Application of multilocus enzyme gel electrophoresis to Haemophilus influenzae

Multilocus enzyme electrophoresis was adapted to the study of Haemophilus influenzae. Protein extracts from sonicated whole bacteria were subjected to starch gel electrophoresis. After staining with substrates, the position of each isoenzyme (electromorph) was registered. Each isolate was assigned an electrophoretic type (ET) by the combination of electromorphs for the enzymes stained. Twenty-seven enzymes were tested; 12 were expressed in H. influenzae. Six enzymes were selected for subsequent study: malate dehydrogenase (MDH), phenylalanylleucine peptidase (PE2), 6-phosphogluconate dehydrogenase (6PG), adenylate kinase (AK), glucose 6-phosphate dehydrogenase (G6P), and phosphoglucose isomerase (PGI). They were polymorphic and occurred in all isolates. Six electromorphs were found for PE2, G6P, and PGI, five for MDH, four for 6PG, and three for AK. PE2, G6P, and PGI contributed most of the ET resolution (48 of 49 ETs). Multilocus enzyme electrophoresis showed several advantages over previous typing techniques. An ET could be assigned to both typable and nontypable (NT) isolates. The technique was powerful in resolving differences among isolates. The 94 isolates comprised 49 ETs, five biotypes, and six capsular types and NT isolates. Strains known to be related expressed the same ET, e.g., RAB b+ and b-, ET12; Ma a+ and a-, ET1. ET variability among type b isolates was low; 26 of 28 clinical isolates expressed ET14; 2 of 28 expressed ET13 and ET15, differing from ET14 by one electromorph each. In contrast, the 47 NT isolates comprised 38 different ETs. No ETs were shared between non-type b capsulated strains and type b or NT strains. Interestingly, five NT isolates expressed the same ET as type b strains. (iv) Strains of the same capsular type but different biotypes expressed different ETs. ET determinations will thus be useful in studying the epidemiology and evolution of H. influenzae.