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Fly-attracting volatiles produced by Rhodococcus fascians and Mycobacterium aurum isolated from myiatic lesions of sheep
Journal of Microbiological Methods 48 (2002) 281 – 287 www.elsevier.com/locate/jmicmeth
Fly-attracting volatiles produced by Rhodococcus fascians and Mycobacterium aurum isolated from myiatic lesions of sheep Jamal M. Khoga a, Erika To´th a, Ka´roly Ma´rialigeti a,*, Jo´zsef Borossay b b
a Department of Microbiology, Eo¨tvo¨s Lora´nd University, Mu´zeum krt. 4/a., 1088 Budapest, Hungary Department of Inorganic Chemistry, Eo¨tvo¨s Lora´nd University, Pa´zma´ny P. stny. 1/a., 1117 Budapest, Hungary
Abstract Bacterial strains isolated from the healthy breech mucosa and myiatic wounds of ewes were tested for their volatile production as fly attractants towards Wohlfahrtia magnifica (Diptera: Sarcophagidae). Cultures were studied as fly baits in field experiments, and strains performing with the best chemotropic effect were selected for further analysis. Static and dynamic headspace samples from shaken cultures were examined by gas chromatography – mass spectrometry (GC – MS). Strains identified as Rhodococcus fascians and Mycobacterium aurum produced various volatile sulfur compounds and benzene, and proved to be the best fly attractants. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Benzene; Mycobacterium aurum; Rhodococcus fascians; Volatile sulfur compounds; Wohlfahrtia magnifica
1. Introduction Flies causing myiasis (infestation of live and/or dead organs and tissues of vertebrates by dipterous larvae) are one of the most deviating insects of the world and are responsible for severe losses in animal husbandry. The fly Wohlfahrtia magnifica is an obligate parasite of live animals, and represents the main agent of traumatic myiasis (wohlfahrtiosis) in the Mediterranean area and in the whole Eurasia (Nedelchev et al., 1988; Martinez et al., 1987; Farkas, 1996). For other screwworm flies (Lucilia cuprina, L. sericata, Cochliomyia hominivorax), there is a wealth of information on the microbiology of the infested body parts (i.e., fleece, preputium), on the natural bacterial partners of the flies, and on the role of bacteria of wounds in fly chemotropism (Hobson, 1935; Cragg *
Corresponding author. Tel./fax: +36-1-266-1148. E-mail address: [email protected] (K. Ma´rialigeti).
and Ramage, 1945; Emmens and Murray, 1983; Dalwitz et al., 1994; Morris et al., 1997). At the same time, scanty data exist on the bacteriology connected to wohlfahrtiosis (Khoga, 1995; To´th et al., 1998), and there is no information concerning the possible role of microorganisms in fly attraction. The aim of the present work was to clarify whether members of the bacterial communities, in or around the myiatic wound, have a role in the attraction of W. magnifica, and to identify the possible chemotropic volatile compounds produced by these bacteria.
2. Materials and methods 2.1. Bacterial strains, culture conditions, identification techniques Numerous bacterial strains (a total of 663) were isolated on King B and Endo agar media, 384 from the
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 ( 0 1 ) 0 0 3 3 0 - X
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healthy breech mucosa, 156 from myiatic wound discharges, and 123 from myiatic wound brim swab samples, each composed of aliquots of subsamples taken from three ewes. Breech is the vulval/anal region of the ewe covered in some parts with a mucous epithelium (mucosa), and in other parts with a corneous epithelium. Myiatic lesions (fly-stricken wounds) occur on both. Wound brim refers to the inflamed edge of a deep lesion. Representative strains (a total of 136) were selected, 66 in the first case, 39 in the second case, and 31 in the third case. The selection was based on colony morphology, microscopic and cultural criteria, and bacteria were identified according to classical phenotypical methods (Khoga, 1995). The best flyattracting bacteria (Rhodococcus fascians and Mycobacterium aurum) were determined by 16 S rDNA partial sequence comparisons using the Ribosomal Database Project sequences and the ABI software package (Strunk and Ludwig, 1995). Bacteria were maintained on King B agar at 10 C.
as a single dose; 1 bowl on a 30 30-cm paper strip). Traps used were wind-oriented, as designed by Broce (1977). Traps with and without bait were allocated to Latin square design (Wishart and Sanders, 1955). The catch was separated according to the taxa and sexes of flies by classical phenotypical methods applied in entomology (Broce, 1985). 2.3. Statistical analysis of the results The catch (n) for each replicate was transformed to log(n + 1) to normalize distributions and to homogenize variances and, provided that less than half of the replicates of each treatment afforded zero catch, data were examined by analysis of variance (ANOVA) to determine the probability associated with the differences between mean catches. The ANOVA analysis was performed by using Microsoft Excel 5.0 and Statgraph 4.0 programs. 2.4. GC analysis of bacterial odors
2.2. Field experiments to check fly chemotropic effect of bacterial cultures Strains for field experiments were selected with respect to (i) their predominance in the bacterial population of the myiatic lesion and (ii) literature records on their possible insect attracting property. Thus, 26 bacterial strains isolated from wound brim, discharge, and healthy mucosa samples were tested together with two reference strains, Pseudomonas aeruginosa CCM 1960 (CCM = Czech Collection of Microorganisms, Brno, Czech Republic; ATCC 10145T) and Bacillus subtilis ATCC 6633 (ATCC = American Type Culture Collection, Manassas, VA, USA), for their fly-attracting volatile production at Mezo¨falva State Farm, Sa´rboga´rd, Hungary. King B agar plates were inoculated in an adequate number of replicates with 0.1 ml of a 24-h-old broth culture of the selected bacterial strains and incubated for 34, 68, 92, 116, 140 and 164 h at 28 C. These cultures, together with uninoculated controls, were used as baits on white sticky papers (one Petri dish culture on a 30 30-cm paper strip as modified by Hogsette et al., 1993). Liquid cultures of the same bacterial strains (shaken cultures in King B broth at 28 C for 24– 48 h) were used in plastic bowls with white sticky papers (20 ml/bowl of attractants applied for 1 h
Eight strains (all belonging to the Corynebacterinae suborder of Actinomycetales) were subjected to gas chromatography – mass spectrometry (GC – MS) to analyze the volatile compounds produced. GC –MS analyses were performed using a Hewlett-Packard (Avondale, PA, USA) HP-5780 A gas chromatograph (carrier gas: He, 35 kPa; direct injection: 150 C, 0.5 min; temperature: 30 C for 1 min, 30– 280 C, 8 C/ min; column: HP Ultra 2 (25 m 0.32 mm i.d., 5% diphenyl- and 95% dimethylsiloxane copolymer, df: 0.14 mm; ref. HP 19091B-012) and a Varian (Darmstadt, Germany) VG 12-250 MS detector (capillary interface connection 200 C; energy: 70 eV, source temperature: 200 C). For this purpose, bacteria were grown either for 48 h (static headspace) or for 24, 48, 72, 96, and 240 h (dynamic headspace) at 28 C as shaken cultures (140 rpm) in 50 ml King B broth supplemented with 0.5 g/l L-cysteine. The medium was distributed into 150-ml screw-capped Erlenmeyer flasks fitted with 3-mm PTFE-coated silicon rubber gaskets. For static headspace analysis, samples (80 ml) were withdrawn from the flasks and were full scan analyzed and also tested in the selected ion monitoring mode. Because of the relatively low concentrations of volatiles, only dynamic headspace analyses were
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disulfide (500 ml) from which samples (1 ml) were injected into the GC – MS system, and analyzed as previously described. To clarify the dynamics of volatile sulfur compound production, a time-course experiment was also designed for R. fascians 2V/35 and M. aurum 2VI/12 in two ways: (i) dynamic headspace analysis was carried out for separate shaken cultures incubated for up to 10 days; (ii) recycled dynamic headspace analysis was performed for shaken cultures after 16, 24, 48, 72, 96, and 240 h of incubation. They were purged in the closed-loop mode (Fig. 1) for 30 min by an electrical Buck pump (Supelco; flow rate 2 l/min). At each purging step carried out on the same flask throughout the study, volatiles were trapped separately and investigated as described previously.
3. Results
Fig. 1. Purging device of the bacterial cultures used in search of volatile compounds as attractants towards W. magnifica (recycled dynamic headspace).
subsequently performed. For that purpose, culture headspaces were purged by high-purity nitrogen gas (2 l/min for 30 min) through a capillary inserted in the culture fluid. Volatiles were trapped on ORBO 32 SM adsorbent tubes (Supelco, Bellefonte, PA, USA). Adsorbed compounds were dissolved in carbon
From the results of the bacterial isolation and identification (Khoga, 1995), it became obvious that there are characteristic differences between healthy and screwworm-infested areas. In the case of wohlfahrtiosis, members of the family Enterobacteriaceae disappeared as the number of bacteria belonging to the order Actinomycetales increased in the sheep vulval skin region samples. The 26 bacterial strains selected as baits all belonged to the Corynebacterinae suborder. Results presented in Table 1 show that cultures of R. fascians and M. aurum isolated from the wound brim
Table 1 Evaluation of the effect of 48-h-old bacterial Petri dish cultures as baits on fly catch in field experiments with wind-oriented white sticky paper traps (selected representative data) Bacterial strain as bait d
R. fascians 2V/35 M. aurum 2VI/12 Aerobic actinobacterial strain 5VI/1K Aerobic actinobacterial strain 9V/8 P. aeruginosa CCM 1960 B. subtilis ATCC 6633 No strain, sterile agar plate only a b c d
Origina
B B A C nd nd –
Daily mean catch ± S.E.b W. magnifica (male and female)
Other flies (male and female)c
11.5 ± 7.7 10.8 ± 6.5 3.0 ± 1.4 2.5 ± 2.2 2.0 ± 1.4 2.0 ± 0.0 3.0 ± 1.4
55.0 ± 12.1 48.6 ± 8.3 31.0 ± 1.4 7.0 ± 0.0 26.0 ± 2.8 9.0 ± 1.4 10.0 ± 1.4
A = wound discharge, B = wound brim, C = healthy breech mucosa, nd = no data. S.E. = standard error. L. cuprina, L. sericata, M. domestica, etc. Strains 2VI/12 and 2V/35 significantly attract more W. magnifica than any other selected bacterial strain ( F = 5.89; df = 3.72; P>0.001).
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Table 2 Volatile compounds detected by GC – MS in the static headspace of 48-h-old shaken cultures of eight selected bacteria isolated from the ewe breech mucosa and from myiatic lesions Bacterial strain
Origina
Volatile sulfur compoundsb
Otherc
R. fascians 2V/35 M. aurum 2VI/12 Aerobic actinobacterial Aerobic actinobacterial Aerobic actinobacterial Aerobic actinobacterial Aerobic actinobacterial Aerobic actinobacterial
B B B A C A A B
DMS, DMDS, DMTS, S DMS, DMDS, DMTS, S MT, DMS, DMDS MT, DMS, DMDS MT, DMS, DMDS MT, DMS, DMDS MT, DMS, DMDS MT, DMS, DMDS
Benzene Benzene
a b c
strain strain strain strain strain strain
5VI/1 5VI/1K 9V/8 12X/12a 1/1BK-27b 1/1AK-1
A = wound discharge, B = wound brim, C = healthy breech mucosa. MT = methanethiol, DMS = dimethyl sulfide, DMDS = dimethyl disulfide, DMTS = dimethyl trisulfide, and S = elemental sulfur (as S8). For dynamic headspace analysis, toluene was detected in all samples and in the control.
were the most effective in the attraction of W. magnifica flies. The 48-h-old plate cultures of both bacteria attracted most flies. Other bacteria were not effective or proved to be attractive only for other fly species (L. cuprina, L. sericata, Musca domestica). For W. magnifica, the aerobic actinobacterial strain 5VI/1K isolated from the wound discharge and the reference strain P. aeruginosa did not show significant differences with controls, though they caught high amounts of other fly species. Other bacteria used as baits gave no significant differences as compared to controls (data not shown). None of the reference strains attracted the adults of W. magnifica. P. aeruginosa CCM 1960 was positive in respect of other flies, though both reference strains were chosen to be fly-attracting for Lucilia species causing fleece rot (Merritt and Watts, 1978). From these experiments, it became obvious that some of the bacteria dominating in the wound brim could be responsible for the production of volatile odorous compounds attracting especially W. magnifica. Results of the static headspace GC analysis indicated that all the strains that were studied mainly produced dimethyl disulfide (DMDS) with dimethyl sulfide (DMS) as a minor component (Table 2). Usually, methanethiol (MT) was present as a minor component. For R. fascians and M. aurum, dimethyl trisulfide (DMTS) and elemental sulfur (as cyclooctasulfur, S8) were detected along with benzene. For the dynamic headspace analysis, results were almost similar in all cases, but toluene was detected in excess in all samples. A characteristic dynamic headspace chromatogram of R. fascians 2V/35 is shown in Fig. 2.
Results of the volatile sulfur compound production in the time-course experiments (dynamic and recycled dynamic headspace analysis) for R. fascians 2V/35 and M. aurum 2VI/12 are given in Table 3. It is clear that compounds containing increasing numbers of S)S bonds increase with time, and that the broadest spectrum of volatile sulfur compounds is produced after 48 h of cultivation. Volatile sulfur compounds accumulate in the headspace, while, by repeated purging, adsorption reduced the amounts and the types of compounds in the recycled dynamic headspace. The main difference between fly-attracting and non-fly-attracting strains was in the production of DMTS, elemental sulfur, and benzene. These com-
Fig. 2. Chromatogram of an ORBO SM 32 adsorbed sample (dynamic headspace) from a 48-h-old shaken culture of R. fascians 2 V/35 isolated from a wound brim sample of a ewe breech infested by W. magnifica maggots. Abbreviations: CS2 = carbon disulfide (extraction solvent), Me-S-Me = dimethyl sulfide (DMS), Me-S-SMe = dimethyl disulfide (DMDS), Me-S-S-S-Me = dimethyl trisulfide (DMTS), S8 = elemental sulfur [as cyclooctasulfur (S)], and X = unknown.
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Table 3 Volatile sulfur compound formation as a function of time in the dynamic headspace of shaken cultures of R. fascians and M. aurum isolated from the brim of myiatic lesions of ewes Time of sampling from inoculation (h)
Volatile sulfur compoundsa Dynamic headspace
16 24 48 72 96 240 a b
Recycled dynamic headspace
R. fascians 2V/35
M. aurum 2VI/12
R. fascians 2V/35
M. aurum 2VI/12
DMDS DMS, DMDS DMS, DMDS, DMTS, S DMDS, DMTS DMTS DMDS, S
DMDS DMS, DMDS DMS, DMDS, DMTS, S DMDS DMDS DMDS
DMDS DMS, DMDS DMS, DMDS DMDS DMDS –
DMDS –b – – – –
DMS = dimethyl sulfide, DMDS = dimethyl disulfide, DMTS = dimethyl trisulfide, and S = elemental sulfur (as S8). No volatile sulfur compounds detected.
pounds could not be detected for other selected actinobacterial strains or for reference strains selected on the basis of literature data and tests (Table 2).
4. Discussion Orientation towards the wind in response to an olfactory stimulus, known as the anemotactic response, is common in insects (Bell et al., 1995). Various sulfur compounds, ammonia, carbon dioxide in elevated levels (Cragg and Ramage, 1945), volatile organic acids, and indolic compounds produced by bacteria of wounds (e.g., P. aeruginosa, B. subtilis, Proteus mirabilis, Enterobacter cloacae) or originating from the disintegration of animal tissues are responsible for the attraction of several screwworm flies (L. cuprina, L. sericata, C. hominivorax). These compounds can also act as ovipository stimuli to myiasis-causing fly species (Emmens and Murray, 1982). For W. magnifica, fly chemoattractivity and larvipositional stimuli have not been examined so far. This question is rather important since W. magnifica is an obligate parasite of live animals, while the other myiatic fly species (also trapped partly in the field experiments of this study) are also able to live on or in necrotic tissues of live animals, in carcasses, and in fouling animal tissues (e.g., blood and liver). R. fascians and M. aurum attracting W. magnifica in field experiments produced benzene and several volatile sulfur compounds. To our knowledge, there is no information on the formation of such compounds within these two genera. The role of other actinobac-
teria, e.g., Brevibacterium linens, in the attraction of other dipteran species causing flystrike disease was shown earlier (Emmens and Murray, 1983; Eiseman, 1995). Volatile sulfur compounds including mercaptans and alkyl sulfides are known to be produced by various microorganisms. MT, which has a strong offensive odor, is commonly found in nature, and is often produced by bacteria (Kadota and Ishida, 1972). Demethiolation of methionine by L-methioninase (Lmethionine methanethiol-lyase) has been demonstrated in several microorganisms (Ruiz-Herrera and Starkey, 1969; Laakso, 1976), such as Escherichia coli, P. vulgaris, Pseudomonas sp. Formation of several volatile sulfur compounds, i.e., MT, ethylene sulfide, and dimethyl mono-, di- and trisulfides by different clostridia was reported by Rimbault (1990). Among actinobacteria, only members of the Arthrobacter – Brevibacterium group of coryneforms are known so far as MT producers (Kadota and Ishida, 1972; Sharpe et al., 1976). B. linens strains that produce MT were isolated from different cheeses and also from the human skin (Sharpe et al., 1977). In the present study, only R. fascians and M. aurum strains did not produce MT in detectable amounts. It is well known that MT spontaneously yields DMDS in the presence of atmospheric oxygen (Kadota and Ishida, 1972). Because MT was present in the static headspace of all but these two bacteria tested, it can be assumed that not only the former process but other, e.g., enzymatic, processes are also involved in DMDS formation. Benzene (detected in the headspace of R. fascians and M. aurum) and toluene can also be
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bacterial metabolites. In the presence of aromatic compounds (i.e., phenylalanine) in the growth medium, the production of benzene is highly probable (Fairlee et al., 1997). This compound was not tested, so far, as a fly attractant. Toluene, the other aromatic compound detected, can be a pollutant from the silicone parts of the experimental device since it was present in all samples. However, bacterial formation of toluene, using labeled L-phenylalanine, is well documented for one Clostridium species (Rimbault, 1990). Results of the time-course experiments for R. fascians 2V/35 and M. aurum 2VI/12 corroborate those of the field experiments as the broadest spectrum of volatile sulfur compounds detected for 2-day-old cultures. Differences between dynamic and recycled dynamic headspace analyses can be explained by the technique of purging itself. In the recycled dynamic headspace analysis, volatiles were extracted from the same culture (for 30 min at 16, 24, 48, 72, 96, and 240 h of incubation) from both the liquid and the gas phases. This process decreases the concentration of volatiles in the cultures, which may alter the dynamics of volatile formation and hinder the reactions of the compounds produced, between each other. To precisely clear up which compounds are responsible for fly attractivity in vivo, artificial mixtures of these compounds must be tested as baits. However, there are several constraints since W. magnifica is not yet culturable and the compounds are extremely volatile. In that perspective, trapping of W. magnifica (mainly females) is of high concern since it could solve the serious problems of myiasis occurring in animal husbandry.
Acknowledgements This work was supported by grants from OTKA (T026599) and FKFP (0537). The helpful remarks and comments of Dr. A. Rimbault (Paris, France) are gratefully acknowledged.
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Chemical Ecology of Insects, vol. 2. Chapman & Hall, New York, pp. 105 – 152. Broce, A.B., 1977. Sexual behavior of screwworm flies stimulated by Swormlure-2. Ann. Entomol. Soc. Am. 33, 386 – 391. Broce, A.B., 1985. Myiasis producing flies. In: Williams, R.E., Hall, R.D., Broce, A.B. (Eds.), Livestock Entomology. Wiley, New York, pp. 83 – 95. Cragg, J.B., Ramage, G.R., 1945. Chemotropic studies on the blowflies Lucilia sericata (Mg.) and Lucilia caesar (L.). Parasitology 36, 168 – 172. Dalwitz, R., Roberts, J.A., Kitching, R.L., 1994. Factors determining the predominance of Lucilia cuprina larvae in blowfly strikes of sheep in Southern New-South Wales. J. Aust. Entomol. Soc. 23, 175 – 177. Eiseman, C.H., 1995. Orientation by gravid Australian sheep blowflies Lucilia cuprina (Diptera: Calliphoridae) to fleece and synthetic chemical attractant in laboratory bioassays. Bull. Entomol. Res. 85, 473 – 477. Emmens, R.L., Murray, M.D., 1982. The role of bacterial odors in oviposition by Lucilia cuprina (Wiedeman) (Diptera: Calliphoridae), the Australian sheep blowfly. Bull. Entomol. Res. 72, 367 – 375. Emmens, R.L., Murray, M.D., 1983. Bacterial odors as oviposition stimulants for Lucilia cuprina (Wiedeman) (Diptera: Calliphoridae), the Australian sheep blowfly. Bull. Entomol. Res. 73, 411 – 415. Fairlee, J.R., Burback, B.L., Perry, J.J., 1997. Biodegradation of groundwater pollutants by a combined culture of Mycobacterium vaccae and Rhodococcus sp. Can. J. Microbiol. 43, 841 – 846. Farkas, R., 1996. Myiasis caused by Wohlfahrtia magnifica. Hung. Vet. J. 51, 349 – 352. Hobson, R.P., 1935. Sheep blowfly investigations: II. Substances which induce Lucilia sericata Mg. to oviposit on sheep. Ann. Appl. Biol. 32, 294 – 299. Hogsette, J.A., Jacobs, R.D., Miller, R.W., 1993. The sticky card: device for studying the distribution of adult housefly (Diptera: Muscidae) populations in closed poultry houses. J. Econ. Entomol. 86, 450 – 457. Kadota, H., Ishida, Y., 1972. Production of volatile sulfur compounds by microorganisms. Annu. Rev. Microbiol. 26, 127 – 138. Khoga, J.M., 1995. Myiasis: a consequence or a cause of skin bacterial alterations. PhD dissertation. Eo¨tvo¨s Lora´nd University, Budapest, pp. 28 – 63. Laakso, S., 1976. The relationship between methionine uptake and demethiolation in a methionine-utilising mutant of Pseudomonas fluorescens UK1. J. Microbiol. 95, 391 – 398. Martinez, R.I., Cruz, S.M.D., Rodriguez, R., Lopez, D.M., Parra, M.S., Navio, F.A., 1987. Myiasis caused by Wohlfahrtia magnifica in Southern Spain. Isr. J. Vet. Med. 43, 34 – 41. Merritt, G.C., Watts, J.E., 1978. An in-vitro technique for studying fleece-rot and flystrike in sheep. Aust. Vet. J. 54, 513 – 522. Morris, M.C., Joyce, M.A., Heath, A.C.G., Rabel, B., Delisle, G.W., 1997. The responses of Lucilia cuprina to odors from sheep, offal and bacterial cultures. Med. Vet. Entomol. 11, 58 – 64. Nedelchev, N., Khalaceva, M., Uruchev, K., 1988. Screwworm. Vet. Sb. 87, 41 – 48.
J.M. Khoga et al. / Journal of Microbiological Methods 48 (2002) 281–287 Rimbault, A., 1990. Headspace analysis by gas chromatographymass spectrometry of volatile organic compounds associated with Clostridium cultures. In: Fox, A., Larsson, L., Odham, G., Morgan, S.L. (Eds.), Analytical Microbiology Methods: Gas Chromatography and Mass Spectrometry. Plenum, New York, pp. 101 – 123. Ruiz-Herrera, J., Starkey, R.L., 1969. Dissimilation of methionine by a demethiolase of Aspergillus species. J. Bacteriol. 99, 764 – 769. Sharpe, M.E., Law, B.A., Phillips, B.A., 1976. Coryneform bacteria producing methanethiol. J. Gen. Microbiol. 94, 430 – 435. Sharpe, M.E., Law, B.A., Phillips, B.A., Pitcher, D.G., 1977. Methanethiol producing coryneform bacteria: strains isolated from
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dairy and human skin sources and Brevibacterium linens. J. Gen. Microbiol. 101, 345 – 349. Strunk, O., Ludwig, W., 1995. ARB—A Software Environment for Sequence Data. Department of Microbiology, Technical University of Munich, Germany. To´th, E., Farkas, R., Ma´rialigeti, K., Mokhtar, I.S., 1998. Bacteriological investigations on wound myiasis of sheep caused by Wohlfahrtia magnifica (Diptera: Sarcophagidae). Acta Vet. Hung. 46, 219 – 229. Wishart, J., Sanders, H.G., 1955. Principles and Practice of Field Experimentation. Commonwealth Agricultural Bureau, Fernham Royal, Cambridge, pp. 35 – 76.
Fly-attracting volatiles produced by Rhodococcus fascians and Mycobacterium aurum isolated from myiatic lesions of sheep Jamal M. Khoga a, Erika To´th a, Ka´roly Ma´rialigeti a,*, Jo´zsef Borossay b b
a Department of Microbiology, Eo¨tvo¨s Lora´nd University, Mu´zeum krt. 4/a., 1088 Budapest, Hungary Department of Inorganic Chemistry, Eo¨tvo¨s Lora´nd University, Pa´zma´ny P. stny. 1/a., 1117 Budapest, Hungary
Abstract Bacterial strains isolated from the healthy breech mucosa and myiatic wounds of ewes were tested for their volatile production as fly attractants towards Wohlfahrtia magnifica (Diptera: Sarcophagidae). Cultures were studied as fly baits in field experiments, and strains performing with the best chemotropic effect were selected for further analysis. Static and dynamic headspace samples from shaken cultures were examined by gas chromatography – mass spectrometry (GC – MS). Strains identified as Rhodococcus fascians and Mycobacterium aurum produced various volatile sulfur compounds and benzene, and proved to be the best fly attractants. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Benzene; Mycobacterium aurum; Rhodococcus fascians; Volatile sulfur compounds; Wohlfahrtia magnifica
1. Introduction Flies causing myiasis (infestation of live and/or dead organs and tissues of vertebrates by dipterous larvae) are one of the most deviating insects of the world and are responsible for severe losses in animal husbandry. The fly Wohlfahrtia magnifica is an obligate parasite of live animals, and represents the main agent of traumatic myiasis (wohlfahrtiosis) in the Mediterranean area and in the whole Eurasia (Nedelchev et al., 1988; Martinez et al., 1987; Farkas, 1996). For other screwworm flies (Lucilia cuprina, L. sericata, Cochliomyia hominivorax), there is a wealth of information on the microbiology of the infested body parts (i.e., fleece, preputium), on the natural bacterial partners of the flies, and on the role of bacteria of wounds in fly chemotropism (Hobson, 1935; Cragg *
Corresponding author. Tel./fax: +36-1-266-1148. E-mail address: [email protected] (K. Ma´rialigeti).
and Ramage, 1945; Emmens and Murray, 1983; Dalwitz et al., 1994; Morris et al., 1997). At the same time, scanty data exist on the bacteriology connected to wohlfahrtiosis (Khoga, 1995; To´th et al., 1998), and there is no information concerning the possible role of microorganisms in fly attraction. The aim of the present work was to clarify whether members of the bacterial communities, in or around the myiatic wound, have a role in the attraction of W. magnifica, and to identify the possible chemotropic volatile compounds produced by these bacteria.
2. Materials and methods 2.1. Bacterial strains, culture conditions, identification techniques Numerous bacterial strains (a total of 663) were isolated on King B and Endo agar media, 384 from the
0167-7012/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 ( 0 1 ) 0 0 3 3 0 - X
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J.M. Khoga et al. / Journal of Microbiological Methods 48 (2002) 281–287
healthy breech mucosa, 156 from myiatic wound discharges, and 123 from myiatic wound brim swab samples, each composed of aliquots of subsamples taken from three ewes. Breech is the vulval/anal region of the ewe covered in some parts with a mucous epithelium (mucosa), and in other parts with a corneous epithelium. Myiatic lesions (fly-stricken wounds) occur on both. Wound brim refers to the inflamed edge of a deep lesion. Representative strains (a total of 136) were selected, 66 in the first case, 39 in the second case, and 31 in the third case. The selection was based on colony morphology, microscopic and cultural criteria, and bacteria were identified according to classical phenotypical methods (Khoga, 1995). The best flyattracting bacteria (Rhodococcus fascians and Mycobacterium aurum) were determined by 16 S rDNA partial sequence comparisons using the Ribosomal Database Project sequences and the ABI software package (Strunk and Ludwig, 1995). Bacteria were maintained on King B agar at 10 C.
as a single dose; 1 bowl on a 30 30-cm paper strip). Traps used were wind-oriented, as designed by Broce (1977). Traps with and without bait were allocated to Latin square design (Wishart and Sanders, 1955). The catch was separated according to the taxa and sexes of flies by classical phenotypical methods applied in entomology (Broce, 1985). 2.3. Statistical analysis of the results The catch (n) for each replicate was transformed to log(n + 1) to normalize distributions and to homogenize variances and, provided that less than half of the replicates of each treatment afforded zero catch, data were examined by analysis of variance (ANOVA) to determine the probability associated with the differences between mean catches. The ANOVA analysis was performed by using Microsoft Excel 5.0 and Statgraph 4.0 programs. 2.4. GC analysis of bacterial odors
2.2. Field experiments to check fly chemotropic effect of bacterial cultures Strains for field experiments were selected with respect to (i) their predominance in the bacterial population of the myiatic lesion and (ii) literature records on their possible insect attracting property. Thus, 26 bacterial strains isolated from wound brim, discharge, and healthy mucosa samples were tested together with two reference strains, Pseudomonas aeruginosa CCM 1960 (CCM = Czech Collection of Microorganisms, Brno, Czech Republic; ATCC 10145T) and Bacillus subtilis ATCC 6633 (ATCC = American Type Culture Collection, Manassas, VA, USA), for their fly-attracting volatile production at Mezo¨falva State Farm, Sa´rboga´rd, Hungary. King B agar plates were inoculated in an adequate number of replicates with 0.1 ml of a 24-h-old broth culture of the selected bacterial strains and incubated for 34, 68, 92, 116, 140 and 164 h at 28 C. These cultures, together with uninoculated controls, were used as baits on white sticky papers (one Petri dish culture on a 30 30-cm paper strip as modified by Hogsette et al., 1993). Liquid cultures of the same bacterial strains (shaken cultures in King B broth at 28 C for 24– 48 h) were used in plastic bowls with white sticky papers (20 ml/bowl of attractants applied for 1 h
Eight strains (all belonging to the Corynebacterinae suborder of Actinomycetales) were subjected to gas chromatography – mass spectrometry (GC – MS) to analyze the volatile compounds produced. GC –MS analyses were performed using a Hewlett-Packard (Avondale, PA, USA) HP-5780 A gas chromatograph (carrier gas: He, 35 kPa; direct injection: 150 C, 0.5 min; temperature: 30 C for 1 min, 30– 280 C, 8 C/ min; column: HP Ultra 2 (25 m 0.32 mm i.d., 5% diphenyl- and 95% dimethylsiloxane copolymer, df: 0.14 mm; ref. HP 19091B-012) and a Varian (Darmstadt, Germany) VG 12-250 MS detector (capillary interface connection 200 C; energy: 70 eV, source temperature: 200 C). For this purpose, bacteria were grown either for 48 h (static headspace) or for 24, 48, 72, 96, and 240 h (dynamic headspace) at 28 C as shaken cultures (140 rpm) in 50 ml King B broth supplemented with 0.5 g/l L-cysteine. The medium was distributed into 150-ml screw-capped Erlenmeyer flasks fitted with 3-mm PTFE-coated silicon rubber gaskets. For static headspace analysis, samples (80 ml) were withdrawn from the flasks and were full scan analyzed and also tested in the selected ion monitoring mode. Because of the relatively low concentrations of volatiles, only dynamic headspace analyses were
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disulfide (500 ml) from which samples (1 ml) were injected into the GC – MS system, and analyzed as previously described. To clarify the dynamics of volatile sulfur compound production, a time-course experiment was also designed for R. fascians 2V/35 and M. aurum 2VI/12 in two ways: (i) dynamic headspace analysis was carried out for separate shaken cultures incubated for up to 10 days; (ii) recycled dynamic headspace analysis was performed for shaken cultures after 16, 24, 48, 72, 96, and 240 h of incubation. They were purged in the closed-loop mode (Fig. 1) for 30 min by an electrical Buck pump (Supelco; flow rate 2 l/min). At each purging step carried out on the same flask throughout the study, volatiles were trapped separately and investigated as described previously.
3. Results
Fig. 1. Purging device of the bacterial cultures used in search of volatile compounds as attractants towards W. magnifica (recycled dynamic headspace).
subsequently performed. For that purpose, culture headspaces were purged by high-purity nitrogen gas (2 l/min for 30 min) through a capillary inserted in the culture fluid. Volatiles were trapped on ORBO 32 SM adsorbent tubes (Supelco, Bellefonte, PA, USA). Adsorbed compounds were dissolved in carbon
From the results of the bacterial isolation and identification (Khoga, 1995), it became obvious that there are characteristic differences between healthy and screwworm-infested areas. In the case of wohlfahrtiosis, members of the family Enterobacteriaceae disappeared as the number of bacteria belonging to the order Actinomycetales increased in the sheep vulval skin region samples. The 26 bacterial strains selected as baits all belonged to the Corynebacterinae suborder. Results presented in Table 1 show that cultures of R. fascians and M. aurum isolated from the wound brim
Table 1 Evaluation of the effect of 48-h-old bacterial Petri dish cultures as baits on fly catch in field experiments with wind-oriented white sticky paper traps (selected representative data) Bacterial strain as bait d
R. fascians 2V/35 M. aurum 2VI/12 Aerobic actinobacterial strain 5VI/1K Aerobic actinobacterial strain 9V/8 P. aeruginosa CCM 1960 B. subtilis ATCC 6633 No strain, sterile agar plate only a b c d
Origina
B B A C nd nd –
Daily mean catch ± S.E.b W. magnifica (male and female)
Other flies (male and female)c
11.5 ± 7.7 10.8 ± 6.5 3.0 ± 1.4 2.5 ± 2.2 2.0 ± 1.4 2.0 ± 0.0 3.0 ± 1.4
55.0 ± 12.1 48.6 ± 8.3 31.0 ± 1.4 7.0 ± 0.0 26.0 ± 2.8 9.0 ± 1.4 10.0 ± 1.4
A = wound discharge, B = wound brim, C = healthy breech mucosa, nd = no data. S.E. = standard error. L. cuprina, L. sericata, M. domestica, etc. Strains 2VI/12 and 2V/35 significantly attract more W. magnifica than any other selected bacterial strain ( F = 5.89; df = 3.72; P>0.001).
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Table 2 Volatile compounds detected by GC – MS in the static headspace of 48-h-old shaken cultures of eight selected bacteria isolated from the ewe breech mucosa and from myiatic lesions Bacterial strain
Origina
Volatile sulfur compoundsb
Otherc
R. fascians 2V/35 M. aurum 2VI/12 Aerobic actinobacterial Aerobic actinobacterial Aerobic actinobacterial Aerobic actinobacterial Aerobic actinobacterial Aerobic actinobacterial
B B B A C A A B
DMS, DMDS, DMTS, S DMS, DMDS, DMTS, S MT, DMS, DMDS MT, DMS, DMDS MT, DMS, DMDS MT, DMS, DMDS MT, DMS, DMDS MT, DMS, DMDS
Benzene Benzene
a b c
strain strain strain strain strain strain
5VI/1 5VI/1K 9V/8 12X/12a 1/1BK-27b 1/1AK-1
A = wound discharge, B = wound brim, C = healthy breech mucosa. MT = methanethiol, DMS = dimethyl sulfide, DMDS = dimethyl disulfide, DMTS = dimethyl trisulfide, and S = elemental sulfur (as S8). For dynamic headspace analysis, toluene was detected in all samples and in the control.
were the most effective in the attraction of W. magnifica flies. The 48-h-old plate cultures of both bacteria attracted most flies. Other bacteria were not effective or proved to be attractive only for other fly species (L. cuprina, L. sericata, Musca domestica). For W. magnifica, the aerobic actinobacterial strain 5VI/1K isolated from the wound discharge and the reference strain P. aeruginosa did not show significant differences with controls, though they caught high amounts of other fly species. Other bacteria used as baits gave no significant differences as compared to controls (data not shown). None of the reference strains attracted the adults of W. magnifica. P. aeruginosa CCM 1960 was positive in respect of other flies, though both reference strains were chosen to be fly-attracting for Lucilia species causing fleece rot (Merritt and Watts, 1978). From these experiments, it became obvious that some of the bacteria dominating in the wound brim could be responsible for the production of volatile odorous compounds attracting especially W. magnifica. Results of the static headspace GC analysis indicated that all the strains that were studied mainly produced dimethyl disulfide (DMDS) with dimethyl sulfide (DMS) as a minor component (Table 2). Usually, methanethiol (MT) was present as a minor component. For R. fascians and M. aurum, dimethyl trisulfide (DMTS) and elemental sulfur (as cyclooctasulfur, S8) were detected along with benzene. For the dynamic headspace analysis, results were almost similar in all cases, but toluene was detected in excess in all samples. A characteristic dynamic headspace chromatogram of R. fascians 2V/35 is shown in Fig. 2.
Results of the volatile sulfur compound production in the time-course experiments (dynamic and recycled dynamic headspace analysis) for R. fascians 2V/35 and M. aurum 2VI/12 are given in Table 3. It is clear that compounds containing increasing numbers of S)S bonds increase with time, and that the broadest spectrum of volatile sulfur compounds is produced after 48 h of cultivation. Volatile sulfur compounds accumulate in the headspace, while, by repeated purging, adsorption reduced the amounts and the types of compounds in the recycled dynamic headspace. The main difference between fly-attracting and non-fly-attracting strains was in the production of DMTS, elemental sulfur, and benzene. These com-
Fig. 2. Chromatogram of an ORBO SM 32 adsorbed sample (dynamic headspace) from a 48-h-old shaken culture of R. fascians 2 V/35 isolated from a wound brim sample of a ewe breech infested by W. magnifica maggots. Abbreviations: CS2 = carbon disulfide (extraction solvent), Me-S-Me = dimethyl sulfide (DMS), Me-S-SMe = dimethyl disulfide (DMDS), Me-S-S-S-Me = dimethyl trisulfide (DMTS), S8 = elemental sulfur [as cyclooctasulfur (S)], and X = unknown.
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Table 3 Volatile sulfur compound formation as a function of time in the dynamic headspace of shaken cultures of R. fascians and M. aurum isolated from the brim of myiatic lesions of ewes Time of sampling from inoculation (h)
Volatile sulfur compoundsa Dynamic headspace
16 24 48 72 96 240 a b
Recycled dynamic headspace
R. fascians 2V/35
M. aurum 2VI/12
R. fascians 2V/35
M. aurum 2VI/12
DMDS DMS, DMDS DMS, DMDS, DMTS, S DMDS, DMTS DMTS DMDS, S
DMDS DMS, DMDS DMS, DMDS, DMTS, S DMDS DMDS DMDS
DMDS DMS, DMDS DMS, DMDS DMDS DMDS –
DMDS –b – – – –
DMS = dimethyl sulfide, DMDS = dimethyl disulfide, DMTS = dimethyl trisulfide, and S = elemental sulfur (as S8). No volatile sulfur compounds detected.
pounds could not be detected for other selected actinobacterial strains or for reference strains selected on the basis of literature data and tests (Table 2).
4. Discussion Orientation towards the wind in response to an olfactory stimulus, known as the anemotactic response, is common in insects (Bell et al., 1995). Various sulfur compounds, ammonia, carbon dioxide in elevated levels (Cragg and Ramage, 1945), volatile organic acids, and indolic compounds produced by bacteria of wounds (e.g., P. aeruginosa, B. subtilis, Proteus mirabilis, Enterobacter cloacae) or originating from the disintegration of animal tissues are responsible for the attraction of several screwworm flies (L. cuprina, L. sericata, C. hominivorax). These compounds can also act as ovipository stimuli to myiasis-causing fly species (Emmens and Murray, 1982). For W. magnifica, fly chemoattractivity and larvipositional stimuli have not been examined so far. This question is rather important since W. magnifica is an obligate parasite of live animals, while the other myiatic fly species (also trapped partly in the field experiments of this study) are also able to live on or in necrotic tissues of live animals, in carcasses, and in fouling animal tissues (e.g., blood and liver). R. fascians and M. aurum attracting W. magnifica in field experiments produced benzene and several volatile sulfur compounds. To our knowledge, there is no information on the formation of such compounds within these two genera. The role of other actinobac-
teria, e.g., Brevibacterium linens, in the attraction of other dipteran species causing flystrike disease was shown earlier (Emmens and Murray, 1983; Eiseman, 1995). Volatile sulfur compounds including mercaptans and alkyl sulfides are known to be produced by various microorganisms. MT, which has a strong offensive odor, is commonly found in nature, and is often produced by bacteria (Kadota and Ishida, 1972). Demethiolation of methionine by L-methioninase (Lmethionine methanethiol-lyase) has been demonstrated in several microorganisms (Ruiz-Herrera and Starkey, 1969; Laakso, 1976), such as Escherichia coli, P. vulgaris, Pseudomonas sp. Formation of several volatile sulfur compounds, i.e., MT, ethylene sulfide, and dimethyl mono-, di- and trisulfides by different clostridia was reported by Rimbault (1990). Among actinobacteria, only members of the Arthrobacter – Brevibacterium group of coryneforms are known so far as MT producers (Kadota and Ishida, 1972; Sharpe et al., 1976). B. linens strains that produce MT were isolated from different cheeses and also from the human skin (Sharpe et al., 1977). In the present study, only R. fascians and M. aurum strains did not produce MT in detectable amounts. It is well known that MT spontaneously yields DMDS in the presence of atmospheric oxygen (Kadota and Ishida, 1972). Because MT was present in the static headspace of all but these two bacteria tested, it can be assumed that not only the former process but other, e.g., enzymatic, processes are also involved in DMDS formation. Benzene (detected in the headspace of R. fascians and M. aurum) and toluene can also be
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bacterial metabolites. In the presence of aromatic compounds (i.e., phenylalanine) in the growth medium, the production of benzene is highly probable (Fairlee et al., 1997). This compound was not tested, so far, as a fly attractant. Toluene, the other aromatic compound detected, can be a pollutant from the silicone parts of the experimental device since it was present in all samples. However, bacterial formation of toluene, using labeled L-phenylalanine, is well documented for one Clostridium species (Rimbault, 1990). Results of the time-course experiments for R. fascians 2V/35 and M. aurum 2VI/12 corroborate those of the field experiments as the broadest spectrum of volatile sulfur compounds detected for 2-day-old cultures. Differences between dynamic and recycled dynamic headspace analyses can be explained by the technique of purging itself. In the recycled dynamic headspace analysis, volatiles were extracted from the same culture (for 30 min at 16, 24, 48, 72, 96, and 240 h of incubation) from both the liquid and the gas phases. This process decreases the concentration of volatiles in the cultures, which may alter the dynamics of volatile formation and hinder the reactions of the compounds produced, between each other. To precisely clear up which compounds are responsible for fly attractivity in vivo, artificial mixtures of these compounds must be tested as baits. However, there are several constraints since W. magnifica is not yet culturable and the compounds are extremely volatile. In that perspective, trapping of W. magnifica (mainly females) is of high concern since it could solve the serious problems of myiasis occurring in animal husbandry.
Acknowledgements This work was supported by grants from OTKA (T026599) and FKFP (0537). The helpful remarks and comments of Dr. A. Rimbault (Paris, France) are gratefully acknowledged.
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