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Studies on the β-Glucuronidase Production of Clostridia
Zbl. Bakt. H yg., LAbt. Orig. A 254, 118-122 (1983)
Department of Clinico -Laboratory Medici ne, Kansai Medical University, 1 Fu rnizono-cho, Moriguchi-shi, Osaka, Japan 570
Studies on the fJ-Glucuronidase Production of Clostridia Untersuchungen tiber di e fJ- Glucoronidas e- P r odukti on v o n Clostridien YOSHIAKI SAKAGUCHI and KEN]IRO MURATA
Received October 8, 1982
Abs tract We investigated the fJ-glucuronidase-producing capacity of 197 strains of 19 different species of Clostridia, using the procedure described by Kilian and Biilow (8). Most of the Hobbs' serot yped strains of Clostridium perfringens (104 out of 109 strains) were found to produce fJ-glucuronidase, whereas non-seroryped strains (21 out of 32 strains) did not . Some strains of other species such as Clostridium beijerinckii (c. beijerinckii ), C. clostridiforme. C. dilficile, C. lim osum , and C. ramosum were found to produce fJ-glucuronidase, while all strains of 13 oth er species which we examined did not.
Zusammen fassung Wir untersuchte n mit Hilfe des von Kilian und Bulow (8) beschriebenen Verfahrens die Fahigkeit zur fJ-Glucuronidase-Bildung bei 197 Stammen von 19 verschiedenen ClostridiumArten . Die meisten der C. periringens-Stiutune, die nach Hobbs serotypisiert worden waren (104 von 109), erwiesen sich als P-Glucuronidase-Bildner, wogegen dies bei den nicht sero typisierren Stammen (21 von 32) nicht der Fall war . Bei einigen Stammen anderer Arten wie C. beiierinckii , C. clostridiforme, C. difficile, C. limosum und C. ramosum wur de fJGluc uronidase-Bildung festgestellt, wahrend 13 andere von uns untersuchte Arten keine solchen Stamme aufwiesen,
Introduction T he bacterial Ii-glucuronidase of Staphylococcus albus, Streptococcus pyogenes, Escherichia coli, and Shigella has been int en sively studied (2, 3, 7 , 8), but the Ii-
glucuronida se production of anaerobes has not yet been studied. We frequently isolated Clostridium perjringens from patients' bile, which was co llected by duod en al sounding. We k new t hat Ii-glucuronidase-producing strains existed among t hese C. perlringens (9).
Stud ies on th e {3-Glucuro nidase Production of Clostridia
119
C. periringens is p art of the n o rm al intes ti nal flora, an d was fo un d in all of the feca l specime ns fr om a gro up of he althy per sons (IGl -109 / g feces) (1). We in vesti gate d the ,8-gluc uron idase-p ro d uc ing ca pacity of 197 st ra in s of Clostridia, iso lat ed fro m va rio us so urces, u sing the proced ure de scribed by Kilian an d Bulow (8). Materials and Methods Clost ridium strains Nineteen different species of Clostridium including 197 strains, were isolated from pa tient's feces, bile, gastr ic juice, blood, secretions, umb ilical cord or urine. Reference strai ns used were Clostridiu m bi fermentans GAl 0209, C. diffi cile GAl 0288, C. perlringens 0084, C. ramosum GAl 2560, C. sordellii GAl 0029, C. sporogenes GAI0005, and C. tetani, Osa ka Associat ion of Medical T echnologists. In th e case of feces, about 0.5 g of feces was dissolved in 5 ml of GAM broth (Nissui Co., T okyo, j apan) and incubated at 60 °C for 30 min. Then one loopful of medium was inocu lated onto GAM agar (Nissui) with egg-yolk. Oth er specimens were inocul ated on both GAM agar and Phenylethylalcohol agar (Nikken Co., Kyot o, j apan ). Anaerob ic cultures were do ne by an anaerobic culture system (Forma Scientific Co., U. S.A. ), and incubated at 37 °C for 48 h. Ident ification of Clostridia isolated from patient's feces, bile, gastric juice, blood , secretio ns, umb ilical cord or urine Bacterial morph ology was checked by Gram 's sta in. a-Toxin (lecithinase) production was detected by wide zones of precipi tate around th e colonies on egg-yolk agar. C. perfringens was identified by a-toxin productio n, sto rmy ferment ation of 10% litmus milk (BBL, U. S. A.), and degradat ion of 1% gelatin, glucose, lactose and DNA (4). C. perfringens strai ns isolated from vario us sources were as foll ows : 113 stra ins from feces, 25 strai ns from bile, and 1 strain each fro m secretions, umb ilical cord and urine. Identificatio n of other Clost ridia was perfo rmed with an API 20 A system (API Laborato ry Produ cts Ltd., France) with reference to the VPI Anaerob e laboratory manual (6). Detection of {J-glucuronidase-producing strains amo ng the Clostridia
Th e grow th medium used to exa mine {J-glucuro nidase activity was PGUA aga r, which was developed by Kilian and BUlow for the detection of Escherichia coli and Shigella among Entero bacteriaceae and Vibrioaceae (8). Th e incub ati on time for PGUA plates was 24- 48 h at 37 °C. {J-Glucuro nidase-pro ducing Clostridia developed a yellow zone aro und each colony. T he seroty pe of C. perjrin gens
Th e stra ins from th e blood agar or egg-yolk plate were typed into the kn own Hobb s' serotypes 1 through 17 by slide aggluti natio n tests using heat-stabl e C. perfringens type A ant i-sera for diagnosi s (Denk a-Seiken, T okyo, japan) (5, 10).
Results and Discussion Nineteen different species of Clostridium were te st ed on PGUA agar for ,8-glucuronidase production. Clos t rid ia l ,8-glucuronidase was the extracellular enzyme produced by 115 of 141 stra ins of C. periringens, 2 of 3 st ra ins of C. beijerinckii, 1 of 2 of C. dijfi cile, 1 of 9 of C. lim osum, an d 1 of 4 of C. ram osum (Table 1).
120
Y.Sakaguchi and K.Murata
Table 1. ,B-Glucuronidase producing capacity of 19 different species of Clostridia on PGUA agar Species
Total strains examined
C. barati C. beijerinckii C. bifermentans C. butyricum C. clostridiforme C. cochlearium C. difficile C. [allax 9 C. in no cum C.limosum C. per jringens C. paraputrificum C. ramosum C. septicum C. sordellii C. sporogenes C. subterminal C. tertium C. tetani
3 3 2 4 1 4 2 6 2 9 141 4 4 1 1 1 7 1 1
Total &Gastric juice.
Number Positive
(1)&
(l)b (l)b
(l) C
197 b Bile.
Number Negative
0 2 0 0 1 0 1 0 0 1 (l )b 115 0 1 0 0 0 0 0 0
3 1 2 4 0 4 (1)& 1 6 2 8 26 4 (l)b 3 1 1 1 7 1 (1) c 1
121
76
CBlood.
Even after sonic disruption of cell suspensions, no n-ji-glucuronidase-producing strains did not have any p-glucuronidase. Therefore the enzyme, if present, was secreted into the growth medium. By the source from which the C. perjringens was isolated, 87 out of 113 strains from feces (77%) were Hobbs' typed strains, as were 20 out of 25 strains from bile Table 2. Clostridium perfringens strains isolated from various sources and their ,B-glucuronidase production Source
Number of strains
Hobbs' typed strains
Nontyped strains
,B-Glucuronidase Positive Nega tive
Feces Bile Secretions Umbilical cord Urine
113 25 1 1 1
87 20 1 0 1
26 5 0 1 0
93 20 1 0 1
20 5 0 1 0
141
109
32
115
26
T ot al
Studies on the p-Glucuronidase Production of Clostridia
121
(80% ) and 1 strain each from secretions and urine, whi le /1-g1ucuronidase production strains existed in 93 out of 113 strains from feces (82%),20 out of 25 strains from bile (80% ) and 1 strain each from secretions and urine (Table 2). When we examined the relationship between C. perjringens serotypes and /1-glucuronidase production, the enzyme was present in 104 out of 109 strains from H obbs' serotyped strains, but 21 of the 32 non-typed strains failed to produce /1-glucuroni dase (Table 3). There is a correlation between serologic type and /1glucuronidase production in C. perfringens except in Hobbs' serotypes 3/4, 4, 6 and 13. The reference strain C. perjringens GAl 0084 was serotyped into Hobbs 6, yet it did not produce /1-glucuronidase. It is very interesting that Hobbs' typed strains, which all cause food poisoning by an endotoxin, also usually produce /1-g1ucuronidase. Table 3. Correlation of serologic types with p-glucuronidase activity in Clostridium perfringens
Hobbs' serotype
Total strains examined
1 2 3 3 /4 4 5 6 7 8 9 10 11
7 3 5 4 15 7 4 7 5 2 11 6 4 5 9 4 9 2 32
12 13 14 15 16 17
N.T. Total a II
Bile. Secretions.
(2)& (2)&
(3) & (2) & (1)& (1)11 (1)& (1)& (1) C (2)& (2) & (1)& (1)& (1)& (1)& (5)& (l)d
141
Positive 7 3 5 3
(2)& (2)&
14 (3)& 7 2 7 5 2 11 6 4 4 9 4 9 2 11
(2) & (1)& (1)11 (1)& (1) & (1) C (2)& (2) & (1)& (1) & (1) & (1) &
115
<
Urine.
p-Glucuronidase Negative 0 0 0 1 1 0 2 0 0 0 0 0 0 1 0 0 0 0 21
(5)' (l)d
26
Others from feces d Umbilical cord.
References 1. Akama, K. and S. Otani: Clostridium perfringens as the flora in the isolation of healthy
persons. Jap. J. Med. Sci. BioI. 23
(1970) 161-175
curonide by bacteria. Nature 162
(1948) 701-702
2. Barber, M., B.W. L. Brooksbank, and G. A. D. Haslewood: Destruction of urinary glu-
122
Y. Sakaguchi an d K. Murata
3. Buehlar, H. J., P. A. Katzman , and E. A. Daisy : Studies on p-glucuronidase from E. coli. Pro c. Soc. expo BioI. 76 (1951) 672-676 4. Doll, W. : Untersuchungen tiber die DNase-Bildung von Clostridien. Zbl. Bakt. H yg., I. Abt. Orig, A 224 (1973) 115-119 5. Hall, H. E., R. Angelotti, K. H . Lewis, and M.]. Fater: Charact eristic of Clostridium perfringens strains associated with food and food-borne disease. J. Bact . 85 (1963) 1094-1103 6. Holdeman, L. V. and W. E. C. M oore: Anaerobe Labor ato ry Manual. Virginia Pol ytechni c Institute and State University Anaerobe Laboratory, Black sburg /Virginia (1977) 7. Jacox, R.F.: Streptococcal p-glucuronidase. J. Bact. 65 (1953) 700-705 8. Kilian, M. and P. BUlow: Rap id identificat ion of Enterobacteriaceae : II. Use of a pglucuronidase detecting agar medium (PGUA agar) for the identification of E. coli in primary cultures of urine samples. Acta pa th. microbiol. scand. Sect . B 87 (1979) 271276 9. Sakaguchi, Y., K. Murata , and M. Kimu ra: Clostridium perfringens and other an aerobes isola ted from bile. J. Clin. Path. (in press) 10. Sutton, R. G. A. an d B. C. Hobbs : Food poisoning caused by heat-sensitive Clostridium welchii. A report of five recent outbreaks . J. Hyg . (Camb.) 66 (1965) 135-145 Pro f. Dr. Kenjiro M urata, Dept. of Clinico-Labora rory Medicine, Kansai Me dica l University, 1 Fumizono-cho, Moriguchi-shi, Osaka, Japan 570
Department of Clinico -Laboratory Medici ne, Kansai Medical University, 1 Fu rnizono-cho, Moriguchi-shi, Osaka, Japan 570
Studies on the fJ-Glucuronidase Production of Clostridia Untersuchungen tiber di e fJ- Glucoronidas e- P r odukti on v o n Clostridien YOSHIAKI SAKAGUCHI and KEN]IRO MURATA
Received October 8, 1982
Abs tract We investigated the fJ-glucuronidase-producing capacity of 197 strains of 19 different species of Clostridia, using the procedure described by Kilian and Biilow (8). Most of the Hobbs' serot yped strains of Clostridium perfringens (104 out of 109 strains) were found to produce fJ-glucuronidase, whereas non-seroryped strains (21 out of 32 strains) did not . Some strains of other species such as Clostridium beijerinckii (c. beijerinckii ), C. clostridiforme. C. dilficile, C. lim osum , and C. ramosum were found to produce fJ-glucuronidase, while all strains of 13 oth er species which we examined did not.
Zusammen fassung Wir untersuchte n mit Hilfe des von Kilian und Bulow (8) beschriebenen Verfahrens die Fahigkeit zur fJ-Glucuronidase-Bildung bei 197 Stammen von 19 verschiedenen ClostridiumArten . Die meisten der C. periringens-Stiutune, die nach Hobbs serotypisiert worden waren (104 von 109), erwiesen sich als P-Glucuronidase-Bildner, wogegen dies bei den nicht sero typisierren Stammen (21 von 32) nicht der Fall war . Bei einigen Stammen anderer Arten wie C. beiierinckii , C. clostridiforme, C. difficile, C. limosum und C. ramosum wur de fJGluc uronidase-Bildung festgestellt, wahrend 13 andere von uns untersuchte Arten keine solchen Stamme aufwiesen,
Introduction T he bacterial Ii-glucuronidase of Staphylococcus albus, Streptococcus pyogenes, Escherichia coli, and Shigella has been int en sively studied (2, 3, 7 , 8), but the Ii-
glucuronida se production of anaerobes has not yet been studied. We frequently isolated Clostridium perjringens from patients' bile, which was co llected by duod en al sounding. We k new t hat Ii-glucuronidase-producing strains existed among t hese C. perlringens (9).
Stud ies on th e {3-Glucuro nidase Production of Clostridia
119
C. periringens is p art of the n o rm al intes ti nal flora, an d was fo un d in all of the feca l specime ns fr om a gro up of he althy per sons (IGl -109 / g feces) (1). We in vesti gate d the ,8-gluc uron idase-p ro d uc ing ca pacity of 197 st ra in s of Clostridia, iso lat ed fro m va rio us so urces, u sing the proced ure de scribed by Kilian an d Bulow (8). Materials and Methods Clost ridium strains Nineteen different species of Clostridium including 197 strains, were isolated from pa tient's feces, bile, gastr ic juice, blood, secretions, umb ilical cord or urine. Reference strai ns used were Clostridiu m bi fermentans GAl 0209, C. diffi cile GAl 0288, C. perlringens 0084, C. ramosum GAl 2560, C. sordellii GAl 0029, C. sporogenes GAI0005, and C. tetani, Osa ka Associat ion of Medical T echnologists. In th e case of feces, about 0.5 g of feces was dissolved in 5 ml of GAM broth (Nissui Co., T okyo, j apan) and incubated at 60 °C for 30 min. Then one loopful of medium was inocu lated onto GAM agar (Nissui) with egg-yolk. Oth er specimens were inocul ated on both GAM agar and Phenylethylalcohol agar (Nikken Co., Kyot o, j apan ). Anaerob ic cultures were do ne by an anaerobic culture system (Forma Scientific Co., U. S.A. ), and incubated at 37 °C for 48 h. Ident ification of Clostridia isolated from patient's feces, bile, gastric juice, blood , secretio ns, umb ilical cord or urine Bacterial morph ology was checked by Gram 's sta in. a-Toxin (lecithinase) production was detected by wide zones of precipi tate around th e colonies on egg-yolk agar. C. perfringens was identified by a-toxin productio n, sto rmy ferment ation of 10% litmus milk (BBL, U. S. A.), and degradat ion of 1% gelatin, glucose, lactose and DNA (4). C. perfringens strai ns isolated from vario us sources were as foll ows : 113 stra ins from feces, 25 strai ns from bile, and 1 strain each fro m secretions, umb ilical cord and urine. Identificatio n of other Clost ridia was perfo rmed with an API 20 A system (API Laborato ry Produ cts Ltd., France) with reference to the VPI Anaerob e laboratory manual (6). Detection of {J-glucuronidase-producing strains amo ng the Clostridia
Th e grow th medium used to exa mine {J-glucuro nidase activity was PGUA aga r, which was developed by Kilian and BUlow for the detection of Escherichia coli and Shigella among Entero bacteriaceae and Vibrioaceae (8). Th e incub ati on time for PGUA plates was 24- 48 h at 37 °C. {J-Glucuro nidase-pro ducing Clostridia developed a yellow zone aro und each colony. T he seroty pe of C. perjrin gens
Th e stra ins from th e blood agar or egg-yolk plate were typed into the kn own Hobb s' serotypes 1 through 17 by slide aggluti natio n tests using heat-stabl e C. perfringens type A ant i-sera for diagnosi s (Denk a-Seiken, T okyo, japan) (5, 10).
Results and Discussion Nineteen different species of Clostridium were te st ed on PGUA agar for ,8-glucuronidase production. Clos t rid ia l ,8-glucuronidase was the extracellular enzyme produced by 115 of 141 stra ins of C. periringens, 2 of 3 st ra ins of C. beijerinckii, 1 of 2 of C. dijfi cile, 1 of 9 of C. lim osum, an d 1 of 4 of C. ram osum (Table 1).
120
Y.Sakaguchi and K.Murata
Table 1. ,B-Glucuronidase producing capacity of 19 different species of Clostridia on PGUA agar Species
Total strains examined
C. barati C. beijerinckii C. bifermentans C. butyricum C. clostridiforme C. cochlearium C. difficile C. [allax 9 C. in no cum C.limosum C. per jringens C. paraputrificum C. ramosum C. septicum C. sordellii C. sporogenes C. subterminal C. tertium C. tetani
3 3 2 4 1 4 2 6 2 9 141 4 4 1 1 1 7 1 1
Total &Gastric juice.
Number Positive
(1)&
(l)b (l)b
(l) C
197 b Bile.
Number Negative
0 2 0 0 1 0 1 0 0 1 (l )b 115 0 1 0 0 0 0 0 0
3 1 2 4 0 4 (1)& 1 6 2 8 26 4 (l)b 3 1 1 1 7 1 (1) c 1
121
76
CBlood.
Even after sonic disruption of cell suspensions, no n-ji-glucuronidase-producing strains did not have any p-glucuronidase. Therefore the enzyme, if present, was secreted into the growth medium. By the source from which the C. perjringens was isolated, 87 out of 113 strains from feces (77%) were Hobbs' typed strains, as were 20 out of 25 strains from bile Table 2. Clostridium perfringens strains isolated from various sources and their ,B-glucuronidase production Source
Number of strains
Hobbs' typed strains
Nontyped strains
,B-Glucuronidase Positive Nega tive
Feces Bile Secretions Umbilical cord Urine
113 25 1 1 1
87 20 1 0 1
26 5 0 1 0
93 20 1 0 1
20 5 0 1 0
141
109
32
115
26
T ot al
Studies on the p-Glucuronidase Production of Clostridia
121
(80% ) and 1 strain each from secretions and urine, whi le /1-g1ucuronidase production strains existed in 93 out of 113 strains from feces (82%),20 out of 25 strains from bile (80% ) and 1 strain each from secretions and urine (Table 2). When we examined the relationship between C. perjringens serotypes and /1-glucuronidase production, the enzyme was present in 104 out of 109 strains from H obbs' serotyped strains, but 21 of the 32 non-typed strains failed to produce /1-glucuroni dase (Table 3). There is a correlation between serologic type and /1glucuronidase production in C. perfringens except in Hobbs' serotypes 3/4, 4, 6 and 13. The reference strain C. perjringens GAl 0084 was serotyped into Hobbs 6, yet it did not produce /1-glucuronidase. It is very interesting that Hobbs' typed strains, which all cause food poisoning by an endotoxin, also usually produce /1-g1ucuronidase. Table 3. Correlation of serologic types with p-glucuronidase activity in Clostridium perfringens
Hobbs' serotype
Total strains examined
1 2 3 3 /4 4 5 6 7 8 9 10 11
7 3 5 4 15 7 4 7 5 2 11 6 4 5 9 4 9 2 32
12 13 14 15 16 17
N.T. Total a II
Bile. Secretions.
(2)& (2)&
(3) & (2) & (1)& (1)11 (1)& (1)& (1) C (2)& (2) & (1)& (1)& (1)& (1)& (5)& (l)d
141
Positive 7 3 5 3
(2)& (2)&
14 (3)& 7 2 7 5 2 11 6 4 4 9 4 9 2 11
(2) & (1)& (1)11 (1)& (1) & (1) C (2)& (2) & (1)& (1) & (1) & (1) &
115
<
Urine.
p-Glucuronidase Negative 0 0 0 1 1 0 2 0 0 0 0 0 0 1 0 0 0 0 21
(5)' (l)d
26
Others from feces d Umbilical cord.
References 1. Akama, K. and S. Otani: Clostridium perfringens as the flora in the isolation of healthy
persons. Jap. J. Med. Sci. BioI. 23
(1970) 161-175
curonide by bacteria. Nature 162
(1948) 701-702
2. Barber, M., B.W. L. Brooksbank, and G. A. D. Haslewood: Destruction of urinary glu-
122
Y. Sakaguchi an d K. Murata
3. Buehlar, H. J., P. A. Katzman , and E. A. Daisy : Studies on p-glucuronidase from E. coli. Pro c. Soc. expo BioI. 76 (1951) 672-676 4. Doll, W. : Untersuchungen tiber die DNase-Bildung von Clostridien. Zbl. Bakt. H yg., I. Abt. Orig, A 224 (1973) 115-119 5. Hall, H. E., R. Angelotti, K. H . Lewis, and M.]. Fater: Charact eristic of Clostridium perfringens strains associated with food and food-borne disease. J. Bact . 85 (1963) 1094-1103 6. Holdeman, L. V. and W. E. C. M oore: Anaerobe Labor ato ry Manual. Virginia Pol ytechni c Institute and State University Anaerobe Laboratory, Black sburg /Virginia (1977) 7. Jacox, R.F.: Streptococcal p-glucuronidase. J. Bact. 65 (1953) 700-705 8. Kilian, M. and P. BUlow: Rap id identificat ion of Enterobacteriaceae : II. Use of a pglucuronidase detecting agar medium (PGUA agar) for the identification of E. coli in primary cultures of urine samples. Acta pa th. microbiol. scand. Sect . B 87 (1979) 271276 9. Sakaguchi, Y., K. Murata , and M. Kimu ra: Clostridium perfringens and other an aerobes isola ted from bile. J. Clin. Path. (in press) 10. Sutton, R. G. A. an d B. C. Hobbs : Food poisoning caused by heat-sensitive Clostridium welchii. A report of five recent outbreaks . J. Hyg . (Camb.) 66 (1965) 135-145 Pro f. Dr. Kenjiro M urata, Dept. of Clinico-Labora rory Medicine, Kansai Me dica l University, 1 Fumizono-cho, Moriguchi-shi, Osaka, Japan 570