& Vol. 105,January February, 1994 47 A LIST OF BACTERIAL FLORA RESIDING IN THE MID AND HINDGUT REGIONS OF SIX SPECIES OF CARRION BEETLES (COLEOPTERA: SILPHIDAE) Glenn Berdela, Bonnie Lustigman,^PaulP. S'uibeck^ ABSTRACT: Forty-eight carrion beetles (Silphidae: Nicrophorus tomentosus, Nicro- phorus orbicollis, Oiceoptoma noveboracense, Oiceoptoma inaequale, Necrophila ameri- cana, Necrodes surinamensis) were dissected and the midgut, hindgut, and associated hemolymph were cultured for bacteria. Eight specimens ofeach ofthe six species above were utilized. Analytical profile index rapid biochemical systems were used for bacterial identifications.Thirty-twobacteriawereidentifiedtospeciesandseventogenuslevel.Six isolations were limited to "group" identifications and eight morphologically distinct bac- teria could not be identified using the rapid biochemical test strips because ofdata base limitations. Manyofthespeciesare known opportunisticpathogens. Most entomologists, and many field naturalists, have been aware of the existence ofcarrion beetles formanyyears and, in fact, the last quar- tercentury hasseen a fair number ofpublications on this family in ento- mological literature. Only recently, however, has a serious effort been made to survey the medically important true bacteria associated with the silphids. In that study (Solter et al. 1989) 36 carrion beetles (Silphidae: Nicrophorus tomentosus Weber, Oiceoptoma noveboracense (Forster), Necrophila americana (L.)) were collected in the Great Swamp National Wildlife Refuge (GSNWR), Basking Ridge, N.J. They were dissected and the midgut, hindgut and associated hemolymph were cultured for bacteria. Nineteen bacteria were identified to species and seven to the genus level. Several of the identified coliform and staphy- lococci bacteria were known opportunistic pathogens. The primary objective of the current study was to expand on Solter et al. (1989), establishing the full spectrum of intestinal bacterial micro- flora harbored among a greater number of silphid species. In our study we were able to collect six of the eight species of silphids found in GSNWR (Shubeck 1983). In addition to the three species studied by Solter et al. (1989) our study included also Nicrophorus orbicollis Say, Oiceoptoma inaequale (F.), and Necrodes surinamensis (F.). Beetle tax- onomy follows Anderson (1982). These six species are common at dif- ferent times throughout the spring and summer months, in a variety of habitats including fields and forests. It was hoped such information 1 ReceivedJune 30, 1993. AcceptedJuly20, 1993 2 Biology Department, MontclairState College,UpperMontclair,NewJersey07043 3 Present Address:6.5 Pleasantview Ave.,New Providence,N.J. 07974 ENT. NEWS 105(1):47-58,January& February, 1994 ENTOMOLOGICALNEWS 48 would help further define an epidemiological role for the carrion beetles. A secondary objective ofthis study was to develop and utilize bacte- riological techniques suitable for this type of study. Efforts to identify the large numbers and varieties of intestinal bacteria of silphid beetles have proven a formidable task. Colonies representing different species may not always be readily distinguishable when viewed among polymi- crobic growth. This approach contrasts with that taken in a clinical microbiology laboratory, where efforts are directed only at identifying orcharacterizingsuspected causitive agentsofinfection. Isolation media commonly employed in clinical applications were used here to assist in the detection of such pathogens as Salmonella, Shigella, Yersinia enter- coloitia, Campylobacter jejuni, Staphylococcus aureus, and pathogenic streptococci and clostridia. These media were also used in conjunction with biochemical tests as a means ofgrouping isolates. Identification of pure culture isolates was accomplished using the API 20E, Rapid-STREPT, STAPH-Trac, STAPH-Ident and AN- IDENT identification systems. Such commercially available systems offer advantages over conventional methods including ease of use, stan- dardized selection of biochemical tests, relatively low cost and quick response. Profile numbers are constructed from the results obtained and compared with those in an established data base. It should be under- stood that data bases supporting each of these systems have been derived primarily from human clinical strains. While systems like API are often used to identify isolates ofnon-human origin, there is a possi- bility ofobtainingprofile numbers either not contained in the data base, or ofthose yieldingincorrect identifications. In spite ofthis, since bacte- ria associated with carrion beetlesmaybe expected to represent those of entomological, veterinaryorotherenvironmental origins, we considered this study ofsufficient importance to be pursued. MATERIALS AND METHODS Collection. Six species of carrion beetles (Silphidae) were collected at the Great Swamp National Wildlife Refuge in Basking Ridge, N.J. Collection was accomplished by the use of 5 ground surface type traps described elsewhere (Shubeck 1976). Three of these traps were placed along a straight line at 5 meter intervals within a mixed-oak forest, one in an adjacent field, and the fifth near a swamp. Each trap was baited with a fresh (1-7 days) and a stale (8-14 days) chicken leg, each in a sep- arate styrofoam cup. Seven collections were made during the summerof 1988. Collectingdateswere;06/06,06/13,06/20,06/27,07/08,08/02,08/08. Vol. 105,January& February, 1994 49 Collected beetles were stored in a refrigerator and dissected within 72 hours. Only those that were alive at the time ofdissection were used. The beetles were killed bycrushing their heads with forceps, then rinsed in 2% Amphyl disinfectant and rinsed twice in sterile distilled water. They were then dissected and the midgut, hindgut and associated hemolymph were removed and placed in vials containing 1.0 ml of Trypticase Soy Broth with 15% glycerol. At least 8specimens ofeach of the following 6 species were prepared: Necrophila americana, Oice- optoma inaequale, Oiceoptoma noveboracense, Nicrophorus orbicollis, Necrodes surinamensis and Nicrophorus tomentosus.The cultures were then stored at or below -60 C. Culture Techniques. The following protocol is based on the work of Solter et al. (1989) but does include some changes which increased the likelihood of identifying additional bacterial species. The contents of each vial were thawed, homogenized and preincubated for 2 hours in lactose broth to facilitatecellularrepair.Ten uLaliquotswere then inoc- ulated into Gram Negative (GN) Broth, Selenite Cysteine Medium and 0.1% sterile peptone water. Two drops ofthe preincubated homogenate were also plated onto Columbia Colistan Naladixic Acid (CNA) Blood Agar Plate (BAP), Mannitol Salt Agar and anerobic media. The anero- bic media included Anerobic Phenylethyl Alcohol Blood Agar Plates (ANA PEA BAP), Anerobic Kanamycin-Vancomycin Blood Agar Plates (ANA KV BAP). These were cultured in a Gas Pak pouch at 30- 35 C. The GN Broth was then plated onto the following agar plates; Salmonella Shigella (SS), Xylose Lysine Deoxycholate (XLD), MacConkey's (MAC), Levine Eosin Methylene Blue (EMB), Bismuth Sulfite (BS), Pseudosel and Cefsulfodin Irgasen Novobiocin Agar Base (Yersinia selective agar) (CIN agar). The inoculated tubes of0.1% pep- tone water were futher serially diluted and plated onto Trypticase Soy Agar (TSA) fortotal aerobiccounts (TACs). Aerobic plateswere incu- bated at 35 C for 24 hours except for the TAC's which were incubated for 72 hours. Representative colonial types from the primary isolation plates were then subcultured onto fresh media to obtain pure cultures. All gram neg- ative organisms were streaked onto TSA, gram positive onto Columbia CNA BAP, and anerobes onto ANA PEA BAP. Stock cultures of the purified isolates were prepared and stored at -6O C. Each gram nega- tive isolate was streaked onto MAC, XLD, BS, and CIN agars. The plates were incubated and the morphological characteristics of the col- onies determined.Tubes of Triple Sugar Iron (TSI), Lysine Iron Agar (LIA),and Urease Broth were inoculated and then incubated. 50 ENTOMOLOGICALNEWS Gram positive bacteria were characterized and grouped on the basis of colonial morphology observed on Columbia CNA BAP, TSA and coagulase and reactions in Oxidation Fermentation (OF) Glucose Bile Esculin andTrypticase SoyBroth (TSB) with 6.5% NaCl and the results were recorded. Anerobic isolates were tested for oxygen tolerance by incubating PEA BAP under both aerobic and anerobic conditions. Final identification was performed using the following API identifi- cation systems; API 2OE for gram negative bacteria, API Staph-Ident and Staph-Trac (catalase positive gram + cocci), API Rapid-Strep (cata- lase negative gram + cocci) and API An-Ident for anerobes. RESULTS Most of the beetles were collected during the four weeks of June, with the exception ofN. tomentosus which was common duringJuly and early August. Nineteen ofthe 48 beetles tested (39.6%) yielded aerobic countson the orderof10 E7 cfu's (colonies) perspecimen,while 22 bee- tles (45.8%) yielded 10 E6 cfu's perspecimen, 6 beetles (12.5%) yielded 10 E5 cfu's per specimen and 1 (2%) yielded 10 E4 cfu's per specimen. There was no observable correlation between count and carrion beetle species. Overall, 607 isolates consisting of at least 42 different strains of bacteria were recovered. Of these isolates 52.1% were gram negative bacteria (21 strains), 21.1% were coagulase-negative staphylococci (5 strains), 8.1% were obligately anaerobic bacteria (7 strains), 7.6% were streptococci (6 strains), 5.4% were Bacillus spp., 4.4% were Aerococcus and less than 1% were coryneform bacteria (3 strains) or Gemella (for- A merly Streptococcus) haemolysans. variety of colony types were recorded as either Staphylococcus spp.. Streptococcus spp., Clostridium spp. or Bacillus spp., so that the total number of varying strains recov- ered was actually more than indicated. Facultativeand AerobicGram Positive Bacteria. Columbia CNA BAP's supported growth ofall recovered gram-pos- itive bacteria, while that of gram-negatives was effectively inhibited. Yeast colonies, although not targeted in this study, also grew on this media. Mannitol Salt Agar was considerably more selective, as only Staphylococcus sciuri, S. xylosus and S. warned were recovered. Biochemical testing performed on the gram-positive isolates was able to distinguish between the mannitol positive and mannitol nega- tive non-agglutinating staphylococci, and between Streptococcus/ Aerococcus spp. and Streptococcus bovis (Table 1). These tests were Vol. 105,January& February, 1994 51 used inconjunctionwithcolonial morphology, togroupthe isolatesprior to attempts at final identification. Coagulase-negative staphylococci accounted for 52.9% of the 242 facultative and aerobic gram-positive bacteria recovered. Included were Staphylococcusxylosus (19.0%), S. sciuri (13.6%,), S. warneri (9.1%), S. saprophyticus (8.3%), and 2.9% identified asStaphylococcusspp. (Table 1). Streptococci accounted for 19.8% of all gram-positive bacteria, 15.7% ofwhich were equally divided between the enterococci and those identified as Streptococcus spp. Also recovered were Streptococcus san- guis (2.1%), 5". lactis (1.7%,), and one S. bovis variant isolate. Two iso- lates were identified as Gemella haemolysans, a species previously assigned to the genus Streptococcus. Another 13.6% of the gram-positive isolates consisted of a number of morphologically distinct catalase-positive, spore-forming rods, all of which were recorded as Bacillus spp.. Finally, a small numberofcoryne- form bacteria (1.7%) were recovered. Anaerobic Bacteria Most of the 49 anaerobic isolates recovered belonged to the genus Clostridium, with 44.4% identified as Clostridium spp., 18.4% as C. barati, 14.3% as C. bifermentans and only one isolate each (2%,) of C. cadaveris and C. innocuum. All clostridia failed to grow, or grew only slightly when incubated under aerobic conditions. The two remaining anaerobes recovered were Peptostreptococcus anaerobius (16.3%) and Bacteroides capillosus (2%) (Table 2). Facultativeand AerobicGram-Negative Bacteria There were 23 different strains ofgram-negative bacteria recovered, 14 ofwhich belong to the family Enterobacteriaceae. The gram-negative bacteria constituted the single largest and most diverse group in this study. There was a total of638 colonies recorded from among the 7 dif- ferent gram-negative isolation plates, representing 514 isolates when only one species per plate is considered. The difference may represent either strain variation, or inadvertent recording (as different colony types) of a single strain more than once from the same plate.When counting each species only once per beetle specimen, the number ofiso- lates drops to 316. There were 23 different taxa and groups of gram-negative bacteria identified by the API 20E system (Table 3). Approximately one half of these constituted almost 90% of the 316 recovered isolates. The single ENTOMOLOGICALNEWS 52 largest group was the so-called Proteeae tribe, including Proteus (26.6%), Providencia (16.5%) and Morganella (6.6%). Other predomi- nant gram-negatives included Serratia (17.1%), Citrobacter freundii (9.5%), Klebsiella (7%) and Hafnia alvd (6.3%). DISCUSSION Bacteria representing most of the major groups, of which approxi- mately 50% are gram-negative rods, were recovered from the beetles studied. These findings are consistent with previous studies of silphids (Solteretal. 1989) and otherinsects (Steinhaus 1941). Eighteenofthe 26 bacterial types recovered from the Solteretal. study (1989) are included among the 45 different types recovered here. Frank pathogens were not recovered from either study with the exception of3 possible Salmonella isolations in this study. On the other hand, many of these isolates are considered to be opportunistic pathogens, recognizing the often tenuous distinction between "harmless" and "pathogenic" microorganisms. In the discussion that follows only species not mentioned in the Solter etal. study (1989) will be covered. GramPositive BacterialIsolates (Table 1). In animals,variants ofStaphylococcus have evolved to be adapted to various hosts. These separate biotypes ofecotypesvarydependent upon the host species. Most of these organisms are coagulase negative and have limited clinical importance (Joklik et al. 1988). Included would be Staphylococcus sciuri which has been isolated from the skin of rodents, ungulates,carnivora and marsupials. It maybe isolated fromothermam- mals and environmental sourcessuch assoil and water. It has only rarely been isolated from humans (Sneath etal. 1986). Streptococcusfaecalis is primarily located in the gastrointestinal tract ofhumans, homothermic and poikilothermic animals and in insects and plants. It is common in many non-sterile foods and its presence is often not related to fecal contamination. It is an opportunistic pathogen agent in urinary tract infections (Sneath etal. 1986). It also has been frequent- ly associated with biliary tract infections, septicemia, wound infection and intraabdominal abcess complicating appendicitis, especially in the elderly or those who are immunologically compromised (Joklik et al. 1988). Streptococcussanguis is one ofa group ofstreptococci that is associ- ated with the oral cavity. These organisms are consistently isolated as a part of the flora of the mouth and are associated, along with several Vol. 105,January& February, 1994 53 other streptococcal species, in the production ofdental plaque and den- tal carries. They have been isolated from the blood and heart valves in some cases of bacterial endocarditis. They are present in low levels in human feces and have been isolated from soil (Sneath et al. 1986). Streptococcusbovis is frequently isolated from the alimentarytract of cows, sheep, and other ruminants and the feces of pigs. It is occasional- ly found in large numbers in human feces. It is one of the acidogenic streptococci found in raw and pasteurized milk, cream and cheese. It has been found occasionally in cases ofendocarditis and is considered to be related to S. faecalis (Sneath et al. 1986). Streptococcus lactis is non-pathogenic and is associated with the pro- duction of acid from large numbers of sugars. It is responsible for the souring ofmilk, and production ofyogurt (Sneath et al. 1986). As the name implies, Streptococcus avium is characteristically isolat- ed from the feces ofchickens and other fowl. These organisms also have been found in the feces ofhumans, dogs and pigs. They have been asso- ciated with appendicitis, otitis and abscesses of the brain (Sneath et al. 1986). Aerococcus viridans is frequently found as a common airborne organism. Another variety is a marine organism which causes disease in lobsters. No pathogenic association is known to exist in humans (Sneath et al. 1986). Gemella haemolysans is considered to be similar to the neissera species. It is not known to be a pathogen. This organism has been isolat- ed from bronchial secretions and human gingiva (Sneath et al 1986). AnaerobicBacterial Isolates (Table2) Bacteroides capillosus is a gram (-) anerobic organism forming tan to black pigments. Bactero'des are indigenous tovarious locations through- out the body including the mouth and thoracic region, intraabdominal and pelvic regions. The infections that they may cause are related to their location in the body (Krieg & Holt 1984). Bacteroides capillosus has been isolated from cysts and wounds in humans, as well as the human mouth and feces. This organism has also been isolated from the intestinal tractsofseveral animals including hogs, & mice and termites. It has alsobeen found in sewage sludge (Krieg Holt 1984). Clostridia are anerobic spore forming bacilli that are usually gram (+). Most species are obligate anerobes.The pathogenicspecies produce soluble toxins some of which are extremely potent. The clostridia are widely distributed in nature and are present in soil and as inhabitants of the intestinal tract of humans and other animals (Joklik et al. 1988). ENTOMOLOGICALNEWS 54 The histotoxic clostridia cause a severe infection of muscle-com- monly called gas gangrene. Because the clostridia are so widely distrib- uted in nature, contamination ofwounds by these bacteria is very com- mon. Often more than one clostridial species is present including both saprophytic and histotoxic species. The most commonly isolated histo- toxic Clostridium is C. perfringens. However, several other species are commonly encountered in soft tissue infections, abscesses, wound infec- tions, anerobiccellulitis and gas gangrene. These organisms maybe con- sidered non-pathogenic, but they can be opportunistic pathogens. Included in this group are C. barati, C. bifermentans, C. innocuum, and C. cadaveris. While these organisms may not produce toxins, they may play a synergistic role in the development of gas gangrene (Joklik et al. 1988). Peptostreptococcus anaerobius is an anerobic gram (+) coccus. These organisms are a part of the normal flora of the mouth, gastrointestinal tract and genital tract. They are particularly important in pleuropul- monarydisease,brain abscesses and obstetric and gynecologicinfections (Sneath etal. 1986). GramNegative BacterialIsolates(Table3) The bulk of the species in this category are members of family Enterobacteriaceae. They are glucose fermenting bacilli or coccobacilli and as the name implies they are enteric bacteria; Serratia marcescens is a prominent opportunistic pathogen affecting hospitalized patients. At one time they were thought to be harmless saprophytes and were used to trace air currents in the environment and in hospitals. In nature they are found widely distributed in soil and water and are found associated with a large number of plants and animals, including insects. Almost all Serratia infections are associated with underlyingdisease.Theycause nosocomial infectionsofthe urinarytract and wound infections, pneumonia and septicemia. Mastitis in cows and other animal infections are caused by this organism (Joklik etal. 1988). Serratia liquifacens has been isolated from clinical specimens. The disease spectrum for this species is similar to S. marcescens (Joklik etal. 1988). Klebsiella pneumoniae is normally found in the intestinal tract of man and animals, but in lower numbers than E. coli. As the name suggests it can cause pneumonia. It can also infect other sites than the respiratory tract. It is the mucoid capsule that determines the patho- genicity. The organism is an opportunistic pathogen and usually causes illness in apatientwho is alreadycompromised. Itcan alsocause urinary Vol. 105,January& February, 1994 55 tract and wound infections, bacteremia and meningitis (Joklik et al. 1988). Klebsiella oxytoca resembles K. pneumoniae in disease spectrum and it is also very similar from a clinical viewpoint (Joklik etal. 1988). Klebsiella oionae causes chronic atrophic rhinitis characterized by a fetid odor. Nasal and pharyngeal infections are primarily seen in people from endemic regions in Eastern Europe and South America. It can also be isolated from urinary tract and soft tissue infections and from sec- ondary bacteremia (Joklik etal. 1988). Enterobacter cloacae is found less frequently than Klebsiella and E. coil. It ismost frequentlyassociated with urinarytract infections in noso- comial patients having other underlying problems. In the 1970's Enterobacter agglomerans and E. cloacae were responsible for 150 bac- teremias and 9 deaths in a nationwide epidemiccaused bycontaminated intravenous fluids (Joklik et al. 1988). Hafnia alvei is found in feces of man and other animals including birds. It is also found in sewage, soil and water. The infections it pro- & duces are similar to those produced by Enterobacter (Krieg Holt 1984). Some serotypesofSalmonella are primarily adapted toone speciesof host or another. Salmonella pullorum is adapted to poultry rather than man and is primarilly transmitted between poultry. Humans can, how- ever, developsalmonellosis fromcontaminated food andwater (Krieg& Holt 1984). Cedacea spp. is an entericgenus that has been isolated from a variety of opportunistic infections (over 50% from the respiratory tract). The organisms are infrequent opportunistic pathogens and constitute only a small percentage of isolates. Very little is known about their ecology, epidemiology or role in human disease (Krieg & Holt 1984). Mollerella wisconsinsis is a recently described organism and was for- merly considered in Enteric Group 46. It has been isolated from feces. The reported isolates have been found in patients with diarrhea but there is no evidence that it can actually cause diarrhea (Farmer et al. 1985). Tatumella ptyseos is similar to other members of the Entero- bacteriaceae. It has been isolated from human clinical specimens-86% from the respiratory tract. It may be a rare opportunistic pathogen, whose epidemiology is not known (Krieg & Holt 1984). The remaining gram negative bacterial isolates are all non-ferment- ing bacilli: Pseudomonasfluorescens is found in soil and water. It is commonly associated with spoilage of foods, such as eggs, meats and it is often iso- 56 ENTOMOLOGICALNEWS & lated from clinical specimens (Krieg Holt 1984). Although these organisms are notetiological agents ofdisease, they may be the cause of opportunistic infections of wounds and the urinary tract (Krieg & Holt 1984). Pseudomonas testosteroni occurs in soil. It is not considered patho- & genic, but like P. fluorescens it may be opportunistic (Krieg Holt 1984). Pasteurella are parasitic on the mucous membrane of the upper res- piratory and digestive tracts of mammals (rarely man) and birds. Some primary diseases include hemorrhagic septicemia of cattle and fowl cholera in chicken, turkeys, ducks, etc. These organisms also cause sec- ondary pneumonia-like illness in cattle and sheep (Krieg & Holt 1984). The most significant source ofmicroorganisms colonizing the silphid gut is the carrion on which they feed. The bacteria recovered in this study should, therefore, be reflective of those commonly found on decayingcarcasses. Most ofthe recovered species are, in fact,widelydis- tributed in nature and capable of existing as free-living organisms. The variety of gram-negative and gram-positive, facultative and anaerobic bacteria recovered from all sixspecies indicatesconditionswhich maybe favorable to at least transient populations of pathogenic bacteria. Such organisms may be acquired exogenously from infected carrion, in which case the silphids could become vectors ofdisease transmission. Table 1. The number of times each identified gram positive bacterial species was isolated from six silphid species. N.a. = Necrophila americana, N.o. = Nicrophorus orbicollis, O.n. = Oiceoptomanoveboracense, O.i. = Oiceoptoniainaequale, N.t. - Nicrophorustomento- sus, N.s. = Necrodessurinamensis. (Silphidae-8specimensperspecies were used.) 688888 GRAM POSITIVE BACTERIAL ISOLATE N.a. N.o6. O.4n. 3O.i.7N.t.5N.s. TOTALS SlaphylococcusxylosusSchleiferand Kloos 563026 46 Staphylococcussciuri Kloos,Schleiferand Smith 8323084 33 Staphylococcus warneri Kloosand Schleifer 11030 22 Staphvlococcussaprophvticus(Fairbrother) 33036 20 Staphylococcusspp. 2301100 7 StreptococcusfaecalisAndrewesand Horder 03 01300 18 StreptococcussanguisWhite and Niven 001000 5 Streptococcuslactis (Lister) Lohnis 10000 4 Streptococcusaviuni Nowlan and Deibel 26533 1 Streptococcusbovisvariant Orla-Jensen 24578 1 Streptococcusspp. 788640 19 AerococcusviridansWilliams, Hirchand Cowan 01 02011 27 Bacillusspp 00010 33 coryneform bacteria 4 Gemellahaeinolysans (Thjotta and Boe) Berger 1 2 TOTALS 39 39 45 31 47 41 242