Gram-positive cocci from apiarian sources

JOURNAL

OF INVERTEBRATE

PATHOLOGY

Gram-Positive

42, 187-195 (1983)

Cocci from Apiarian

Sources1

MARTHA GILLIAM AND BRENDA J. LORENZ U.S. Department

of Agriculture,

Agricultural 2000 E. Allen

Research Service, Carl T. Hayden Road, Tucson, Arizona 85719

Bee Research

Center,

Received July 12. 1982; accepted November 23, 1982 Frass from the greater wax moth, Galleriu me//one//a, obtained from feral colonies of honey bees, Apis mellifera; from domesticated (managed) honey bee colonies; and from a laboratory culture of the wax moth was sampled for Gram-positive cocci. One hundred twenty-three of these organisms were isolated and identified. Frass from domesticated colonies yielded only one isolate. Equal numbers of isolates (61) were obtained from frass from feral bee colonies and from the wax moth culture. Catalase-negative cocci were predominant in frass from feral colonies, whereas catalase-positive cocci were the most common isolates from frass from the wax moth culture. Catalase-positive cocci were identified as Stuphylococcus epidermidis and Micrococcus sp. Catalase-negative cocci were Streptococcus fuecalis var. ,fueca/is and S. fuecium. These results are discussed in relation to the rarity of Gram-positive cocci associated with honey bees, pollen, and nectar in Arizona and the frequency of association with honey bees and wax moth frass of bacteria resembling Arthrobacter spp. that appear as Gram-positive cocci during one stage of the life cycle. KEY WORDS: Apis mellifera, honey bee; Galleriu me//one//u, wax moth; frass; Gram-positive cocci.

INTRODUCTION and Fedorova, 1963; Tysset and Durand. 1968; Tysset and Rousseau, 1968; Tysset et For some years, we have been studying al., 1969; Vecchi, 1959). Streptococcus pluthe microflora (bacteria, molds, and yeasts) ton is known to cause European foulbrood of the intestinal content of worker honey of honey bee larvae (Bailey, 1963a, and S. bees, Apis mellifera (Gilliam and Prest, faecalis, a common secondary invader in 1972; Gilliam and Valentine. 1974, 1976; the disease (White, 1920). is ubiquitous, ocGilliam et al., 1977), nectar (Gilliam, 1975), curring on many plants and their flowers, in and pollen (Gilliam, 1979a, b). In these studsoils (Mundt, 1961), and on insects (Eaves ies, Gram-positive cocci were rare. From and Mundt, 1960). the thousands of platings that we have done Eaves and Mundt (1960) concluded that from these various sources at different streptococci are rarely mutualists or comtimes of the year, only 17 Gram-positive mensals in insects but rather are transient cocci were isolated from the intestinal conresidents that enter the digestive tract durtent of adult worker bees, Staphylococcus ing feeding or are transferred mechanically spp. were isolated from nectar of saguaro to external surfaces. Gram-positive cocci cactus, and 6 Gram-positive cocci were isoform a realtively small percent of the total lated from almond pollen. microflora of honey bees (El-Leithy and ElGram-positive cocci have been isolated Sibaei, 1972; Trilenko and Fedorova. 1963; from healthy honey bees by other researchers (Brown, 1928; Eaves and Mundt. 1960; Tysset et al., 1969). Tysset et al. (1969) conEl-Leithy and El-Sibaei, 1972; Fekl, 1956: cluded that group D streptococci found in bees are temporary residents of higher Kvasnikov et al., 1971; Martin and Mundt, plants, particularly of the floral organs, and 1972; Rizzo, 1959; Trilenko, 1965; Trilenko are in transit in the digestive tract of the healthy bee that becomes infected while 7 Mention of a proprietary product or company name does not imply endorsement by the U.S. De- collecting nectar and pollen. Thus, the abpartment of Agriculture. sence or paucity of these microbes in nectar 187 0022-2011183 $1.50 Copyright All rights

0 1983 by Academic Press. Inc. of reproductionin any form rexwed.

188

GILLIAM

and on pollen from Arizona may explain their rarity in the honey bees we have examined. In contrast, the gut of all stages of laboratory-reared greater wax moths, Galferia mellonella, characteristically contains S. faecalis, a bacterium that is specially adapted to survive and multiply in the gut. Transmission to filial populations occurs by contamination of the surfaces of eggs and oviposition substrates with meconial and fecal deposits of adults (Bucher, 1963). Bucher (1963) found that when young larvae began to feed, they acquired a bacterial flora of several species, but after a week they contained pure populations of S. faecalis. The presence in young larvae of Micrococcus was considered a result of air contamination of the rearing medium, eggs, and honey comb. Gram-positive cocci other than S. faecalis, such as Staphylococcus albus, S. aureus, and Micrococcus spp. appeared sporadically in cultured wax moths (Bucher and Williams, 1967). Bucher (1963) showed that about one-tenth of the S. faecafis organisms remained in the gut when its contents were voided by mature larvae before spinning and that about the same proportion was retained in the adult gut after the meconium was excreted at emergence. He considered that staphylococci and micrococci probably came from the skin of the experimenters and suggested that S. faecalis inhibited the growth of other microorganisms in larvae. Dudziak (1975a) isolated over 500 strains of microorganisms from the alimentary tract of laboratory-reared wax moth larvae, pupae, and imagoes. Among these organisms were S. faecalis, Micrococcus, Staphylococcus, and Sarcina. S. faecalis was the most common organism in all stages and was not always associated with pure beeswax but was always associated with wax from the laboratory culture of wax moths. Dudziak assumed that the wax on which the larvae feed becomes contaminated with excreta and thus with S. faecalis.

AND

LORENZ

Dickman (1933) indicated that esterases produced by the intestinal microbes of wax moth larvae cause changes in beeswax in vitro and may play an intermediate role in the digestion of wax by larvae. However, Dudziak (1975b) isolated no bacteria from wax moth larvae that decomposed wax or palmitic acid and suggested that the intestinal bacterial flora may utilize the indirect products of lipid metabolism. Waterhouse (1959) obtained results with axenic cultures of the greater and lesser wax moths which suggested that larvae, unaided by microbes. can digest some, but not all, of the wax constitutents. Jarosz (1975) found that the lysozyme from S. faecalis was particularly strong against Micrococcus, Sarcina, and Bacillus, the species of microorganisms that constituted the main admixture in S. faecalis populations in larvae. S. faecalis suppresses bacteria ingested with food by producing bacteriocin, an antibiotic-like substance with a narrow range of bactericidal activity, and by releasing a lysozymelike enzyme. These results suggested that the latent form of an autoiytic enzyme released from the cell walls of S. faecalis was activated in the presence of insect proteases to produce L-forms of the bacteria, which are sensitive to the killing action of both bacteriocin and lysozyme (Jarosz. 1979). The purpose of the present investigation was to sample for Gram-positive cocci from additional apiarian sources other than those that we had examined previously to determine whether these bacteria are indeed rare associates of honey bee colonies in Arizona. For this purpose, we examined wax moth frass from feral honey bee colonies, domesticated (managed) honey bee colonies, and laboratory-reared wax moth cultures. MATERIALS

AND

METHODS

Wax moth frass was obtained from feral honey bee colonies living in rock caves near Cottonwood, Arizona (Taber, 1979),

GRAM-POSITIVE

COCCI

from domesticated honey bee colonies near the Carl Hayden Bee Research Center in Tucson, and from a laboratory culture of greater wax moths that were fed a diet of pablum and glycerin (Dutky et al., 1962). Ten samples from each source were collected during September and October. A 0.15-g sample of frass was placed in 5 ml of sterile distilled water in a sterile test tube. The frass was broken up with a sterile applicator stick. A loopful of this mixture was then streaked onto duplicate plates of brain heart infusion agar (Difco), nutrient agar (Difco), and malt extract-yeast extract agar containing 1% glucose (Wickerham, 1951). Our experience had shown that most bacteria from apiarian sources can be isolated by use of these media. One set of plates of each medium was incubated under aerobic conditions at 37°C and the other at 25°C for 10 days.

FROM

Selected colonies were transferred to nutrient agar plates to test for purity and were stained by the Gram reaction. All Grampositive cocci were maintained on slants of nutrient agar, and colonies were separated into groups on the basis of their microscopic morphology and the catalase reaction. All Gram-positive, catalase-positive cocci were identified according to the BairdParker (1963, 1966) and MacFaddin (1976) schemes (Table 1) by the use of the following tests: pigmentation of colonies on nutrient agar, sugar fermentation and acid production (International Subcommittee on Staphylococci and Micrococci, 1965), growth in brain heart infusion broth containing 15% NaCl, coagulase production, acetoin production in Difco VP medium, DNase production on Difco DNase medium with methyl green, nitrate reduction, lique-

TABLE SCHEME

FOR TESTING

AND

IDENTIFICATION

Group Test Pigment” Acid from glucose Aerobic Anaerobic Acid from mannitol Aerobic Anaerobic Acid (aerobic) from Lactose Maltose L-Arabinose Coagulase Acetoin pH in VP medium DNase Gelatin liquefaction Arginine decarboxylase Phosphatase Nitrate reduction Growth in 15% NaCl

I

189

APIARIES

I

OF GRAM-POSITIVE

CATALASE-POSITIVE

1 (Staphy1ococcu.s) Subgrouph 11

111

Group

COCCI”

2 (Micrococcrts) Subgroup<

IV

v

VI

I

2

3

4

5

6

7

8

VI

v2

VI.2

VI,2

v2

VI.2

v2

2

v2

2

v2

VI

VI

v3

+ +

+ +

+ +

+ +

+ +

+ +

+

+

•t

+

-t

+

v

v

+ +

-

-

-

-

+ -

V + -

V + -

V -

+ 4.4 -

-

v +

+ + f

v v v

+

+v+v+-f v f + - + + +

v v -

+ + -

v

v ~ v

5.0 -

4.5 -

4.4 -

4.6 -

4.6

5.1

5.5

5.3

6.5

6.6

V V + V V

+ + + + V

v + -

v v -

v + -

v

v

-v-vv+ v v

v

v +

v -

v -

v +

v v

v v

vvvvvvvv v

v

+

+

v

-

+ + 4.4 + + + + + V

-

c1Compiled from Baird-Parker (1963. 1966) and MacFaddin (1976). h I = S. aureus, II-VI = S. epidermidis. 1’ 7 = M. luteus, 8 = M. roseus. 4 V = variable; I = golden to yellow, 2 = white, 3 = pink.

++v+--

v

5.0

5.2

+

190

GILLIAM

faction of gelatin using Difco thioglycollate medium without indicator containing 5% gelatin, production of arginine decarboxylase using Moller decarboxylase base medium (MacFaddin, 1976), and phosphatase production on PDP nutrient agar medium (MacFaddin, 1976). Catalase-negative cocci were identified by a combination of schemes from Shatoch (195.5), Facklam (1972), Jelinkova and Rotta (1978), and MacFaddin (1976) (Table 2) by use of the following tests: hemolysis on blood agar, pigmentation of colonies on nutrient agar, growth in brain heart infusion broth containing 6.5% NaCl, bile solubility, optochin sensitivity, fermentation in KF streptococcal broth (BBL), acid production from sugars (Jelinkova and Rotta. 1978). bacitracin sensitivity, reaction in litmus milk, bile-esculin, Lancefield grouping using Streptex (Wellcome Reagents), production of tyrosine decarboxylase (Jelinkova and Rotta, 1978). liquefaction of gelatin as described above, production of arginine decarboxylase as described above, hydrolysis of hippurate (Gordon et al., 1973), hydrolysis of starch, and reduction of tetrazolium (Facklam. 1972). Additional information on enzymatic activity (Table 3) was obtained by testing ten representative isolates with the API ZYM system (Analytab Products) according to the manufacturer’s directions. At least one of each identified species was tested. RESULTS

One hundred twenty-three Gram-positive cocci were isolated and identified from wax moth frass (Table 4). Staphylococcus epidermidis subgroups II, V, and VI and Micrococcus sp. subgroup 5 were the catalase-positive organisms isolated. A total of 50 catalase-positive cocci were found in the samples examined. We isolated 73 catalase-negative cocci, all of the enterococcus group. None of these organisms was isolated from domesticated bee colonies.

AND

LORENZ

Catalase-negative cocci were more common than catalase-positive cocci in wax moth frass. Although Gram-positive cocci were rare in frass from domesticated bee colonies (there was only one isolation), they were isolated from all ten frass samples from feral bee colonies and from the wax moth culture. Equal numbers of isolates (61) were obtained from frass from feral bee colonies and from the wax moth culture. Catalase-negative cocci were isolated from all samples of frass from feral colonies, but catalase-positive cocci were isolated from only four of the samples. Catalase-positive cocci were the most common isolates from frass from the wax moth culture; they were found in all samples. Catalase-negative cocci were found in only seven samples. The enzymatic activities of the representative isolates as determined by the API ZYM system are listed in Table 3. All isolates were negative for alkaline phosphatase, trypsin, a-galactosidase, a-mannosidase, and d-fucosidase and positive for butyrate esterase and caprylate esterase-lipase. Acid phosphatase, chymotrypsin, the aminopeptidases, phosphoamidase, and the other glycosidases were produced by some of the cocci. Myristate lipase activity was found consistently only in Micrococcus sp. DISCUSSION

In examining Gram-positive cocci from apiarian sources, we found that we needed to devise schemes for identifying the catalase-positive and catalase-negative organisms we isolated. The available literature on these organisms is confusing, particularly regarding catalase-positive cocci. Therefore, we believe that the information we compiled from the literature (Tables 1, 2) will be useful to other insect microbiologists, though we recognize that some clinical microbiologists may consider it an oversimplification. However, our experience in microbial taxonomy of isolates associated with honey bees has demonstrated

GRAM-POSITIVE

COCCI

FROM

APIARIES

191

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AND LORENZ TABLE

ENZYMES

PRODUCED

3

BY GRAM-POSITIVE

Sfreptococcus faecalis var. faecalis

Enzyme Alkaline phosphatase Butyrate esterase Caprylate esterase-lipase Myristate lipase Leucine aminopeptidase Valine aminopeptidase Cystine aminopeptidase Trypsin Chymotrypsin Acid phosphatase Phosphoamidase a-Galactosidase B-Galactosidase B-Glucuronidase a-Glucosidase B-Glucosidase N-Acetyl-B-glucosaminidase a-Mannosidase a-Fucosidase

COCCI

Micrococcus

+ +

sp.

Staphylococcus epidermidis

+ +

Vl’ + + + + + + -

+ + + + + + -

+ + -

+ +

+ + + + + -

0 V = variable.

that more variability exists within species than has usually been reported in the literature dealing primarily with isolates from mammalian sources. Hence we list as variable those test results that are generally considered almost always positive or negative but are recognized as variable in a small number of cases. As more organisms from diverse sources are examined, more variability will undoubtedly be reported. We prefer to recognize this variability

rather than assigning new names to isolates associated with insects which may differ from recognized species in only one or two biochemical reactions. From our work that is cited and reviewed under Introduction, we conclude that Gram-positive cocci are rarely found in honey bees, nectar, or pollen from Arizona. This may be due to climate, vegetation, or other environmental or ecological factors. However, honey bees in Arizona do con-

TABLE ISOLATIONS

OF GRAM-POSITIVE,

4

CATALASE-POSITIVE FROM WAX MOTH

AND FRASS

CATALASE-NEGATIVE

Source

Organism and subgroup

COCCI Number of isolates

Catalase-positive cocci Staphylococcus

S. epidermidis. S. epidermidis. Micrococcus

epidermidis.

V VI sp., 5

II

Feral bee colony Domesticated bee colony Wax moth culture Feral bee colony Wax moth culture Feral bee colony Wax moth culture

3 6 I 37

Feral bee colony Wax moth culture Wax moth culture

56 6 II

Catalase-negative cocci Streptococcus var. faecalis S. .frecirrm

faecalis

GRAM-POSITIVE

COCCI

tain a diverse intestinal microflora represented by Bacillus spp., Enterobacteriaceae, molds, and under certain conditions, yeasts. Other common microbial associates of honey bees in our area are bacteria resembling Arthrobacter spp., highly pleomorphic organisms that appear as Grampositive cocci in one stage of the life cycle. On the basis of microscopic morphology, these bacteria can be mistaken for Grampositive cocci if Gram stains are not periodically performed on growing cultures during extended incubation. From wax moth frass, we isolated 82 of these cultures, 52 from frass from feral bee colonies, and 30 from the wax moth culture. It is possible that other researchers have not reported these bacteria from apiarian sources because of the difficulty of culturing them on common microbiological media, although these organisms may have been referred to as Bacterium eurydice (Achromobacter eurydice) (Bailey, 1963b). In the present work, Gram-positive cocci were rare in wax moth frass from domesticated honey bee colonies but were isolated from frass of a wax moth culture and from feral honey bee colonies. Catalase-negative cocci were more common in frass from feral bee colonies, but catalase-positive cocci were isolated more often from frass from the wax moth culture. These differences may be due to diet since the cultured wax moths received no beeswax. It is also possible that some S. epidermidis and MicrococC’US sp. in frass from the cultured wax moths originated from the hands of the experimenters, although we used sterile forceps for collection and transfer of the samples. Another difference in our isolates that may be due to diet is the ability of streptococci from frass from the wax moth culture to metabolize glycerol, and the inability, or only weak ability, of streptococci from frass from feral bee colonies to do so. This may be due to an inducible enzyme that was activated by the presence of glycerol in the diet of the wax moth culture.

FROM

APIARIES

193

Honey bees from feral colonies and from domesticated or managed colonies are probably very similar, although differences between feral and domesticated colonies in domicile, geographical location, and surrounding vegetation may have influenced the microbial complement of frass. Our results may also be influenced by the time of the year (September and October) that the frass samples were collected and by the fact that we were able to collect frass from only one laboratory culture of the wax moth. Other researchers have emphasized microbial examination of the gut of wax moth larvae, pupae, and adults. Our results presented in this paper and our unpublished data indicate that frass, which is much easier to obtain and to handle, may well reflect the microbiological complement of the alimentary tract. In fact, our data from frass are in close agreement with data obtained by Dudziak (1975a) from the alimentary tract. Results of the enzyme tests showed that esterases and lipases are produced by the cocci isolated. These microbial enzymes may aid in digestion of wax by larvae as suggested by Dickman (1933), or they may allow the bacteria to utilize the indirect products of lipid metabolism (Dudziak, 1975b). A wide range of enzymes capable of acting on a variety of substrates was produced by the Gram-positive cocci as evidenced by results of both the taxonomic tests and the API ZYM system. The involvement of these enzymes in biological events in vivo has yet to be determined. However, many of the bacterial enzymes detected in the present study were also found in the intestine of G. mellonella by Plantevin and Nardon (1972). ACKNOWLEDGMENT We thank Dr. H. G. Spangler of our laboratory providing the frass samples from the wax moth ture.

for cui-

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194

GILLIAM

AND

BAILEY, L. 1963b. The habitat of ‘Bacterium eurydice.‘J. Gen. Microbial., 31, 147-150. BAIRD-PARKER, A. C. 1963. A classification of micrococci and staphylococci based on physiological and biochemical tests. J. Gen. Microbial.. 30. 409-427. BAIRD-PARKER, A. C. 1966. Methods for classifying staphylococci and micrococci. In “identification Methods for Microbiologists” (B. M. Gibbs and F. A. Skinner, ed.), Part A, pp. 59-64. Academic Press, New York/London. BROWN, F. M. 1928. Enzymes and bacteria in the honey bee. Amer. Mus. Novit., 304. BUCHER, G. E. 1963. Survival of populations of Streptococcus faecalis Andrewes and Horder in the gut of Galleria mellonella (Linnaeus) during metamorphosis and transmission of the bacteria to the filial generation of the host. J. Insect Pathol.. 5, 336-343. BUCHER, G. E.. AND WILLIAMS. R. 1967. The microbial flora of laboratory cultures of the greater wax moth and its effect on rearing parasites. J. Invertebr. Pathol..

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WATERHOUSE,

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WICKERHAM.

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Bull.,

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