INFECTION AND IMMUNITY, Mar. 1983, p. 1441-1456 Vol. 39, No. 3 0019-9567/83/031441-16$02.00/0 Copyright © 1983, American Society for Microbiology Cross-Reactions Between Legionella pneumophila (Serogroup 1) and Twenty-Eight Other Bacterial Species, Including Other Members of the Family Legionellaceae MICHAEL T. COLLINS,lt* FRANK ESPERSEN,2 NIELS H0IBY,' SANG-NAE CHO,3 ALICE FRIIS M0LLER,1 AND JOHN S. REIF3 State Serum Institute, Department of Clinical Microbiology, Rigshospitalet,1 and Department of Clinical Microbiology, Hvidovre Hospital,2 Copenhagen, Denmark, and Department of Microbiology, Colorado State University, Fort Collins, Colorado 805233 Received 20 September 1982/Accepted 10 December 1982 Cross-reactions between Legionella pneumophila serogroup 1 and 28 other bacterial species were studied by various quantitative immunoelectrophoretic techniques. A sonicated L. pneumophila antigen and purified homologous rabbit antibody were used as a reference system. Few antigens (0 to 6) cross-reacted with non-Legionellaceae, but two were found in nearly all gram-negative bacteria tested (antigens no. 1 and 66). Antigen no. 66 of the L. pneumophila reference system was shown to be antigenically similar to the "common antigen" of Pseudomonas aeruginosa reported in many gram-negative bacteria. Greater than 85% of the antigens from L. pneumophila serogroup 1 cross-reacted with the other six serogroups of L. pneumophila. By contrast, Fluoribacter (Legionella) boze- manae, F. (L.) dumoffii, F. (L.) gormanii, and Tatlockia (Legionella) micdadei cross-reacted with only 45, 53, 39, and 43% of the reference system antigens, respectively. The antigenic relatedness of members of the Legionellaceae, expressed as a matching coefficient, is discussed in terms of its taxonomic significance. Serogroup-, genus-, and family-specific antigens are identified in the L. pneumophila reference system. Several serological studies have described such an antigen or antigens becomes increasing- cross-reactions between serogroups (SGs) of ly important for development of a rapid, accu- Legionella pneumophila and between L. pneu- rate diagnostic test to screen patients for legion- mophila and other bacteria, leading to specula- ellosis. tions concerning SG-specific antigens, Legion- Analysis of L. pneumophila antigens by ella-common antigens and antigens common to crossed immunoelectrophoresis (XIE) in our Legionella spp. and other bacterial species (13, laboratory was previously described, and a ref- 39, 49, 65-67). SG-specific antigens have been erence system with 82 antigens was established demonstrated by five different groups of investi- (11). XIE techniques are well-suited for the gators, all using precipitation reactions, but each study of cross-reactions between bacterial spe- using different antigen extraction methods (11, cies and have been widely used for this purpose 16, 38, 56, 58). These studies indicated that the (10, 18, 20, 25, 30, 32, 34, 54, 63). Joly and SG-specific antigen of L. pneumophila was a Kenny (38) used the XIE technique to study high-molecular-weight, heat-stable surface anti- cross-reactions between L. pneumophila SGs 1, gen with the physiochemical properties of lipo- 2, 3, and 4 and Tatlockia (Legionella) micdadei. polysaccharide. They found type-specific antigens for each spe- Antigens common to several members of the cies and SG, and reported finding several anti- Legionellaceae also have been demonstrated gens in common among all five organisms stud- (15, 38, 68), but Legionella-common antigen or ied. No measure of cross-reactivity between antigens, which are present in all Legionella members of the Legionellaceae and other bacte- spp. and not cross-reactive with other bacteria, ria was attempted by Joly and Kenny, however. have not been reported. As the number of anti- The purpose of this study was to quantitative- genic types of Legionellaceae continues to ly determine the degree of cross-reaction be- grow, the need for identification and isolation of tween L. pneumophila SG 1 (Lpl) and other antigenic types of Legionellaceae and other non- t Present address: Department of Microbiology, Colorado Legionellaceae bacteria. Furthermore, we at- State University, Fort Collins, CO 80523. tempted to identify those antigens of the Lpl 1441 1442 COLLINS ET AL. INFECT. IMMUN. TABLE 1. Organisms used for cross-reaction studies Species Strain Culture collection no. Reference(s) Legionella pneumophila SG 1 Philadelphia 1 ATCC 33152 7, 8, 11, 422, 43, 48 Legionella pneumophila SG 1 Knoxville 1 ATCC 33153 43 Legionella pneumophila SG 2 Togus 1 ATCC 33154 43, 44 Legionella pneumophila SG 3 Bloomington 2 ATCC 33155 43, 46, 53 Legionella pneumophila SG 4 Los Angeles 1 ATCC 33156 14, 43 Legionella pneumophila SG 5 Dallas 1E ATCC 33216 17 Legionella pneumophila SG 6 Chicago 2 ATCC 33215 45 Fluoribacter bozemanae WIGA ATCC 33217 4, 6, 22, 26, 41, 60 Fluoribacter dumoffii NY-23 ATCC 33279 6, 9 Fluoribacter gormanii LA-13 ATCC 33297 9, 12, 47 Tatlockia micdadei Tatlock ATCC 33218 22, 26, 27, 50, 51 Staphylococcus aureus Phage type 47/53/54/83A E 1369" 55 Staphylococcus epidermidis 4 biotypes A 1271/76" 18 A 1338/76" A 1394/76" A 1389/76" Streptococcus faecalis 4 subspecies Clinical isolates 23 (XIE analysis; Gutschick, unpublished data) Neisseria meningitidis Groups A, B, and C ATCC 13077 (group A) 29 ATCC 13090 (group B) ATCC 13102 (group C) 33 Escherichia coli Type 021:H27 Clinical isolate 33 (XIE analysis; HOiby, unpublished data) Serratia marcescens Type 013:H4 CDC 3607-60b 24 (XIE analysis; Gutschick, unpublished data) Type 06/014:H3 Clinical isolate designated SM 55 Type 05:H2 Clinical isolate designated SM 104 Salmonella typhi 9, 12, -, - 58" 19 Haemophilus influenzae Biotype 1 Clinical isolate 54 type b Bordetella pertussis 4 strains 3803" 28 3825" 3843" 3860" Pseudomonas aeruginosa 4 strains; 0 groups 3, 5, Clincal isolates 34 6, 11 Pseudomonas pseudomallei 3 strains NCTC 4845 S (XIE analysis; Hoiby, unpublished data) NCTC 10274 NCTC 10276 Pseudomonas cepacia 2 strains ATCC 17559 S (XIE analysis, Hoiby, unpublished data) ATCC 17560 Bacteroides fragilis subsp. 1 strain Clinical isolate 33 fragilis Actinobacillus hominis 6 strains Clinical isolates 21 Actinobacillus equuli 1 strain NCTC 8529 21 Actinobacillus ligniersii 1 strain NCTC 4189 21 Pasteurella haemolytica 1 strain P-557" 21 Pasteurella multocida 1 strain NCTC 10322 21 a State Serum Institute, Copenhagen, Denmark. b Enterobacteriology Unit, Center for Disease Control, Atlanta, Georgia. reference system (11) which were SG-specific, MATERIALS AND METHODS those which were common only to members of Lpl reference system. The Lpl XIE reference sys- the Legionellaceae, and those which were cross- tem has been described previously, in a study in which reactive with other bacteria. 82 anodic-migrating immunoprecipitates were also VOL. 39, 1983 L. PNEUMOPHILA CROSS-REACTIONS 1443 TABLE 2. Cross-reactions between Lpl, Philadelphia 1, strain antigens and 28 other bacteria, using the Lpl reference system Species Total no. Antigen identity and percentage cross-reactivity Matching of antigens 100% <100 to 50%' } <50 to 25% coefficient L. pneumophila SG 1b I51 Alli51 antigens ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I1.00 L. pneumophila SG 2 50 All but no. 61 0.98 L. pneumophila SG 3 49 All but nos. 41 and 61 0.96 L. pneumophila SG 4 47 All but nos. 41, 61, 76, 0.92 and 78 L. pneumophila SG 5 44 All but nos. 7, 9, 41, 61, 0.86 74, 76, and 78 L. pneumophila SG 6 50 All but no. 61 0.98 F. bozemanae 23 Nos. 1, 3, 7, 8, 29, 33, 0.45 36, 41, 66, and 80' F. dumoffi 27 Nos. 1, 3, 7, 8, 9, 33, 36, 0.53 41, 66, and 80c F. gormanii 20 Nos. 1, 3, 7, 8, 9, 29, 33, 0.39 34, 41, 65, 66, and 80' T. micdadei 22 Nos. 1, 3, 7, 8, 29, 33, 41, and 66' 0.43 S. aureus 1 No. 41 0.02 S. epidermidis 1 No. 41 0.02 S. faecalis 0 0.00 N. meningitidis 4 No. 1 No. 33 Nos. 41 and 66 0.08 E. coli 5 No. 1 No. 33 Nos. 29, 41, and 66 0.10 S. marcescens 5 No. 1 and 36 Nos. 29, 33, and 66 0.10 S. typhi 6 No. 1 No. 33 Nos. 29, 41, 43, 0.12 and 66 H. influenzae type b 4 No. 1 No. 33 Nos. 41 and 66 0.08 B. pertussis 3 No. 1 Nos. 33 and 66 0.06 P. aeruginosa 5 No. 1 Nos. 33, 41, and 43 No. 66 0.10 P. pseudomallei 3 No. 1 No. 33 No. 66 0.06 P. cepacia 5 No. 1 Nos. 33 and 66 Nos. 7 and 41 0.10 B. fragilis as fragilis 0 0.00 A. hominis 5 Nos. 1 and 41 No. 33 Nos. 10 and 66 0.10 A. equuli 4 Nos. 1 and 41 No. 33 No. 66 0.08 A. ligniersii 4 Nos. 1 and 41 No. 33 No. 66 0.08 P. multocida 3 No. 1 Nos. 41 and 66 0.06 P. haemolytica 4 Nos. 1 and 41 No. 33 No. 66 0.08 a No antigens showed 75 to 100% cross-reactivity; therefore, this grouping was combined with 50 to 75%. b Knoxville strain. c Remaining cross-reactive antigens not identifiable by antigen number owing to complexity of XLIE pattern. enumerated (11). Fifty-one of these precipitates were Preparation of antigens from 18 other bacterial spe- regularly visible, and these antigens are, therefore, the cies. The antigens from seven bacterial species have subject of the present study. The remaining precipi- been described previously (Table 1). Antigens from 11 tates could be detected only when the antigen/anti- additional organisms were prepared by the same soni- body ratio was changed. The cathodic-migrating anti- cation methods described previously (32, 63). The gens were not investigated in this study.