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Journal of Stored Products Research 44 (2008) 198–203

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Diversity and antibiotic resistance of enterococci associatedwith stored-product insects collected from feed mills$

Zeb Larsona, Bhadriraju Subramanyama,�, Ludek Zurekb, Tim Herrmana,1

aDepartment of Grain Science and Industry, Kansas State University, Manhattan, KS 66506, USAbDepartment of Entomology, Kansas State University, Manhattan, KS 66506, USA

Accepted 27 August 2007

Abstract

Diversity and antibiotic resistance of enterococci associated with stored-product insects were assessed by screening 298 live adults from

nine insect species collected from six feed mills in the midwestern United States. The eight antibiotics tested were tetracycline, ampicillin,

erythromycin, vancomycin, chloramphenicol, ciprofloxacin, streptomycin, and neomycin. Enterococci were isolated from seven out of

nine insect species collected. A majority of enterococci and aerococci were isolated from the red flour beetle, Tribolium castaneum

(Herbst). Approximately 18% of the insects tested positive for bacteria on mEnterococcus agar, and the mean colony forming units

ranged from 2� 101 to 1.3� 105 per insect. Bacteria isolated on mEnterococcus agar included Enterococcus faecium, Enterococcus

gallinarum, and Aerococcus viridans 3. Isolates of E. faecium displayed complete or intermediate resistance, most frequently to neomycin,

tetracycline, and erythromycin. In addition, many E. faecium isolates exhibited intermediate resistance to vancomycin. The majority of

A. viridans 3 isolates were susceptible to all antibiotics, while isolates of E. gallinarum displayed resistance to neomycin. Enterococci and

aerococci are nosocomial human pathogens and are implicated in secondary infections, mainly in immuno-compromised individuals.

Additionally, enterococci are considered an important reservoir of antibiotic resistance genes that can be horizontally transferred to

other bacteria, including serious human pathogens. Our data reinforce the need for pest management to reduce the availability of vectors

(insects) for dissemination of microorganisms carrying antibiotic resistance genes in the feed mill environment.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Stored-product insects; Enterococci; Antibiotic resistance; Feed safety

1. Introduction

Commercial feed mills produce formulated feeds forcattle, horses, poultry, and swine, as well as speciality feedsfor animals such as geese, goats, moose, and buffalo. In theUnited States, approximately 3000 feed mills produce121million tons of feed for various animal species (Feed-stuffs, 2003). The use of antibiotics as growth promotersand for prophylaxis is common in livestock production inthe United States, but the European Union has banned

ee front matter r 2007 Elsevier Ltd. All rights reserved.

r.2007.08.007

a trade name in this paper does not constitute an endorsement

ansas State University or Texas A&M University.

ing author. Tel.: +1785 532 4092; fax: +1 785 532 7010.

ess: sbhadrir@ksu.edu (B. Subramanyam).

ress: State Chemist and Director, Office of the Texas State

A&M University, 445 Agronomy Road, College Station,

.

much of this practice. One reason for this ban is concernregarding the use of antibiotics in feed for livestockproduction and development of resistance in bacterialstrains of clinical importance (Franz et al., 1999).Poor sanitary conditions, high temperatures, and ample

food resources throughout the year make feed mills wellsuited for insect incidence, survival, and population explo-sions (Mills, 1992). Several stored-product insect species areassociated with feed mills in the United States (Rilett andWeigel, 1956; Triplehorn, 1965; Loschiavo and Okumura,1979; Pellitteri and Boush, 1983), Canada (Mills and White,1993), Japan (Kiritani et al., 1963), and Greece (Buchelos andKatopodis, 1995). However, very few studies have addressedthe medical and veterinary importance of these insects in thestored-product environment, including feed mills.Stored-product insects have been reported to harbor

many potentially pathogenic bacteria. For example, the

ARTICLE IN PRESSZ. Larson et al. / Journal of Stored Products Research 44 (2008) 198–203 199

lesser mealworm, Alphitobius diaperinus (Panzer) frompoultry brooder houses was reported to carry Salmonella

spp., Escherichia coli (Harein et al., 1970), Micrococcus

spp., Streptococcus spp., and Bacillus subtilis (de las Casaset al., 1972). Alphitobius diaperinus sampled from turkeybrooder houses were positive for Streptococcus spp. andB. subtilis (Harein et al., 1972). The granary weevil,Sitophilus granarius (L.), from laboratory colonies andgrain storage facilities was identified as a potentialreservoir for Escherichia intermedia, Proteus rettgeri,Proteus vulgaris, B. subtilis, Serratia marcescens, Strepto-

coccus spp., Micrococcus spp., and members of theKlebsiella-Aerobacter group (Harein and de las Casas,1968). In addition, S. granarius has been shown to transferSalmonella montevideo from contaminated to uncontami-nated wheat (Husted et al., 1969).

To our knowledge, there are no studies addressing theassociation between stored-product insects in feed millsand enterococci. Enterococci are Gram-positive, catalase-negative cocci that are ubiquitous in the environment andoccur in the digestive tract of animals, soil, contaminatedwater, animal feces, and food products derived fromanimals (Franz et al., 1999). These bacteria are notregarded as primary pathogens, but they are generallyrecognized as nosocomial pathogens worldwide (Lindenand Miller, 1999), especially in immuno-compromisedpeople (Awada et al., 1992). Moreover, enterococci areconsidered an important reservoir for antibiotic resistancegenes that can be spread by horizontal transfer to moreserious human pathogens (Devriese et al., 1992).

Enterococci are common symbionts in the gastro-intestinal tracts of domestic animals including cattle, swine,and poultry (Kuhn et al., 2003), and the use of antibioticsin feed for domestic animals has led to the selection ofantibiotic resistant strains (Quednau et al., 1998). Enter-

ococcus faecalis, Enterococcus faecium, and Enterococcus

hirae have been isolated from feed samples in Sweden andSpain, and E. faecium has been identified from feedsamples in the UK (Kuhn et al., 2003). However, littleemphasis has been placed on enterococci in the feed millenvironment. Our objectives were to determine diversity,concentration, and antibiotic resistance of enterococciassociated with stored-product insects collected from feedmills in the midwestern United States.

2. Materials and methods

2.1. Insect collection

From March through November 2003, 298 live adultstored-product insects were collected from six feed millslocated in Kansas, Missouri, Iowa, Nebraska, and Okla-homa in the midwestern United States. Insects wereindividually collected using sterile forceps and placed intosterile plastic vials for transportation and processing in alaboratory at Kansas State University (Manhatttan, KS).Between 22 and 65 individuals from a variety of stored-

product insect species, A. diaperinus, Cryptolestes spp.,Palorus ratzeburgi (Wissmann) (the small-eyed flourbeetle); Rhyzopertha dominica (F.) (lesser grain borer);Sitophilus zeamais Motschulsky (maize weevil); Stegobium

paniceum (L.) (drugstore beetle); Tribolium castaneum

(Herbst) (red flour beetle); Trogoderma variabile Ballion(warehouse beetle); and Tribolium confusum Jacquelin DuVal (confused flour beetle), were sampled directly frommills and product samples taken from those mills. Insectsamples were placed in a refrigerator (4 1C) after collectionand processed within 1–5 day after collection.

2.2. Bacterial screening

Individual insects of each species were homogenized in300 ml of phosphate buffer saline (PBS) (ICN Biomedicals,OH) and serially diluted (two-, 10-fold dilutions). Eachdilution was drop-plated (33 ml) onto mEnterococcus agar(mENT; Difco, Sparks, MD), trypticase soy broth agar(TSBA; BBL, Sparks, MD) with cycloheximide (36mg per1 l to inhibit fungi), and MacConkey agar (MAC; Difco,Sparks, MD). TSBA was used to determine the diversity ofaerobic bacteria on and within the insect. Enterococci(red and pink colonies) were enumerated on mENT, whichis a selective medium for this genus. MAC was used as aselective medium for Enterobacteriaceae. Plates wereallowed to dry and then placed into an incubator at37 1C (mENT, MAC) or 28 1C (TSBA). The colonyforming units (CFU) were counted 24, 48, and 72 h afterplating.

2.3. Bacterial identification and antibiotic resistance of

enterococci

After the enumeration of colonies on mENT, 1–6 red-pink colonies were selected and streaked for isolation onTSBA. Individual isolates were identified by the APIs 20STREP (Biomerioux, Vitek, MO) test following manufac-turer’s instructions. Antibiotic resistance was determinedby the diffusion disk test technique using Muller-Hintonagar (Difco, Sparks, MD). The eight antibiotics and discconcentrations tested were tetracycline (30 mg), ampicillin(10 mg), erythromycin (15 mg), vancomycin (30 mg), chlor-amphenicol (30 mg), ciprofloxacin (5 mg), streptomycin(300 mg), and neomycin (30 mg) (all BBL, Sparks, MD).All plates were incubated at 37 1C. After 24 h of incubation,the zone of inhibition was measured to differentiateresistant, intermediate, or susceptible isolates.

3. Results

Insects from mills 1 and 5 accounted for 94% of the 53samples that were positive for growth on mENT (Table 1).In addition, about 85% of the 53 insects sampled from mill5 were observed to be positive for colonies on mENT.The number of insect samples positive for growth onmENT ranged from 1 in each of the mills 3, 4, and 6, to 45

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Table 1

Number of beetles sampled from each mill and number of colony forming units (CFU) per beetle on three media

Mill ID Na mEnterococcus agar (mENT) Trypticase soy broth agar (TSBA) MacConkey agar (MAC)

n (%)b Mean CFU7S.E. n (%)b Mean CFU7S.E. n (%)b Mean CFU7S.E.

1 57 5 (8.7) 1.7� 10579.0� 104 12 (21.1) 3.0� 10473.0� 104 1 (1.8) 3.5� 103

2 22 0 0 11 (50.0) 5.9� 10274.8� 102 1 (4.5) 2.4� 103

3 65 1 (1.5) 4.8� 103 42 (64.6) 3.1� 10371.7 103 4 (6.2) 6.0� 10372.7� 103

4 45 1 (2.2) 1.2� 101 9 (20.0) 4.1� 10274.0� 102 1 (2.2) 3.6� 102

5 53 45 (84.9) 6.5� 10372.0� 103 43 (81.1) 1.6� 10472.6� 103 10 (18.9) 2.4� 10278.4� 101

6 56 1 (1.8) 8.4� 103 23 (41.1) 2.4� 10471.6� 104 2 (3.6) 3.5� 10373.4� 103

aN, total number of insects sampled from each mill.bn, number of positive samples.

Table 2

Species of beetles sampled and number of colony forming units (CFU) per beetle on three media

Insect species Na mEnterococcus agar (mENT) Trypticase soy broth agar (TSBA) MacConkey agar (MAC)

n (%)b Mean CFU7S.E. n (%)b Mean cfu7S.E. n (%)b Mean CFU7S.E.

A. diaperinus 4 4 (100.0) 1.3� 10571.1� 105 NTc NTc 1 (25.0) 3.5� 103

Cryptolestes spp. 17 9 (52.9) 4.7� 10374.7� 103 9 (52.9) 6.7� 10374.2� 103 1 (5.9) 1.3� 104

P. ratzeburgi 1 0 NAd 0 NAd 0 NAd

R. dominica 11 9 (81.8) 3.4� 10373.0� 103 8 (72.7) 6.1� 10374.3� 103 5 (45.4) 1.6� 10279.7� 101

S. zeamais 2 1 (50.0) 4.7� 103 1 (50.0) 3.0� 104 1 (50.0) 4.4� 103

S. paniceum 12 0 NAd 5 (41.7) 7.5� 10277.1� 102 1 (8.3) 3.6� 102

T. castaneum 115 28 (24.3) 1.9� 10471.1� 104 59 (51.3) 2.5� 10478.6� 103 6 (5.2) 3.0� 10271.1� 102

T. confusum 116 1 (0.9) 1.2� 101 46 (39.7) 2.2� 10271.2� 102 2 (1.7) 4.3� 10371.9� 103

T. variabile 20 1 (5) 1.1� 103 12 (60.0) 6.0� 10374.0� 103 2 (10.0) 3.5� 10373.4� 103

aN, total number of insects sampled from each mill.bn, number of positive samples.cNT, not tested.dNA, not applicable.

Z. Larson et al. / Journal of Stored Products Research 44 (2008) 198–203200

in mill 5. The mean CFU per beetle ranged from of1.2� 101 for insects from mill 4 to 1.7� 105 for insects inmill 1 (Table 1). On TSBA, 65% of the insects from mill 3and 81% of insects from mill 5 were positive for bacterialgrowth. Insects from mills 1 and 4 had the lowestpercentage of positive samples (o21%) on TSBA agar.Insects from mills 1, 5, and 6 had the greatest mean CFUper beetle (41.6� 104) (Table 1). On the MAC medium,between 1 and 10 positive samples per mill were observed.Mean CFU per beetle ranged from 2.4� 102 (mill 5 insects)to 6.0� 103 (mill 3 insects). The highest percentage ofpositives samples for growth on MAC was found in mill 5,equaling 19% of the 53 insects sampled (Table 1).

The number of beetles that were positive on mENTranged from 1 to 28 (Table 2). Slightly more than 24% ofT. castaneum were positive for Enterococcus spp. orAerococcus spp., with a mean CFU of 1.9� 104. MeanCFU per beetle on mENT ranged from 1.2� 101 to1.3� 105 (T. confusum and A. diaperinus, respectively).Alphitobius diaperinus accounted for 4 of the 298 insectssampled, but recorded 100% positive growth and the

greatest mean CFU per beetle on mENT. Seven insectspecies, represented by 1–59 individuals, were positive forbacteria on TSBA. The mean CFU per beetle amongspecies on TSBA ranged from 2.2� 102 (T. confusum)to 3.0� 104 (S. zeamais). Between 50% and 53% ofT. castaneum, Cryptolestes spp., and S. zeamais sampleswere positive for bacterial growth on TSBA (Table 2).Rhizopertha dominica yielded the highest percentage ofpositive samples of all the insect species. Nearly 73% of the11 individual insects of this species were positive for growthon TSBA. Mean CFU per beetle observed on MAC rangedfrom 1.6� 102 for R. dominica to 1.3� 104 for Cryptolestes

spp. One to six insect samples were positive for growthon MAC medium. About 46% of R. dominica and 50% ofS. zeamais samples were positive for growth on MAC(Table 2).Three bacterial species were identified from mENT

(Table 3). They were Aerococcus viridans 3, Enterococcus

faecium, and Enterococcus gallinarum. Aerococcus

viridans 3 accounted for 63% of the isolates identified,and was recovered from three insect species—T. castaneum,

ARTICLE IN PRESSZ. Larson et al. / Journal of Stored Products Research 44 (2008) 198–203 201

R. dominica, and Cryptolestes spp. Enterococcus faecium

accounted for 33% of the isolates identified from fourspecies of insects: T. castaneum, S. zeamais, T. confusum,and T. variabile. Enterococcus gallinarum represented lessthan 5% of the total isolates identified with one isolateeach from T. variabile and S. zeamais. Isolates fromT. castaneum accounted for 79% of the 43 isolatesidentified.

Antibiotic resistance tests were carried out on 32 of the43 isolates that were identified to species (Table 4). Thesewere 17 isolates of A. viridans 3, 13 of E. faecium, and twoof E. gallinarum. Most of the A. viridans 3 isolates weresusceptible to all antibiotics; only a few isolates tested wereintermediately resistant to erythromycin and streptomycin.In contrast, 62% and 85% of E. faecium isolates showedresistance to tetracycline and neomycin, respectively.Enterococcus faecium also showed resistance to erythro-mycin and ampicillin with 23% and 8% of the isolatesexhibiting complete resistance, respectively (Table 4).Enterococcus faecium isolates also displayed intermediateresistance to chloramphenicol (15% of isolates), strepto-mycin (15%), neomycin (15%), tetracycline (8%), cipro-floxacin (23%) erythromycin (31%), and vancomycin(61.5%). Enterococcus gallinarum isolates were resistantto streptomycin (one of two isolates), neomycin (two oftwo isolates), and tetracycline (one of two isolates), and

Table 3

Incidence of identified bacteria in observed species of insects

Bacterial species Insect species No. of identified isolates

A. viridans 3 Cryptolestes spp. 3

R. dominica 1

T. castaneum 23

E. faecium S. zeamais 1

T. castaneum 11

T. confusum 1

T. variabile 1

E. gallinarum S. zeamais 1

T. variabile 1

Table 4

Occurrence of antibiotic resistance of three species of bacteria isolated from in

A. viridans 3 (n ¼ 17)a E. faecium

Resistant (%) Intermediate (%) Resistant

Erythromycin 0 3 (17.6) 3 (23.1)

Chloramphenicol 0 0 0

Ampicillin 0 0 1 (7.7)

Streptomycin 0 4 (23.5) 0

Vancomycin 0 0 0

Neomycin 0 0 11 (84.6)

Tetracycline 0 0 8 (61.5)

Ciprofloxacin 0 0 0

an, number of isolates.

this species also showed intermediate resistance to strepto-mycin and vancomycin.

4. Discussion

This is the first survey to document and quantifybacterial populations in stored-product insects collectedfrom feed mills. This is also the first report on antibioticresistance profiles of enterococci and aerococci isolatedfrom stored-product insects. Enterococcus spp. are intrin-sically resistant to several antibiotics, such as cephalospor-ins, b-lactams, sulfonamides, and low levels of clindamycinand aminoglycosides. Enterococci have the ability todevelop or acquire resistance to chloramphenicol, erythro-mycin, clindamycin, aminoglycosides, tetracyclines,b-lactams, fluoroquinolones, and glycopeptides such asvancomycin (Franz et al., 2003; Landman and Quale,1997).Antibiotic resistance profiles showed that enterococci

isolated from insects in feed mills displayed varyingresistance levels to antibiotics used in this study. ManyE. faecium isolates displayed resistance to neomycin,tetracycline, and erythromycin. In addition, eightE. faecium isolates showed intermediate resistance tovancomycin. Enterococcus gallinarum represented by twoisolates only was resistant to streptomycin, neomycin, andtetracycline. The majority of aerococci were susceptible toall antibiotics.Resistance of E. faecium to several antibiotics, including

an intermediate resistance to vancomycin, indicates apotential health risk related to horizontal transfer ofantibiotic resistance genes to other bacteria, includinghuman pathogens such as Staphylococcus aureus (Franzet al., 1999; Weigel et al., 2003).Selection for vancomycin-resistant enterococci probably

occurs because of the use of avoparcin as a growthpromoter in animal feed (Witte, 2000). However, the use ofavoparcin as a growth promoter in animal feed has neverbeen allowed in the United States. Therefore, plausiblereasons for the development of vancomycin resistanceobserved in our study are unclear and warrant further

sects collected in feed mills

(n ¼ 13)a E. gallinarum (n ¼ 2)a

(%) Intermediate (%) Resistant (%) Intermediate (%)

4 (30.8) 0 0

2 (15.4) 0 0

0 0 0

2 (15.4) 1 (50.0) 1 (50.0)

8 (61.5) 0 1 (50.0)

2 (15.4) 2 (100.0) 0

1 (7.7) 1 (50.0) 0

3 (23.1) 0 0

ARTICLE IN PRESSZ. Larson et al. / Journal of Stored Products Research 44 (2008) 198–203202

investigation. Antibiotics commonly used in feed in theUnited States include penicillin, neomycin, tetracyclines,and erythromycin. The use of these antibiotics likely selectsfor bacteria with antibiotic resistance genes, and conse-quently increases the potential for the spread of resistanceto other bacteria.

Aerococci are ubiquitous in the environment and arefrequently observed in air and water and on the skin ofhumans. Several studies noted the clinical importance ofthis genus related to endocarditis and bacteremia (Uhet al., 2002). In France, Buu-hoı et al. (1989) observedseveral strains of A. viridans of animal origin that wereresistant to erythromycin, chloramphenicol, streptomycin,and tetracycline, whereas A. viridans 3 isolates in our studywere susceptible to the antibiotics tested.

TSBA was used to determine the general aerobicbacterial population of the insects collected. Growth onthis agar suggests that insects carry a large microbialcommunity. Growth on MAC was minimal, which was notsurprising, because high numbers of enteric bacteria areusually not associated with cereal grains or their by-products. A few studies report Salmonella associated withanimal by-products, such as meat and bone meal, whichwas used in all of the mills. About 67% of meat and bonemeal samples observed in a British survey were positive(Shrimpton, 1989). In addition, Salmonella is reported tobe more frequently present in dust that has accumulatedover time than in the moving mill stock (Whyte et al.,2003).

Stored-product insects are all highly mobile and prolificin the feed mill environment, and many species are capableof flight (e.g., T. castaneum, R. dominica, and T. variabile).The potential for these insects to become vectors forenterococci is possible given the high mean CFU per beetleon mENT. In addition, research performed with otherinsect and bacterial species displayed the potential ofstored-product insects as vectors of other pathogenicbacteria (Harein and de las Casas, 1968; Husted et al.,1969; de las Casas et al., 1972). Hald et al. (1998)conclusively showed transmission of Salmonella enterica

serovar infantis to chicks in poultry brooder houses by thehairy fungus beetle, Typhaea stercorea (L.).

Tribolium castaneum was the second most commonlycollected insect species in this study. This insect is one ofthe most frequently encountered species in feed mill, flourmill, and stored grain environments (Loschiavo andOkumura, 1979; Roesli et al., 2003; Trematerra andSciarretta, 2004). Tribolium castaneum carried a largebacterial population (CFU per beetle on TSBA andmENT), including A. viridans 3 and E. faecium. Samplesof T. castaneum that were positive for growth on mENTmost frequently came from mill 5. Based on the observa-tions of this study, T. castaneum could be considered themost competent vector for antibiotic resistant enterococci.In contrast, T. confusum, which was sampled in compar-able numbers to T. castaneum, did not carry high numbersof enterococci. Tribolium confusum and T. castaneum are

similar with respect to their feeding and morphologicalappearance, with the major difference being that the lattercan fly. The reason for the observed difference in theenterococcal community between these closely relatedinsect species is not known. It is possible, however, thatT. castaneum, due to its ability to fly, is more frequentlyexposed to various environments and this increases itschance to become contaminated by different microbes,including enterococci. This is also supported by the greaterprevalence and higher number of CFU on TSBA fromT. castaneum than that from T. confusum.Although only four samples of A. diaperinus were

processed, all these samples were positive for bacteriaand these insects carried the highest mean CFU per beetleon mENT of 1.3� 105. This species is the largest beetletested, which would explain the larger bacterial population.All samples of this species were obtained from mill 1.Cryptolestes spp. was another insect species that wasfrequently positive for growth on mENT. All nine of thepositive beetles were collected from mill 5.Stored-product insects are cosmopolitan in distribution

and occur in feed mills in the United States and around theworld. Microbes associated with these insects have notbeen well documented or studied. Based on our observa-tions and previous research, it is our conclusion thatstored-product insects are of medical and veterinaryimportance because they can serve as vectors for humanpathogens and antibiotic-resistant strains. While entero-cocci are not primary pathogens, they can spread the genesof antibiotic resistance to more pathogenic bacteria.Clearly, additional studies focused on assessment ofantibiotic resistance of more bacterial isolates are neededto fully evaluate the medical and veterinary importance ofinsects as vectors for antibiotic resistant strains as well as toaddress the concerns of antibiotic use in feed mills. Ourfindings reinforce the need for pest management to reducethe availability of vectors (insects) for microbial dissemina-tion. It is also recommended that antibiotics important inclinical settings are not used as growth promoters and forprophylaxis in animal feed.

Acknowledgments

We thank the participating feed mills and mill managersfor their cooperation in this survey. This research wassupported by grant from USDA-CSREES (IFSIS), andpartly by CSREES-USDA (RAMP) under Agreement No.00-51101-9674. This paper is contribution number 07-198-Jof the Kansas State University Agricultural ExperimentStation, Manhattan, KS.

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