APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1992, p. 1524-1529
Vol. 58, No. 5
0099-2240/92/051524-06$02.00/0
Rapid Detection of Members of the Family Enterobacteriaceae by a Monoclonal Antibody STEPHANE LEVASSEUR,1 MARIE-ODILE HUSSON,2 RAMONE LEITZ,3
FRANQOISE MERLIN,'
FRANCK LAURENT,1 FABRICE PELADAN,3 JEAN-LOUIS DROCOURT,1* HENRI LECLERC,2 AND MICHEL VAN HOEGAERDEN1t Chemune-x S. A., 41 rue du 11 Novembre 1918, 94700 Maisons-Alfort, 1 Laboratoire de Bacteiologie A, Facult6 de Medecine, 59045 Lille Cede ,2 and TEPRAL S. A., 67200 Strasbourg,3 France Received 28 October 1991/Accepted 9 March 1992
Six monoclonal antibodies directed against enterobacteria were produced and characterized. The specificity of one of these antibodies (CX9/15; immunoglobulin G2a) was studied by indirect immunofluorescence against 259 enterobacterial strains and 125 other gram-negative bacteria. All of the enterobacteria were specifically recognized, the only exception being Erwinia chrysanthemi (one strain tested). Bacteria not belonging to members of the family Enterobacteriaceae were not detected, except for Plesiomonas shigelloides (two strains tested), Aeromonas hydrophila (five strains tested), and Aeromonas sobria (one strain tested). This recognition spectrum strongly suggested that CX9/15 recognized the enterobacterial common antigen. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) experiments, the six antienterobacteria antibodies presented similar specificities; they all revealed only one band with an apparent molecular weight of about 20,000 from the crude extract of an enterobacterium. The six monoclonal antibodies, and especially CX9/15, can be used to develop new tests for rapid and specific detection of enterobacteria. MATERUILS AND METHODS Bacterial strains and cultivation. The strains used in this study are listed in Tables 1 and 2. Escherichia coli 014:K7 (ATCC 19110) was used for immunization and for screening of hybridoma supernatant fluids. The following six strains were used to determine MAb specificity in an enzyme-linked immunosorbent assay (ELISA): E. coli 014:K7, Proteus hauseri, Citrobacter freundii, Serratia marcescens, Enterobacter agglomerans, and Pseudomonas maltophilia. The strains were cultivated overnight at 37°C in peptone broth. They were identified as previously described by Krieg and Holt (4). Immunizations. The antigen was prepared as follows. A suspension of E. coli 014:K7, grown for 18 h at 37°C in peptone broth, was adjusted to 1010 cells per ml in 0.15 M NaCl. The suspension was heated for 1 h at 100°C as described by Suzuki et al. (15) and then centrifuged for 10 min at 2,500 x g. The supernatant was emulsified with an equal volume of complete Freund's adjuvant, and 200 ,ul of emulsion was injected subcutaneously into two 6-weekold BALB/c mice. After 28 days, 100 ,ul of the heated supernatant, emulsified with an equal volume of incomplete Freund's adjuvant, was injected in the same manner. After a further 2 months, the final boost was performed by intravenous injection of 100 ,u of heated supernatant from a suspension of 109 E. coli cells per ml. Fusion was performed three and a half days later. MAb production. Fusions of splenocytes from immunized mice and NS1 myeloma cells were effected as described by Kohler and Milstein (3) by using 41% polyethylene glycol (PM, 1500; Merck Laboratories). Secreting hybrids were cloned according to the limiting dilutions procedure and were cultivated in RPMI medium containing 10 to 20% fetal calf serum with feeder cells. Specificity testing of MAb. Bacterial cells were washed three times with a filtered (0.22-,um pore size) 0.15 M NaCl aqueous solution. Bacterial suspensions were adjusted to 5 x 108 cells per ml and introduced alive (50 ,ul per well) into
Contamination of food or pharmaceutical products by some members of the group termed coliforms (Escherichia, Citrobacter, Enterobacter, and Klebsiella species) or by Salmonella and Shigella species may cause severe intoxication or sepsis. Therefore, the enumeration of these bacteria, all belonging to the family Enterobacteriaceae, is one of the most important quality control analyses carried out in the microbiology laboratory. Their presence is often considered an indicator of unhygienic practices in production processes. The current technique for the enumeration of enterobacteria is the classical microbiological method based on culture in selective media. However, false-positive results resulting from growth of nonenterobacteria as well as false-negative results that arise from no growth of enterobacteria may occur. Culture techniques are also time consuming (24 to 48 h). This delay removes the possibility of intervention and corrective action during production and can have serious economic consequences. For this reason, easy and rapid techniques are needed. One promising approach for rapid detection of bacteria is the development of immunochemical techniques involving the use of specific antibodies. Initially for medical purposes, Peters et al. (13) have produced and characterized two monoclonal antibodies (MAb) raised against the enterobacterial common antigen (ECA), also called Kunin's antigen, a polysaccharide which contains a trisaccharide repeating unit (9, 10). The first use of these MAb to specifically detect a wide range of Enterobacteriaceae in water (12) was not satisfactory because of the requirement of a 10- to 14-h pre-enrichment and the solubilization of the ECA. We describe here the production of MAb selected for their ability to detect rapidly all Enterobacteriaceae in their intact form. One of these antibodies was extensively studied for specific detection of whole enterobacteria by immunofluorescence. *
Corresponding author.
t Present address: B.S.N., 7 rue de Teheran, 75008 Paris, France. 1524
VOL. 58, 1992
RAPID DETECTION OF ENTEROBACTERIA BY MAb
1525
TABLE 1. Bacterial strains recognized by MAb CX9/15 Family and genus
Enterobacteriaceae Escherichia spp .............
Species and strain(s)a
E. coli ATCC 10536T, ATCC 19110, ATCC 12806, ATCC 25922, ATCC 4157, CIP 52170, NCTC 9855, NCTC 8621, NCTC 8623, NCTC 8959, 014:K7 E. coli (20 medical strains) E. coli (1 environmental strain) E. fergusonii CDC 568-73T, CDC 3458-74, CUETM 8582 E. hernannii ATCC 33650T, CUETM 84-01, CUETM 84-96, CUETM 84-52, CUETM 87-11 E. vulneris ATCC 33821T, CUETM 86-159, CUETM 86-01, CUETM 86-05 E. adecarboxylata CUETM 78251 Shigella spp ............. S. flexneri (2 medical strains) S. sonnei (2 medical strains) Salmonella spp ............. S. enteritidis ATCC 13076T S. ententidis (1 medical strain) S. typhi (1 medical strain) S. paratyphi A (1 medical strain) S. typhimurium (5 medical strains) S. agona (1 medical strain) Levinea spp ............. L. malonatica ATCC 27156, CUETM 82-78, CUETM 82-76, CUETM 82-85, CUETM 77-10, CUETM 82-80, CUETM 77-12 L. amalonatica ATCC 25406, ATCC 25405, CUETM 77-26, CUETM 77-09, CUETM 77-19 Citrobacter spp .............. C. freundii ATCC 8090T, CUETM 86-86 C. freundii (1 medical strain) C. freundii (3 environmental strains) Klebsiella spp ............. K. pneumoniae ATCC 13883T K pneumoniae (8 medical strains) K pneumoniae (2 environmental strains) K oxytoca ATCC 13182T K oxytoca (1 medical strain) K oxytoca (1 environmental strain) K ozaenae ATCC 11296T K ozaenae (2 medical strains) K rhinoscleromatis ATCC 13884T K terigena CUETM 78-152, CUETM 78-143, CUETM 78-142, CUETM 78-130 K planticola ATCC 33538T, CUETM 78-122, CUETM 78-113, CUETM 78-92, CUETM 78-118, CUETM 78-115 Enterobacter spp ............. E. cloacae ATCC 13047T E. cloacae (4 medical strains) E. cloacae (5 environmental strains) E. aerogenes ATCC 13048T E. aerogenes (2 environmental strains) E. amnigenus CUETM 87-21, CUETM 78-70, CUETM 78-78, CUETM 78-84, CUETM 78-83, CUETM 88-58, CUETM 77-132, CUETM 78-98, CUETM 78-88, CUETM 78-65, CUETM 78-79, CUETM 77-114, CIP 33072 E. intermedium ATCC 33110T, CUETM 77-127, CUETM 77-145, CUETM 77-123, CUETM 77-139, CUETM 77-147 E. agglomerans ATCC 27155T, CDC 1461-67, CUETM 82-84, CUETM 79-146, CUETM 83-13, CUETM 78-97, CUETM 79-16, CUETM 78-161, CUETM 83-21, CUETM 77-118 E. sakazakii CUETM 79-103 E. asbuviae ATCC 35953T, CDC 3117-86, CDC 114-85 E. gergoviae ATCC 33028T Serratia spp ............. S. marcescens (4 medical strains) S. marcescens (3 environmental strains) S. liquefaciens ATCC 27592T S. liquefaciens (3 medical strains) S. liquefaciens (1 environmental strain) S. fonticola ATCC 29844T, ATCC 29846, ATCC 29845, CUETM 78-14, CUETM 78-45, CUETM 78-22, CUETM 78-25, CUETM 78-07, CUETM 78-12 S. plymuthica ATCC 183T, CUETM 77-52, CUETM 78-219, CUETM 78-222, CUETM 78-170 S. rubidea ATCC 27593T, CUETM 83-31, CUETM 82-75, CUETM 82-20, CUETM 83-35, CUETM 83-29 S. ficaria ATCC 33105T, ATCC 33106 S. odorifera ATCC 33077T Hafnia sp........... H. alvei ATCC 13337T,TCUETM 78-313, CUETM 78-302, CUETM 77-144, CUETM 78-322, CUETM 78-305 R. aquatilis ATCC 33991, CUETM 77-131, CUETM 78-85, CUETM 88-69, CUETM 87-08, Rahnella sp ............. CUETM 88-71 Proteus spp .............P . mirabilis ATCC 29906T P. mirabilis (11 medical strains) Continued on following page
1526
APPL. ENVIRON. MICROBIOL.
LEVASSEUR ET AL.
TABLE 1-Continued Family and genus
Species and strain(s)a P. vulganis ATCC 13315T P. vulgaris (1 medical strain) Providencia spp .................. P. stuartii ATCC 29914T P. stuartii (1 medical strain) P. rettgeri ATCC 29944T P. alcalifaciens ATCC 9886T M. morganii (4 medical strains) Morganella sp . Yersinia spp .................. Y pseudotuberculosis ATCC 29833T, CUETM 23-207, CUETM 84-44 Y enterocolitica ATCC 9610T, CUETM 82-52 Y ruckeri CUETM 82-64, CUETM 82-62 Y frederiksenii CIP 8029T Y. intermedia CIP 8028 Y aldovae ATCC 35236T, CUETM 84-182, CUETM 84-181, CUETM 84-185 .................
Buttiauxella sp .................
B. agrestis CUETM 78-41, CUETM 77-163, CUETM 78-08, CUETM 78-03 K cryocrescens ATCC 33435T, ATCC 14238 K ascorbata ATCC 33434T, ATCC 14236, CUETM 82-09, CUETM 82-33, CUETM 82-35 Budvicia sp ..................B. aquatica CUETM 84-70 Erwinia spp ................. E. herbicola NCPPB 2971T E. cypripedi (1 environmental strain)
Kluyvera spp .
.................
E. ananas (1 environmental strain) E. tarda ATCC 15947T, CUETM 85-55 E. hoshinae ATCC 33379T, CUETM 84-74, CUETM 84-73 P. fontium CGUG 18077T, CCUG 18074 Pragia sp ................. Obesumbacterium sp .................0. proteus ATCC 12841T
Edwardsiella spp .................
Non-Enterobacteniaceae Plesiomonas sp .................
Aeromonas spp .................
P. shigelloides ATCC
14029T, CUETM 84-238
A. hydrophila CUETM 84-231, CUETM 77-94, CUETM 77-95, CUETM 84-235, CUETM 84-232 A. sobria 84-233 CUETM.
a ATCC, American Type Culture Collection, Rockville, Md.; CIP, Collection de l'Institut Pasteur, Paris, France; NCTC, National Collection of Type Cultures, London, England; CDC, Centers for Disease Control or Communicable Disease Center, Atlanta, Ga.; CUETM, Collection de l'Unite Ecotoxicologie Microbienne, Villeneuve d'Ascq, France; NCPPB, National Collection of Plant Pathogenic Bacteria, Harpenden, England; CGUG, Culture Collection of Universitat Goteborg, Goteborg, Sweden.
polystyrene titration plates (Costar). Coating of the wells with bacteria was achieved overnight at 4°C. The wells were washed three times with 200 RI of phosphate-buffered saline (PBS; 10 mM, pH 7.1). Saturation was performed for 1 h at 37°C by using 100 pI of PBS supplemented with gelatin (0.25% [wt/vol]) and Tween 20 (0.1% [vol/vol]) (PGT). Aliquots (50 ,ul) of culture supernatants, diluted twofold in RPMI medium, were introduced into each well and incubated for 2 h at 37°C. After five washings, 50 ,ul of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Amersham Laboratories), diluted according to the recommendations of the manufacturer, was introduced into each well and incubated for 1 h at 37°C. After five washings, peroxidase activity was detected colorimetrically by adding azino-bis (3-ethyl-benzthiazoline-6 sulfonic acid; 1.1 mg/ml) in 0.6% (vol/vol) acetic acid (pH 4.7) containing 0.015% (vol/vol) hydrogen peroxide. Colorimetric staining was performed for 15 to 30 min in the dark, and the optical densities were read at 405 nm. Each culture supernatant was tested against five enterobacterial strains from different genera and species and one strain of the species P. maltophilia as a negative control. Hybridoma culture supernatants recognizing all five enterobacteria strains which did not react against the Pseudomonas strain were selected. After confirmation, the corresponding cells were cloned and amplified and the subsequent antibody-enriched supernatants were tested again by ELISA as described above. They were also tested by indirect immunofluorescence on slides as follows. A 20-j,l volume of bacterial suspensions was dried onto an immunofluorescence slide (Dynatech) and fixed for 5 min with 70% ethanol. The slides were washed with PGT, 20 ,u of pure clone
supernatant was added, and the plates were then incubated for 2 h at 37°C. The plates were washed and successively incubated for 30 min at 37°C with 20 ,ul of biotinylated sheep anti-mouse immunoglobulin G (heavy plus light chains) antibodies washed and incubated for 15 min with 20 ,u of fluoresceinated streptavidin (Amersham). The same indirect immunofluorescence technique was applied for the extensive study of the recognition spectra of MAb CX9/15 (ascites fluid diluted 1:500 in PGT). Electrophoresis and immunoblot analysis. Antigen extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed as described by Peters et al. (13). Briefly, 100 ,ul of buffer (62.5 mM; Tris-hydrochloride 2-mercaptoethanol [5%], SDS [2%], glycerol [12.5%]; pH 6.8) was added to a pellet containing 5 x 108 bacteria washed in saline (0.15 M NaCl). The bacterial suspensions were heated for 10 min at 100°C. SDS-PAGE was performed with the equivalent of 108 bacteria (20 RI). The gels were run in duplicate; one was developed by silver staining, the other was subjected to a 1-h electric transfer onto nitrocellulose membranes with an LKB Novablot apparatus and a field of 0.8 mA/cm2. Membranes were saturated by incubation for 1 h in PGT and then incubated for 1 h with antienterobacterial MAb (ascites fluid) diluted 1:500 in PGT. The nitrocellulose filters were then washed three times (for 10 min each) with 0.1% PBS-Tween and subsequently incubated for 1 h with horseradish peroxidaseconjugated goat anti-mouse immunoglobulin at 2.5 ,ug/ml in PGT. Three washings were performed as described above, followed by a final washing with PBS. The membranes were developed in 50 ml of PBS containing 9 mg of 4-chloronaphthol (previously solubilized in 3 ml of methanol) and
VOL. 58, 1992
RAPID DETECTION OF ENTEROBACTERIA BY MAb
TABLE 2. Bacterial strains that were not recognized by MAb CX9/15 Family and genus Species and strain(s)a Enterobacteriaceae Erwinia sp .................E. chrysanthemi ATCC 11663T Non-Enterobacteriaceae Pseudomonas spp. P. aeruginosa ATCC 27853T ,CIP A22 P. aeruginosa (1 environmental strain) P. aeruginosa (5 medical strains) P. fluorescens ATCC 13525T, ATCC 17397 P. fluorescens (13 environmental strains) P. fluorescens (1 medical strain) P. aureofaciens (1 environmental strain) P. chloraphis (4 environmental strains) P. putida ATCC 12633T P. putida (22 environmental strains) P. mendocina ATCC 25411T ,ATCC 25413, ATCC 5411, NCTC 10897 P. stutzeri ATCC 17588T, CUETM 8639, CUETM 86-37 P. vesicularis (5 environmental strains) P. acidovorans ATCC 15668T CUETM 83-76, CUETM 84-104, CUETM 83-152 P. pseudomallei (1 environmental strain) P. picketti ATCC 27511T P. pickettii (1 environmental strain) P. paucimobilis ATCC 6518 P. paucimobilis (3 environmental strains) P. cepacia ATCC 25609 Xanthomonas spp ...... X maltophilia ATCC 13637T X. maltophilia (7 medical strains) Acinetobacter spp ........ A. calcoaceticus (1 medical strain) A. haemolyticus (6 medical strains) A. junii (4 medical strains) A. johnsonii (1 medical strain) A. iwoffii (5 medical strains) A. baumanii (2 medical strains) Achromobacter spp ...... A. xylososidans CUETM 84-134, CUETM 84-143, CUETM 83-149, CUETM 84-115 A. xylososidans (4 medical strains) Alcaligenes sp ............. A. denitrificans ATCC 15173T Flavobacterium sp. F. odoratum ATCC 4651 Pasteurella sp ..............P. haemolytica (3 medical strains) Haemophilus sp .............H. influenzae (1 medical strain) Vibrio sp ................. (1 medical strain) Agrobacter sp. ............. A. radiobacter CUETM 77-93 .........
.
........
aSee footnote to Table 1 for abbreviations.
0.03% (vol/vol) hydrogen peroxide. Migration markers were the low-molecular-mass markers (14.4 to 92 kDa) from Pharmacia Laboratories. RESULTS AND DISCUSSION
Among the six clones produced, the MAb CX9/15 (immunoglobulin G2a) was selected, and its specificity was studied by the indirect immunofluorescence technique against 259 Enterobacteriaceae strains and 125 other gram-negative bacteria. These results are summarized in Table 1 for positive reactions and in Table 2 for negative ones. The MAb
1527
CX9/15 recognized all species of the Enterobacteriaceae family except Erwinia chrysanthemi (one strain tested). This recognition spectrum corresponded to the distribution of ECA in wild-type strains of gram-negative bacteria (2, 5-8, 14). It agreed with previous reports of the absence of ECA at the surface of the species E. chrysanthemi (2, 8). MAb CX9/15 did not react against 117 of 125 gramnegative bacteria not belonging to the Enterobacteniaceae but cross-reacted with the species Plesiomonas shigelloides (two strains tested), Aeromonas hydrophila (five strains tested) and Aeromonas sobria (one strain tested). The recognition of P. shigelloides by anti-ECA antibodies had already been reported elsewhere (1, 14). This gram-negative bacterium, which is closely related to the Enterobacteriaceae, presented an ECA antigen identical to the enterobacterial one (5). The species A. hydrophila has been previously reported to be devoid of ECA (8); however, one strain (209A) has been found to contain ECA (Nacescu and Ciufecu; cited in reference 11). Our strain of A. sobria was detected by CX9/15, which was an unexpected result, since the only strain tested previously was found to be ECA negative (14). This discrepancy needs further investigation and may be due to differences in the techniques used, since Ramia et al. used hemagglutination with heated supernatants (14). ECA has also been detected in Tatumella ptyseos and Xenorhabdus species (14); we have not yet tested these bacteria. Western blot (immunoblot) studies have been performed to determine the characteristics of the antigen recognized by six of our antienterobacterial MAb. Heated supernatants from four enterobacteria and a P. maltophilia strain were analyzed by SDS-PAGE, and staining on nitrocellulose with MAb CX9/15 was also studied (Fig. 1). The pattern obtained with the four enterobacteria was similar; a single band with an apparent molecular weight of about 20,000 appeared. Only minor and nonsignificant migration differences between the four antigens revealed existed. We believe that CX9/15 is directed against the ECA for two reasons. (i) The enterobacterium that we have used as the source of antigen was E. coli 014:K7, known as a mutant presenting an immunogenic form of ECA (7). The crude extract used for immunization of mice was obtained after the first step of an ECA purification process (15). (ii) The recognition spectrum of MAb CX9/15 closely resembled the distribution of ECA described by Kuhn et al. (6). Our six antienterobacterial MAb presented similar reactivities (Fig. 2); all of the immunoblots presented a single band. This result was different from the ladderlike patterns presented by Kuhn et al. (5) and Peters et al. (13) using their anti-ECA MAb (MAb 898 and 865). The enterobacteria (particularly E. coli 014:K7) and the conditions of sample treatment and electrophoresis were the same as those described by Peters et al. (13). We conclude that our antibodies either do not recognize the same epitope as MAb 898 and 865 or recognize different forms of ECA. Two main differences may explain these different reactivities. (i) Peter et al. immunized mice against formalinized E. coli K-12 (13), whereas we used heated supernatant from E. coli 014:K7, and (ii) our screening procedure by ELISA, which has been performed elsewhere with glutaraldehyde-treated enterobacteria (and extracts of heated bacteria) for 898 and 865 (13), used intact untreated bacteria. Previous immunizations with intact whole enterobacteria (UV inactivated) have also been performed. However, the antibodies produced have not been able to recognize the five enterobacteria chosen for the screening. These observations
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Vol. 58, No. 5
0099-2240/92/051524-06$02.00/0
Rapid Detection of Members of the Family Enterobacteriaceae by a Monoclonal Antibody STEPHANE LEVASSEUR,1 MARIE-ODILE HUSSON,2 RAMONE LEITZ,3
FRANQOISE MERLIN,'
FRANCK LAURENT,1 FABRICE PELADAN,3 JEAN-LOUIS DROCOURT,1* HENRI LECLERC,2 AND MICHEL VAN HOEGAERDEN1t Chemune-x S. A., 41 rue du 11 Novembre 1918, 94700 Maisons-Alfort, 1 Laboratoire de Bacteiologie A, Facult6 de Medecine, 59045 Lille Cede ,2 and TEPRAL S. A., 67200 Strasbourg,3 France Received 28 October 1991/Accepted 9 March 1992
Six monoclonal antibodies directed against enterobacteria were produced and characterized. The specificity of one of these antibodies (CX9/15; immunoglobulin G2a) was studied by indirect immunofluorescence against 259 enterobacterial strains and 125 other gram-negative bacteria. All of the enterobacteria were specifically recognized, the only exception being Erwinia chrysanthemi (one strain tested). Bacteria not belonging to members of the family Enterobacteriaceae were not detected, except for Plesiomonas shigelloides (two strains tested), Aeromonas hydrophila (five strains tested), and Aeromonas sobria (one strain tested). This recognition spectrum strongly suggested that CX9/15 recognized the enterobacterial common antigen. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) experiments, the six antienterobacteria antibodies presented similar specificities; they all revealed only one band with an apparent molecular weight of about 20,000 from the crude extract of an enterobacterium. The six monoclonal antibodies, and especially CX9/15, can be used to develop new tests for rapid and specific detection of enterobacteria. MATERUILS AND METHODS Bacterial strains and cultivation. The strains used in this study are listed in Tables 1 and 2. Escherichia coli 014:K7 (ATCC 19110) was used for immunization and for screening of hybridoma supernatant fluids. The following six strains were used to determine MAb specificity in an enzyme-linked immunosorbent assay (ELISA): E. coli 014:K7, Proteus hauseri, Citrobacter freundii, Serratia marcescens, Enterobacter agglomerans, and Pseudomonas maltophilia. The strains were cultivated overnight at 37°C in peptone broth. They were identified as previously described by Krieg and Holt (4). Immunizations. The antigen was prepared as follows. A suspension of E. coli 014:K7, grown for 18 h at 37°C in peptone broth, was adjusted to 1010 cells per ml in 0.15 M NaCl. The suspension was heated for 1 h at 100°C as described by Suzuki et al. (15) and then centrifuged for 10 min at 2,500 x g. The supernatant was emulsified with an equal volume of complete Freund's adjuvant, and 200 ,ul of emulsion was injected subcutaneously into two 6-weekold BALB/c mice. After 28 days, 100 ,ul of the heated supernatant, emulsified with an equal volume of incomplete Freund's adjuvant, was injected in the same manner. After a further 2 months, the final boost was performed by intravenous injection of 100 ,u of heated supernatant from a suspension of 109 E. coli cells per ml. Fusion was performed three and a half days later. MAb production. Fusions of splenocytes from immunized mice and NS1 myeloma cells were effected as described by Kohler and Milstein (3) by using 41% polyethylene glycol (PM, 1500; Merck Laboratories). Secreting hybrids were cloned according to the limiting dilutions procedure and were cultivated in RPMI medium containing 10 to 20% fetal calf serum with feeder cells. Specificity testing of MAb. Bacterial cells were washed three times with a filtered (0.22-,um pore size) 0.15 M NaCl aqueous solution. Bacterial suspensions were adjusted to 5 x 108 cells per ml and introduced alive (50 ,ul per well) into
Contamination of food or pharmaceutical products by some members of the group termed coliforms (Escherichia, Citrobacter, Enterobacter, and Klebsiella species) or by Salmonella and Shigella species may cause severe intoxication or sepsis. Therefore, the enumeration of these bacteria, all belonging to the family Enterobacteriaceae, is one of the most important quality control analyses carried out in the microbiology laboratory. Their presence is often considered an indicator of unhygienic practices in production processes. The current technique for the enumeration of enterobacteria is the classical microbiological method based on culture in selective media. However, false-positive results resulting from growth of nonenterobacteria as well as false-negative results that arise from no growth of enterobacteria may occur. Culture techniques are also time consuming (24 to 48 h). This delay removes the possibility of intervention and corrective action during production and can have serious economic consequences. For this reason, easy and rapid techniques are needed. One promising approach for rapid detection of bacteria is the development of immunochemical techniques involving the use of specific antibodies. Initially for medical purposes, Peters et al. (13) have produced and characterized two monoclonal antibodies (MAb) raised against the enterobacterial common antigen (ECA), also called Kunin's antigen, a polysaccharide which contains a trisaccharide repeating unit (9, 10). The first use of these MAb to specifically detect a wide range of Enterobacteriaceae in water (12) was not satisfactory because of the requirement of a 10- to 14-h pre-enrichment and the solubilization of the ECA. We describe here the production of MAb selected for their ability to detect rapidly all Enterobacteriaceae in their intact form. One of these antibodies was extensively studied for specific detection of whole enterobacteria by immunofluorescence. *
Corresponding author.
t Present address: B.S.N., 7 rue de Teheran, 75008 Paris, France. 1524
VOL. 58, 1992
RAPID DETECTION OF ENTEROBACTERIA BY MAb
1525
TABLE 1. Bacterial strains recognized by MAb CX9/15 Family and genus
Enterobacteriaceae Escherichia spp .............
Species and strain(s)a
E. coli ATCC 10536T, ATCC 19110, ATCC 12806, ATCC 25922, ATCC 4157, CIP 52170, NCTC 9855, NCTC 8621, NCTC 8623, NCTC 8959, 014:K7 E. coli (20 medical strains) E. coli (1 environmental strain) E. fergusonii CDC 568-73T, CDC 3458-74, CUETM 8582 E. hernannii ATCC 33650T, CUETM 84-01, CUETM 84-96, CUETM 84-52, CUETM 87-11 E. vulneris ATCC 33821T, CUETM 86-159, CUETM 86-01, CUETM 86-05 E. adecarboxylata CUETM 78251 Shigella spp ............. S. flexneri (2 medical strains) S. sonnei (2 medical strains) Salmonella spp ............. S. enteritidis ATCC 13076T S. ententidis (1 medical strain) S. typhi (1 medical strain) S. paratyphi A (1 medical strain) S. typhimurium (5 medical strains) S. agona (1 medical strain) Levinea spp ............. L. malonatica ATCC 27156, CUETM 82-78, CUETM 82-76, CUETM 82-85, CUETM 77-10, CUETM 82-80, CUETM 77-12 L. amalonatica ATCC 25406, ATCC 25405, CUETM 77-26, CUETM 77-09, CUETM 77-19 Citrobacter spp .............. C. freundii ATCC 8090T, CUETM 86-86 C. freundii (1 medical strain) C. freundii (3 environmental strains) Klebsiella spp ............. K. pneumoniae ATCC 13883T K pneumoniae (8 medical strains) K pneumoniae (2 environmental strains) K oxytoca ATCC 13182T K oxytoca (1 medical strain) K oxytoca (1 environmental strain) K ozaenae ATCC 11296T K ozaenae (2 medical strains) K rhinoscleromatis ATCC 13884T K terigena CUETM 78-152, CUETM 78-143, CUETM 78-142, CUETM 78-130 K planticola ATCC 33538T, CUETM 78-122, CUETM 78-113, CUETM 78-92, CUETM 78-118, CUETM 78-115 Enterobacter spp ............. E. cloacae ATCC 13047T E. cloacae (4 medical strains) E. cloacae (5 environmental strains) E. aerogenes ATCC 13048T E. aerogenes (2 environmental strains) E. amnigenus CUETM 87-21, CUETM 78-70, CUETM 78-78, CUETM 78-84, CUETM 78-83, CUETM 88-58, CUETM 77-132, CUETM 78-98, CUETM 78-88, CUETM 78-65, CUETM 78-79, CUETM 77-114, CIP 33072 E. intermedium ATCC 33110T, CUETM 77-127, CUETM 77-145, CUETM 77-123, CUETM 77-139, CUETM 77-147 E. agglomerans ATCC 27155T, CDC 1461-67, CUETM 82-84, CUETM 79-146, CUETM 83-13, CUETM 78-97, CUETM 79-16, CUETM 78-161, CUETM 83-21, CUETM 77-118 E. sakazakii CUETM 79-103 E. asbuviae ATCC 35953T, CDC 3117-86, CDC 114-85 E. gergoviae ATCC 33028T Serratia spp ............. S. marcescens (4 medical strains) S. marcescens (3 environmental strains) S. liquefaciens ATCC 27592T S. liquefaciens (3 medical strains) S. liquefaciens (1 environmental strain) S. fonticola ATCC 29844T, ATCC 29846, ATCC 29845, CUETM 78-14, CUETM 78-45, CUETM 78-22, CUETM 78-25, CUETM 78-07, CUETM 78-12 S. plymuthica ATCC 183T, CUETM 77-52, CUETM 78-219, CUETM 78-222, CUETM 78-170 S. rubidea ATCC 27593T, CUETM 83-31, CUETM 82-75, CUETM 82-20, CUETM 83-35, CUETM 83-29 S. ficaria ATCC 33105T, ATCC 33106 S. odorifera ATCC 33077T Hafnia sp........... H. alvei ATCC 13337T,TCUETM 78-313, CUETM 78-302, CUETM 77-144, CUETM 78-322, CUETM 78-305 R. aquatilis ATCC 33991, CUETM 77-131, CUETM 78-85, CUETM 88-69, CUETM 87-08, Rahnella sp ............. CUETM 88-71 Proteus spp .............P . mirabilis ATCC 29906T P. mirabilis (11 medical strains) Continued on following page
1526
APPL. ENVIRON. MICROBIOL.
LEVASSEUR ET AL.
TABLE 1-Continued Family and genus
Species and strain(s)a P. vulganis ATCC 13315T P. vulgaris (1 medical strain) Providencia spp .................. P. stuartii ATCC 29914T P. stuartii (1 medical strain) P. rettgeri ATCC 29944T P. alcalifaciens ATCC 9886T M. morganii (4 medical strains) Morganella sp . Yersinia spp .................. Y pseudotuberculosis ATCC 29833T, CUETM 23-207, CUETM 84-44 Y enterocolitica ATCC 9610T, CUETM 82-52 Y ruckeri CUETM 82-64, CUETM 82-62 Y frederiksenii CIP 8029T Y. intermedia CIP 8028 Y aldovae ATCC 35236T, CUETM 84-182, CUETM 84-181, CUETM 84-185 .................
Buttiauxella sp .................
B. agrestis CUETM 78-41, CUETM 77-163, CUETM 78-08, CUETM 78-03 K cryocrescens ATCC 33435T, ATCC 14238 K ascorbata ATCC 33434T, ATCC 14236, CUETM 82-09, CUETM 82-33, CUETM 82-35 Budvicia sp ..................B. aquatica CUETM 84-70 Erwinia spp ................. E. herbicola NCPPB 2971T E. cypripedi (1 environmental strain)
Kluyvera spp .
.................
E. ananas (1 environmental strain) E. tarda ATCC 15947T, CUETM 85-55 E. hoshinae ATCC 33379T, CUETM 84-74, CUETM 84-73 P. fontium CGUG 18077T, CCUG 18074 Pragia sp ................. Obesumbacterium sp .................0. proteus ATCC 12841T
Edwardsiella spp .................
Non-Enterobacteniaceae Plesiomonas sp .................
Aeromonas spp .................
P. shigelloides ATCC
14029T, CUETM 84-238
A. hydrophila CUETM 84-231, CUETM 77-94, CUETM 77-95, CUETM 84-235, CUETM 84-232 A. sobria 84-233 CUETM.
a ATCC, American Type Culture Collection, Rockville, Md.; CIP, Collection de l'Institut Pasteur, Paris, France; NCTC, National Collection of Type Cultures, London, England; CDC, Centers for Disease Control or Communicable Disease Center, Atlanta, Ga.; CUETM, Collection de l'Unite Ecotoxicologie Microbienne, Villeneuve d'Ascq, France; NCPPB, National Collection of Plant Pathogenic Bacteria, Harpenden, England; CGUG, Culture Collection of Universitat Goteborg, Goteborg, Sweden.
polystyrene titration plates (Costar). Coating of the wells with bacteria was achieved overnight at 4°C. The wells were washed three times with 200 RI of phosphate-buffered saline (PBS; 10 mM, pH 7.1). Saturation was performed for 1 h at 37°C by using 100 pI of PBS supplemented with gelatin (0.25% [wt/vol]) and Tween 20 (0.1% [vol/vol]) (PGT). Aliquots (50 ,ul) of culture supernatants, diluted twofold in RPMI medium, were introduced into each well and incubated for 2 h at 37°C. After five washings, 50 ,ul of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Amersham Laboratories), diluted according to the recommendations of the manufacturer, was introduced into each well and incubated for 1 h at 37°C. After five washings, peroxidase activity was detected colorimetrically by adding azino-bis (3-ethyl-benzthiazoline-6 sulfonic acid; 1.1 mg/ml) in 0.6% (vol/vol) acetic acid (pH 4.7) containing 0.015% (vol/vol) hydrogen peroxide. Colorimetric staining was performed for 15 to 30 min in the dark, and the optical densities were read at 405 nm. Each culture supernatant was tested against five enterobacterial strains from different genera and species and one strain of the species P. maltophilia as a negative control. Hybridoma culture supernatants recognizing all five enterobacteria strains which did not react against the Pseudomonas strain were selected. After confirmation, the corresponding cells were cloned and amplified and the subsequent antibody-enriched supernatants were tested again by ELISA as described above. They were also tested by indirect immunofluorescence on slides as follows. A 20-j,l volume of bacterial suspensions was dried onto an immunofluorescence slide (Dynatech) and fixed for 5 min with 70% ethanol. The slides were washed with PGT, 20 ,u of pure clone
supernatant was added, and the plates were then incubated for 2 h at 37°C. The plates were washed and successively incubated for 30 min at 37°C with 20 ,ul of biotinylated sheep anti-mouse immunoglobulin G (heavy plus light chains) antibodies washed and incubated for 15 min with 20 ,u of fluoresceinated streptavidin (Amersham). The same indirect immunofluorescence technique was applied for the extensive study of the recognition spectra of MAb CX9/15 (ascites fluid diluted 1:500 in PGT). Electrophoresis and immunoblot analysis. Antigen extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed as described by Peters et al. (13). Briefly, 100 ,ul of buffer (62.5 mM; Tris-hydrochloride 2-mercaptoethanol [5%], SDS [2%], glycerol [12.5%]; pH 6.8) was added to a pellet containing 5 x 108 bacteria washed in saline (0.15 M NaCl). The bacterial suspensions were heated for 10 min at 100°C. SDS-PAGE was performed with the equivalent of 108 bacteria (20 RI). The gels were run in duplicate; one was developed by silver staining, the other was subjected to a 1-h electric transfer onto nitrocellulose membranes with an LKB Novablot apparatus and a field of 0.8 mA/cm2. Membranes were saturated by incubation for 1 h in PGT and then incubated for 1 h with antienterobacterial MAb (ascites fluid) diluted 1:500 in PGT. The nitrocellulose filters were then washed three times (for 10 min each) with 0.1% PBS-Tween and subsequently incubated for 1 h with horseradish peroxidaseconjugated goat anti-mouse immunoglobulin at 2.5 ,ug/ml in PGT. Three washings were performed as described above, followed by a final washing with PBS. The membranes were developed in 50 ml of PBS containing 9 mg of 4-chloronaphthol (previously solubilized in 3 ml of methanol) and
VOL. 58, 1992
RAPID DETECTION OF ENTEROBACTERIA BY MAb
TABLE 2. Bacterial strains that were not recognized by MAb CX9/15 Family and genus Species and strain(s)a Enterobacteriaceae Erwinia sp .................E. chrysanthemi ATCC 11663T Non-Enterobacteriaceae Pseudomonas spp. P. aeruginosa ATCC 27853T ,CIP A22 P. aeruginosa (1 environmental strain) P. aeruginosa (5 medical strains) P. fluorescens ATCC 13525T, ATCC 17397 P. fluorescens (13 environmental strains) P. fluorescens (1 medical strain) P. aureofaciens (1 environmental strain) P. chloraphis (4 environmental strains) P. putida ATCC 12633T P. putida (22 environmental strains) P. mendocina ATCC 25411T ,ATCC 25413, ATCC 5411, NCTC 10897 P. stutzeri ATCC 17588T, CUETM 8639, CUETM 86-37 P. vesicularis (5 environmental strains) P. acidovorans ATCC 15668T CUETM 83-76, CUETM 84-104, CUETM 83-152 P. pseudomallei (1 environmental strain) P. picketti ATCC 27511T P. pickettii (1 environmental strain) P. paucimobilis ATCC 6518 P. paucimobilis (3 environmental strains) P. cepacia ATCC 25609 Xanthomonas spp ...... X maltophilia ATCC 13637T X. maltophilia (7 medical strains) Acinetobacter spp ........ A. calcoaceticus (1 medical strain) A. haemolyticus (6 medical strains) A. junii (4 medical strains) A. johnsonii (1 medical strain) A. iwoffii (5 medical strains) A. baumanii (2 medical strains) Achromobacter spp ...... A. xylososidans CUETM 84-134, CUETM 84-143, CUETM 83-149, CUETM 84-115 A. xylososidans (4 medical strains) Alcaligenes sp ............. A. denitrificans ATCC 15173T Flavobacterium sp. F. odoratum ATCC 4651 Pasteurella sp ..............P. haemolytica (3 medical strains) Haemophilus sp .............H. influenzae (1 medical strain) Vibrio sp ................. (1 medical strain) Agrobacter sp. ............. A. radiobacter CUETM 77-93 .........
.
........
aSee footnote to Table 1 for abbreviations.
0.03% (vol/vol) hydrogen peroxide. Migration markers were the low-molecular-mass markers (14.4 to 92 kDa) from Pharmacia Laboratories. RESULTS AND DISCUSSION
Among the six clones produced, the MAb CX9/15 (immunoglobulin G2a) was selected, and its specificity was studied by the indirect immunofluorescence technique against 259 Enterobacteriaceae strains and 125 other gram-negative bacteria. These results are summarized in Table 1 for positive reactions and in Table 2 for negative ones. The MAb
1527
CX9/15 recognized all species of the Enterobacteriaceae family except Erwinia chrysanthemi (one strain tested). This recognition spectrum corresponded to the distribution of ECA in wild-type strains of gram-negative bacteria (2, 5-8, 14). It agreed with previous reports of the absence of ECA at the surface of the species E. chrysanthemi (2, 8). MAb CX9/15 did not react against 117 of 125 gramnegative bacteria not belonging to the Enterobacteniaceae but cross-reacted with the species Plesiomonas shigelloides (two strains tested), Aeromonas hydrophila (five strains tested) and Aeromonas sobria (one strain tested). The recognition of P. shigelloides by anti-ECA antibodies had already been reported elsewhere (1, 14). This gram-negative bacterium, which is closely related to the Enterobacteriaceae, presented an ECA antigen identical to the enterobacterial one (5). The species A. hydrophila has been previously reported to be devoid of ECA (8); however, one strain (209A) has been found to contain ECA (Nacescu and Ciufecu; cited in reference 11). Our strain of A. sobria was detected by CX9/15, which was an unexpected result, since the only strain tested previously was found to be ECA negative (14). This discrepancy needs further investigation and may be due to differences in the techniques used, since Ramia et al. used hemagglutination with heated supernatants (14). ECA has also been detected in Tatumella ptyseos and Xenorhabdus species (14); we have not yet tested these bacteria. Western blot (immunoblot) studies have been performed to determine the characteristics of the antigen recognized by six of our antienterobacterial MAb. Heated supernatants from four enterobacteria and a P. maltophilia strain were analyzed by SDS-PAGE, and staining on nitrocellulose with MAb CX9/15 was also studied (Fig. 1). The pattern obtained with the four enterobacteria was similar; a single band with an apparent molecular weight of about 20,000 appeared. Only minor and nonsignificant migration differences between the four antigens revealed existed. We believe that CX9/15 is directed against the ECA for two reasons. (i) The enterobacterium that we have used as the source of antigen was E. coli 014:K7, known as a mutant presenting an immunogenic form of ECA (7). The crude extract used for immunization of mice was obtained after the first step of an ECA purification process (15). (ii) The recognition spectrum of MAb CX9/15 closely resembled the distribution of ECA described by Kuhn et al. (6). Our six antienterobacterial MAb presented similar reactivities (Fig. 2); all of the immunoblots presented a single band. This result was different from the ladderlike patterns presented by Kuhn et al. (5) and Peters et al. (13) using their anti-ECA MAb (MAb 898 and 865). The enterobacteria (particularly E. coli 014:K7) and the conditions of sample treatment and electrophoresis were the same as those described by Peters et al. (13). We conclude that our antibodies either do not recognize the same epitope as MAb 898 and 865 or recognize different forms of ECA. Two main differences may explain these different reactivities. (i) Peter et al. immunized mice against formalinized E. coli K-12 (13), whereas we used heated supernatant from E. coli 014:K7, and (ii) our screening procedure by ELISA, which has been performed elsewhere with glutaraldehyde-treated enterobacteria (and extracts of heated bacteria) for 898 and 865 (13), used intact untreated bacteria. Previous immunizations with intact whole enterobacteria (UV inactivated) have also been performed. However, the antibodies produced have not been able to recognize the five enterobacteria chosen for the screening. These observations
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