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The question of whether gut microflora of the millipede Ommatoiulus sabulosus could function as a threshold to food infections
Pedobiologia 44, 705–708 (2000) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/pedo
Short Communication
The question of whether gut microflora of the millipede Ommatoiulus sabulosus could function as a threshold to food infections Jan Jarosz1 and Grzegorz Kania2 Department of Insect Pathology, Marie Curie-Skl/odowska University, Akademicka 19, 20033 Lublin, Poland 2 Department of Biology and Parasitology, University of Medical Sciences, Radziwil//lowska 11, 20080 Lublin, Poland 1
Accepted: 20. March 2000
Summary The specific composition of bacterial flora in the intestines of O. sabulosus cannot be explained simply by antibiotic inhibition of contaminating microflora since the predominant types of bacteria present are unable to produce any bacteriolytic activity of lysozyme-like enzymes, bacteriocins or other antimicrobial molecules. The lack of microbial contaminators could rather result from the unfavourable biochemical niche in the midgut enzymes and little or no competition for the enteric bacteria group predominant in the millipede alimentary tract. In other words, the rapid colonization and overgrowth of the Ommatoiulus intestines eliminate adventitious bacteria, yeasts and moulds ingested with food. Key words: Millipede, intestinal microflora, enteric bacteria group, antimicrobial activities, competition mechanisms, Ommatoiulus sabulosus
Introduction A number of investigators, beginning with Bucher (1963), have claimed that the intestinal microflora of an arthropod may be needed by the host for some physiological processes to extend its own metabolic faculties. Garber (1956), discussing a nutritionCorrespondence to: Grzegorz Kania, e-mail: [email protected] 0031–4056/00/44/06–705 $ 15.00/0
706
Jan Jarosz and Grzegorz Kania
inhibition hypothesis, emphasized that pathogenicity must always be defined as a two-component system: the host-parasite relationship. Only the combination of adequate nutritional and infective inhibitory environments can result in virulence of a pathogen to the susceptible host. The presence of antimicrobial substances in the insect gut might be detrimental in development and multiplication of the ingested bacteria. The sterilizing effect due to Streptococcus faecalis in the gut of the greater wax moth Galleria mellonella, especially with regard to the production of bacteriocins and the release of lysozyme-like activity by bacterial symbiont preventing the proliferation of other bacteria in insect intestines, was documented by Jarosz (1975, 1979). Since diplopods are characterized by biochemical and physiological similarities to insects it was of interest to determine if a similar barrier to food infections could occur in the millipede Ommatoiulus sabulosus.
Materials and Methods The millipede O. sabulosus L. (Diplopoda: Julidae) lives within woodland litter and soil as its natural habitat. In the laboratory, the specimens caged in glass vessels were fed plant litter in varying stages of decomposition. In order to isolate the bacterial strains, the alimentary canals that had been dissected with needles were isolated under sterile conditions and the intestinal contents were collected in 10 ml of physiological saline, subsequently serial 1/10 dilutions were made, and the viable cell counts per individual were determined. Three days were allowed to develop the intestinal bacteria on nutrient agar. Predominating colonies were transferred to nutrient agar plates to test for purity and were identified using API tests. The antibiotic-producing ability of intestinal isolates was bioassayed by the double agar layer technique of Gratia (Hamon 1957) in a stab modification. Two day old colonies (5 replicates/plate) were killed by exposing them to chloroform for at least 30 min, and them were covered with overnight broth cultures of each indicator bacterium suspended in a soft (0.6 %) nutrient agar in the proportion of 1:100. Lysozyme-like bactericidal activity (EC.3.2.1.17; endoβ-(1-4)-N-acetylmuramide glucanohydrolase) was determined in an inhibition zone assay around the bacterial colonies, using heat-killed freeze-dried Micrococcus luteus (Sigma) incorporated into nutrient agar at a concentration of 1.0 mg/ml. The method of Brock et al. (1963) was employed to demonstrate the bacteriocin activity of enteric bacteria isolated from the Ommatoiulus intestinal tract.
Results and Discussion A typical bacterial microflora harboured in the millipede guts and assay for ability to produce antibacterial activities by predominant bacterial species are presented in Table 1. Evidence obtained in this study indicates that an enteric bacteria group predominated in the intestinal track of most Ommatoiulus individuals. The gut content taken from the test animals revealed no significant differences in the number and types of bacterial species present: Escherichia coli, Enterobacter agglomerans and Klebsiella sp. occurred frequently in large populations (from 3.84 × 108 E. coli/individual to 1.09 × 108 E. agglomerans/individual) in the millipede guts. Accidental microflora represented by Pseudomonas fluorescens, Sarcina, Micrococcus and spore-forming bacilli belonging to the Bacillus sp. group were not numerous. Occasionally, yeast-
Mean bacterial count/ individual
15.0 0
0 0
21.5 8.0
Antibiotic activity –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Bacillus subtilis Escherichia coli Sarcina lutea –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Growth zone inhibition diam. (mm) 0 0 0 0 0 0 0 0 0 0 0 0 0 11.0
0 0 0 0
Lysozyme-like activity
* Isolates originating from the digestive tract of Galleria mellonella (Insecta: Lepidoptera); 0, no antimicrobial activity. Bacterial count/Ommatoiulus gut was calculated from 17 specimens
Enterobacter agglomerans 1.09 × 108 Escherichia coli 3.84 × 108 Klebsiella sp. 1.08 × 108 Pseudomonas fluorescens 5.15 × 106 Standard bacterial species*: Bacillus cereus No8 Streptococcus faecium G7
Intestinal bacterium
Table 1. Indigenous gut microflora of the millipede Ommatoiulus sabulosus and summary of effects of assay for the ability to produce antibacterial activities by predominant bacterial isolates
Gut microflora of millipedes a threshold? 707
708
Jan Jarosz and Grzegorz Kania
like fungi and moulds were detected at low populations only. Surprisingly, when the individual contained larger populations of Enterobacteriaceae, other morphologically different forms constituted only a very limited percentage of microorganisms. This specific composition of microflora suggested that intestinal bacteria have the capacity to produce one or more compounds with antibacterial activity. By the double agar layer technique of Gratia (Hamon 1957), our observations unequivocally did not confirm the ability to produce an antibiotic compound(s) released into nutrient agar during multiplication of the bacteria. Moreover, the predominant types of bacteria present in the guts of O. sabulosus were unable to produce any bacteriolytic activity contributing to lysozyme-like enzymes, colicins (or other bacteriocins) or other antimicrobial molecules. Some other competition mechanisms must, therefore, be active since the predominand of enteric bacteria in Ommatoiulus intestines cannot be explained simply by antibiosis. The lack of contaminating microflora could rather result from the unfavourable biochemical environment of the midgut enzymes and little or no competition for the predominant bacteria that by a rapid colonization and overgrowth of the millipede intestines eliminate microbial contaminators adsorbed with food.
Acknowledgements I am grateful to Professor Jan Jarosz for his support during the research of “Immune defence responses of Ommatoiulus sabulosus”. This study is the result of cooperation with Professor Jan Jarosz, who died tragically at the car accident on 3 th January 2000. Grzegorz Kania
References Brock, T.D., Peacher, B., Pierson, D. (1963) Survey of the bacteriocins of enterococci. Journal of Bacteriology 86, 702–707. Bucher, G.E. (1963) Survival of populations of Streptococcus faecalis Andrewes and Horder in the gut of Galleria mellonella (Linnaeus) during metamorphosis and transmission of the bacteria to the filial generation of the host. Journal of Insect Pathology 5, 336–343. Garber, E.D. (1956) A nutrition-inhibition hypothesis of pathogenicity. American Naturalist 90, 183–194. Hamon, J. (1957) Etude des images de sensibilite des Escherichia coli pathogenes pour le nourisson a diverses colicenes types. Annales de L’Institute Pasteur 95, 117–123. Jarosz, J. (1975) Lysozyme-like lytic enzyme of Streptococcus faecalis and its role in the larval development of wax moth, Galleria mellonella. Journal of Invertebrate Pathology 26, 275–281. Jarosz, J. (1979) Gut flora of Galleria mellonella suppressing ingested bacteria. Journal of Invertebrate Pathology 34, 192–198.
Short Communication
The question of whether gut microflora of the millipede Ommatoiulus sabulosus could function as a threshold to food infections Jan Jarosz1 and Grzegorz Kania2 Department of Insect Pathology, Marie Curie-Skl/odowska University, Akademicka 19, 20033 Lublin, Poland 2 Department of Biology and Parasitology, University of Medical Sciences, Radziwil//lowska 11, 20080 Lublin, Poland 1
Accepted: 20. March 2000
Summary The specific composition of bacterial flora in the intestines of O. sabulosus cannot be explained simply by antibiotic inhibition of contaminating microflora since the predominant types of bacteria present are unable to produce any bacteriolytic activity of lysozyme-like enzymes, bacteriocins or other antimicrobial molecules. The lack of microbial contaminators could rather result from the unfavourable biochemical niche in the midgut enzymes and little or no competition for the enteric bacteria group predominant in the millipede alimentary tract. In other words, the rapid colonization and overgrowth of the Ommatoiulus intestines eliminate adventitious bacteria, yeasts and moulds ingested with food. Key words: Millipede, intestinal microflora, enteric bacteria group, antimicrobial activities, competition mechanisms, Ommatoiulus sabulosus
Introduction A number of investigators, beginning with Bucher (1963), have claimed that the intestinal microflora of an arthropod may be needed by the host for some physiological processes to extend its own metabolic faculties. Garber (1956), discussing a nutritionCorrespondence to: Grzegorz Kania, e-mail: [email protected] 0031–4056/00/44/06–705 $ 15.00/0
706
Jan Jarosz and Grzegorz Kania
inhibition hypothesis, emphasized that pathogenicity must always be defined as a two-component system: the host-parasite relationship. Only the combination of adequate nutritional and infective inhibitory environments can result in virulence of a pathogen to the susceptible host. The presence of antimicrobial substances in the insect gut might be detrimental in development and multiplication of the ingested bacteria. The sterilizing effect due to Streptococcus faecalis in the gut of the greater wax moth Galleria mellonella, especially with regard to the production of bacteriocins and the release of lysozyme-like activity by bacterial symbiont preventing the proliferation of other bacteria in insect intestines, was documented by Jarosz (1975, 1979). Since diplopods are characterized by biochemical and physiological similarities to insects it was of interest to determine if a similar barrier to food infections could occur in the millipede Ommatoiulus sabulosus.
Materials and Methods The millipede O. sabulosus L. (Diplopoda: Julidae) lives within woodland litter and soil as its natural habitat. In the laboratory, the specimens caged in glass vessels were fed plant litter in varying stages of decomposition. In order to isolate the bacterial strains, the alimentary canals that had been dissected with needles were isolated under sterile conditions and the intestinal contents were collected in 10 ml of physiological saline, subsequently serial 1/10 dilutions were made, and the viable cell counts per individual were determined. Three days were allowed to develop the intestinal bacteria on nutrient agar. Predominating colonies were transferred to nutrient agar plates to test for purity and were identified using API tests. The antibiotic-producing ability of intestinal isolates was bioassayed by the double agar layer technique of Gratia (Hamon 1957) in a stab modification. Two day old colonies (5 replicates/plate) were killed by exposing them to chloroform for at least 30 min, and them were covered with overnight broth cultures of each indicator bacterium suspended in a soft (0.6 %) nutrient agar in the proportion of 1:100. Lysozyme-like bactericidal activity (EC.3.2.1.17; endoβ-(1-4)-N-acetylmuramide glucanohydrolase) was determined in an inhibition zone assay around the bacterial colonies, using heat-killed freeze-dried Micrococcus luteus (Sigma) incorporated into nutrient agar at a concentration of 1.0 mg/ml. The method of Brock et al. (1963) was employed to demonstrate the bacteriocin activity of enteric bacteria isolated from the Ommatoiulus intestinal tract.
Results and Discussion A typical bacterial microflora harboured in the millipede guts and assay for ability to produce antibacterial activities by predominant bacterial species are presented in Table 1. Evidence obtained in this study indicates that an enteric bacteria group predominated in the intestinal track of most Ommatoiulus individuals. The gut content taken from the test animals revealed no significant differences in the number and types of bacterial species present: Escherichia coli, Enterobacter agglomerans and Klebsiella sp. occurred frequently in large populations (from 3.84 × 108 E. coli/individual to 1.09 × 108 E. agglomerans/individual) in the millipede guts. Accidental microflora represented by Pseudomonas fluorescens, Sarcina, Micrococcus and spore-forming bacilli belonging to the Bacillus sp. group were not numerous. Occasionally, yeast-
Mean bacterial count/ individual
15.0 0
0 0
21.5 8.0
Antibiotic activity –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Bacillus subtilis Escherichia coli Sarcina lutea –––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Growth zone inhibition diam. (mm) 0 0 0 0 0 0 0 0 0 0 0 0 0 11.0
0 0 0 0
Lysozyme-like activity
* Isolates originating from the digestive tract of Galleria mellonella (Insecta: Lepidoptera); 0, no antimicrobial activity. Bacterial count/Ommatoiulus gut was calculated from 17 specimens
Enterobacter agglomerans 1.09 × 108 Escherichia coli 3.84 × 108 Klebsiella sp. 1.08 × 108 Pseudomonas fluorescens 5.15 × 106 Standard bacterial species*: Bacillus cereus No8 Streptococcus faecium G7
Intestinal bacterium
Table 1. Indigenous gut microflora of the millipede Ommatoiulus sabulosus and summary of effects of assay for the ability to produce antibacterial activities by predominant bacterial isolates
Gut microflora of millipedes a threshold? 707
708
Jan Jarosz and Grzegorz Kania
like fungi and moulds were detected at low populations only. Surprisingly, when the individual contained larger populations of Enterobacteriaceae, other morphologically different forms constituted only a very limited percentage of microorganisms. This specific composition of microflora suggested that intestinal bacteria have the capacity to produce one or more compounds with antibacterial activity. By the double agar layer technique of Gratia (Hamon 1957), our observations unequivocally did not confirm the ability to produce an antibiotic compound(s) released into nutrient agar during multiplication of the bacteria. Moreover, the predominant types of bacteria present in the guts of O. sabulosus were unable to produce any bacteriolytic activity contributing to lysozyme-like enzymes, colicins (or other bacteriocins) or other antimicrobial molecules. Some other competition mechanisms must, therefore, be active since the predominand of enteric bacteria in Ommatoiulus intestines cannot be explained simply by antibiosis. The lack of contaminating microflora could rather result from the unfavourable biochemical environment of the midgut enzymes and little or no competition for the predominant bacteria that by a rapid colonization and overgrowth of the millipede intestines eliminate microbial contaminators adsorbed with food.
Acknowledgements I am grateful to Professor Jan Jarosz for his support during the research of “Immune defence responses of Ommatoiulus sabulosus”. This study is the result of cooperation with Professor Jan Jarosz, who died tragically at the car accident on 3 th January 2000. Grzegorz Kania
References Brock, T.D., Peacher, B., Pierson, D. (1963) Survey of the bacteriocins of enterococci. Journal of Bacteriology 86, 702–707. Bucher, G.E. (1963) Survival of populations of Streptococcus faecalis Andrewes and Horder in the gut of Galleria mellonella (Linnaeus) during metamorphosis and transmission of the bacteria to the filial generation of the host. Journal of Insect Pathology 5, 336–343. Garber, E.D. (1956) A nutrition-inhibition hypothesis of pathogenicity. American Naturalist 90, 183–194. Hamon, J. (1957) Etude des images de sensibilite des Escherichia coli pathogenes pour le nourisson a diverses colicenes types. Annales de L’Institute Pasteur 95, 117–123. Jarosz, J. (1975) Lysozyme-like lytic enzyme of Streptococcus faecalis and its role in the larval development of wax moth, Galleria mellonella. Journal of Invertebrate Pathology 26, 275–281. Jarosz, J. (1979) Gut flora of Galleria mellonella suppressing ingested bacteria. Journal of Invertebrate Pathology 34, 192–198.