ASSOCIATED

very far. Many of the slugs were collected in a suburb about one block from a farm, but no pigs were raised on the farm. Slug 10 was particularly interesting since it carried 5 different genera of possible pathogens for man. One generally thinks of E. aerogenes as being saprophytic, but they have been incriminated as pathogens (Bailey and Scott, 1966). In the study of the intestinal flora of the black slug, Arion ater, Shrewsbury and Barson (1947) found that the dominant flora was composed of gram-negative rods which they arranged into 5 groups based on biochemical activity, and which they did not consider to be human pathogens. They also found a few clostridia in the slugs they analyzed but did not confirm which species they were. Although it is known that C. perfringens is widely distributed in soil, feces, and sewage, this paper documents another way in which the organism could be spread. Hypothetically, slugs can spread the organism to vegetables which might be made into dehydrated soups and sauces. Nakamura and Kelly (1968) analyzed 55 samples of dehydrated soups and sauces and found C. perfringens in 18.2% of the samples. Shrewsbury and Barson’s (1947) results suggest that they isolated S. faecalis from the intestinal contents of the slug A. titer, although the biochemical tests used were not the usual tests that are run to distinguish the organism. In this study, further evidence for the identification of S. faecalis was that their growth in tellurite broth in still culture produced black cells, which agreed with the observations of Tucker et al. (1966). They found S. faecium tended to produce gray cells, The growth on MBA agar substantiated the fact that the orga. nisms were fecal enterococci (Smith and Bodily, 1967). In this work the isolation of S. faecalis from the slug was not surprising, since the organism has been found in the intestinal tract of both man and animals.

WITH

311

SLUG

The organism has been encountered occasionally in subacute bacterial endocarditis and, possibly more frequently, in peritonitis, wound infections, and infections of the urinary tract ( Deibel, 1964). The habitat of A. aurescerw appears to be the soil. Since the slug feeds from the soil and plants, it seems reasonable that this organism would be found on the surface and in the gut of the slug. The slug is at least somewhat involved in the dissemination of this organism. The finding that P. aeruginosa is carried on the surface of slugs suggests the closeness of this slug species to man’s environment. Hoadley and McCoy (1968), in studying the ecology of P. aeruginosa, found the carrier state of calves for P. aeruginosa is influenced by the calves exposure to a reservoir of the organism such as a human carrier or contaminated water or milk. Through the observations described, it is confirmed that the slug Limax maximus, which is common in this area, is a potential carrier of pathogenic bacteria in its digestive system and on its skin. ACKNOWLEDGMENTS The capable technical assistance of Martha Schell is acknowledged. This study was supported by a research grant from the Faculty Research Committee of Western Kentucky University.

REFERENCES AMERICAN PUBLIC HEALTH “Standard Methods for Water and Wastewater.” Public Health Association,

ASXKUTION. 19%. the Examination of 12 ed. American New York. 769 pp.

ANGELOTTI, R., HALL, H. E., FOTER, M. J., AND 1962. Quantitation of CloLEWIS, K. H. stridium perfringens in foods, Appt. Microbiol. 10, 193-199. BAILEY, W. R., AND SCOTT, E. G. 1966. “Diagnostic Microbiology,” 2nd ed. 342 pp. C. V. Mosby, St. Louis.

312

ELLIOTI-

BARNETT, H. L. 1960. “Illustrated Genera of Imperfect Fungi.” 2nd ed. 225 pp. Burgess, Minneapolis. BREED, R. S., MURRAY, E. G. D., AND SMITH, N. R. 1957. “Bergey’s Manual of Determinative Bacteriology.” 7th ed. 1094 pp. Williams and Wilkins, Baltimore. 1968. Tetrahymenid ciliates as BROOKS, W. M. parasites of the gray garden slug. Hilgurdia 39, 205-276. DEIBEL, R. H. 1964. The group D streptococci. Bacterial. Reu. 28, 330-366. EDWARDS, P. R., AND EWING, W. H. 1962. “Identification of Enterobacteriaceae.” 258 pp. Burgess, Minneapolis. GLANTZ, P. J. 1968. Identification of unclassified Escherichiu coli strains. Appl. Microbial. 16, 417-418. GLANTZ, P. J., AND JACKS, T. M. 1968. An evaluation of the use of Escherichiu coli serogroups as a means of tracing microbial pollution in water. Water Resources Res. 4, 625638. HAUSCHILD, A. H. W., ERDMAN, I. E., HILSHEIMER, R., AND THATCHER, F. S. 1967. Variations in recovery of Clostridium peffringens on commercial sulfite-polymyxin-sulfadiazine ( SPS ) agar. J. Food Sci. 32, 469473. HOADLEY, A. W., AND MCCOY, E. 1968. Some observations on the ecology of Pseudomonas aeruginosa and its occurrence in the intestinal tracts of animals. Cornell Vet. 58, 354-363. NAKAMURA, M., AND KELLY, K. D. 1968. Clos-

tridium perfringens in dehydrated soups and sauces. J. Food Sci. 33, 424-426. PRATT, H. S. 1916. “A Manual of the Common Invertebrate Animals.” 737 pp. A. C. McClurg, Chicago. SGUROS, P. L. 1955. Microbial transformations of the ,tobacco alkaloids. I. Cultural and morphological characteristics of a nicotinophile. J. Bacterial. 69, 28-36. SHREWSBURY, J. F. D., AND BARSON, G. J. 1947. A contribution to the study of the bacterial flora of Arion ater. Proc. Sot. Appl. Bacterial. 2, 70-76. SMITH, R. F., AND BODILY, H. L. 1967. Use of a methylene blue azide medium for isolation of enterococci. Appl. Microbial. 15, 108’71090. SOCIETY OF AMERICAN BACTERIOLOGISTS. 1957. “Manual of Microbiological Methods.” 315 pp. McGraw-Hill Book Co., New York. SUTTER, V. L. 1968. Identification of Pseudomonus species from hospital environment and human sources. Appl. Microbial. 16, 15321538. TUCKER, F. L., THOMAS, J. W., APPLEMAN, M. D., GOODMAN, S. H., AND DONOWE, J. 1966. X-ray diffraction studies on metal deposition in group D streptococci. J. Bacterial. 92, 1311-1314. YAMADA, G., YONEMOTO, S. MATWMOTO, H., TODA, N., AND IBUSIJKI, 0. 1960. Studies on slugs as vectors of pathogenic microbes. J. Osoku City Med. Center 2, 707-717.