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Variability among 29 clinical and environmental strains of Legionella dumoffii was investigated by multilocus enzyme analysis by use of starch gel electrophoresis. Based on results of analysis at 20 enzyme loci, the strains were separated into five closely related electrophoretic types (ETs), which were clearly distinguished from 53 strains representing 53 ETs of L. pneumophila. DNA hybridization results (hydroxyapatite method, 60 and 75 degrees C) for representative strains confirmed that all L. dumoffii ETs were a single genetic species. Although multilocus enzyme analysis indicated that L. dumoffii was genetically a quite uniform species, sufficient variability existed to warrant electromorph fingerprinting for epidemiologic studies.

To determine the morbidity and mortality due to listeriosis in the United States, the authors undertook an active surveillance project in 1986 to identify all cases in which Listeria monocytogenes was isolated from cultures of ordinarily sterile sites in a population of 34 million persons. The authors estimated that at least 1,700 cases of listeriosis and 450 deaths occurred in the United States in 1986; 27% of these cases occurred in pregnant women, with 22% of perinatal cases resulting in stillbirths or neonatal deaths. The risk of listeriosis in adults (0.5 per 100,000 population) was similar in all regions studied; the incidence of perinatal listeriosis was three times higher in Los Angeles County, California, than in the other areas (24.3/100,000 live births vs. 7.8/100,000 live births). Geographic variation may have resulted from underdiagnosis of perinatal listeriosis in five of the study areas. Multilocus electrophoretic enzyme typing was useful for elucidating the molecular epidemiology of L. monocytogenes; perinatal listeriosis was significantly associated with one group of related strains. Multilocus electrophoretic enzyme typing also identified three clusters representing possible common-source outbreaks. These findings document the substantial morbidity due to listeriosis in the United States; to the extent that sporadic listeriosis is foodborne, this morbidity could be reduced by appropriate preventive measures, particularly in persons known to be at increased risk of infection.

We developed an enzyme-linked immunosorbent assay (ELISA) system to measure human immunoglobulin G (IgG) and IgM response to the major outer membrane proteins of Brucella melitensis. The ELISA was more sensitive in detecting antibody than a standard microagglutination (MA) test with B. abortus antigen. Of 101 sera from persons with suspected brucellosis, 79 (78.2%) gave ELISA IgM titers greater than or equal to the B. abortus MA titer without 2-mercaptoethanol (2ME), which measures both IgM and IgG. Of the 101 sera, 97% gave ELISA IgG titers greater than or equal to the MA with 2ME titer. A total of 58 sera, drawn from 11 human patients from 1 to 29 weeks after onset of brucellosis, gave higher geometric mean titers for the ELISA IgG test than for the MA with 2ME test. These 58 sera also gave ELISA IgM geometric mean titers that were greater than or within one doubling dilution of the geometric mean titers of MA without 2ME. In addition to detecting antibody response to B. abortus, B. melitensis, and B. suis, the ELISA was sensitive to antibody response to human and canine infections with B. canis. The B. canis antibody response is not detected by the MA test with B. abortus antigen. The ELISA, with a standard preparation of major outer membrane proteins of B. melitensis as antigen, appears to be useful in measuring antibody response in humans to infections by all species of Brucella known to infect humans.

A panel of monoclonal antibodies to Legionella pneumophila serogroup 1 and a subclassification scheme were developed in a collaborative project among three laboratories. The seven most useful monoclonal antibodies were selected from three previously developed panels on the basis of indirect fluorescent antibody patterns with 83 strains of L. pneumophila serogroup 1 that were obtained from widely distributed geographic locations. The isolates were divided into 10 major subgroups on the basis of reactivity patterns that can be readily reproduced in any laboratory and are not subject to major inconsistencies of interpretation of staining intensity. A standard protocol for the indirect fluorescent antibody procedure was also developed.

Urine and serum specimens from three patients with pneumonia caused by Legionella pneumophila serogroup 1 (Lp1) were tested by enzyme-linked immunosorbent assay (ELISA) for Lp1-soluble antigen. A three-layer direct ELISA with polyclonal antibodies and a four-layer indirect ELISA with both polyclonal and monoclonal antibodies were used. Lp1 antigen was detected in both urine and serum from the three patients. As determined by ELISA, the concentration of antigen was 30- to 100-fold less in serum than in urine collected on the same day. In some instances the indirect ELISA was more sensitive than the direct ELISA, but in others it was less sensitive, depending on the monoclonal antibody used. The subgroup of the infecting Lp1 organism was determined based on antigenic determinants expressed in the urine. This study illustrates the use of serum as well as urine as an antigen reservoir in the laboratory diagnosis of legionellosis by ELISA and the potential for developing more sensitive antigen detection systems by the judicious use of monoclonal antibodies.

Six isolates, five from water samples and one from a human tracheal swab taken at autopsy, reacted strongly with working dilutions of Legionella fluorescent-antibody conjugates. Of these, two isolates of Pseudomonas fluorescens (EB and CDC93), one isolate of the Flavobacterium-Xanthomonas group (CDC65), and one isolate of P. alcaligenes (CDC11) reacted with Legionella pneumophila serogroup 1 conjugate. P. alcaligenes ABB 50 reacted with an L. pneumophila serogroup 3 conjugate and of P. maltophilia reacted with the L. micdadei conjugate. Antisera and labeled conjugates were prepared for these new cross-reacting isolates, and their relationships to the legionellae were examined by direct fluorescent-antibody and immunodiffusion tests. A nonreciprocal cross-reaction existed between L. micdadei and P. maltophilia and also between serogroups 3 of L. pneumophila and P. alcaligenes ABB50. Of the four isolates that reacted with serogroup 1 of L. pneumophila, P. fluorescens CDC93 had the strongest relationship, and the other three had only minor relationships. Although cross-reactivity among non-legionellae and legionellae has not been a major problem, these findings are relevant to the interpretation of direct fluorescent-antibody tests for detecting these bacteria.

Listeria monocytogenes NCTC 7973 and this same strain deposited as ATCC 35152 contain two phenotypes: hemolytic virulent colonies and nonvirulent colonies that show no zones of hemolysis when streaked on heart infusion agar containing 5% rabbit blood. Results of examinations of these virulent and nonvirulent strains by investigators at the Centers for Disease Control, Atlanta, Ga., the Pasteur Institute, Paris, France, and the University of Würzburg, Federal Republic of Germany, support the conclusion that the avirulent strain is a nonhemolytic mutant of the virulent strain and that hemolysin is a virulence factor for L. monocytogenes.

Antigenic typing of strains of Legionella pneumophila serogroup 1 (Lp1) has been shown to be useful in epidemiological studies of outbreaks of legionellosis. Selective absorption of rabbit antibodies produced against five strains of Lp1 resulted in the recognition of 17 somatic types among the 176 strains tested. A comparison was made of our results and those obtained by McKinney et al. (Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 255:91-95, 1983) and Joly et al. (J. Clin. Microbiol. 18:1040-1046, 1983), who used monoclonal antibodies to subgroup Lp1 strains. The results indicate that antigens are present in Lp1 strains that were undetected by either system. The data presented in this study may be helpful in selecting for the production of additional monoclonal or absorbed antibodies for diagnostic purposes or epidemiological studies.

Although direct fluorescent-antibody (DFA) testing has been used successfully for a number of years to detect legionellae in clinical specimens, the number of known species and serogroups of Legionella has now increased to such an extent that the performance of DFA testing for all serological variants is impractical. Lung homogenates that were submitted to the Centers for Disease Control, Atlanta, Ga., from patients with suspected legionellosis, from November 1977 through May 1982, were originally screened by DFA testing. In our study 498 of these lung homogenates were screened by indirect fluorescent-antibody (IFA) testing, using a panvalent antiserum pool containing antibodies to 25 serological variants of Legionella spp. Fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin was used as the second antibody of the sandwich system. For positive homogenates, i.e., those containing Legionella organisms, species and serogroup identification was made by IFA staining with polyvalent serum pools and then with monovalent antiserum. Of the 498 homogenates screened, 39 (7.8%) were positive by IFA testing. Four (0.8% of total; 10.3% of positive homogenates) of these had previously been negative by DFA testing, but subsequent testing showed that they contained Legionella organisms for which DFA reagents were not available at the initial screening. All specimens that were positive by DFA testing were also positive by IFA testing. IFA testing with polyvalent antisera is a simple, efficient method which is at least as sensitive as DFA testing and which can be used by clinical laboratories to cope with the increasing number of known serological variants of Legionella spp.

Representative strains of each of the named species of Legionella were examined for isoprenoid quinones by reverse-phase thin-layer chromatography. All strains contained three or more ubiquinones (Q9, Q10, Q11, Q12, Q13) which were useful for placing the species into one of three distinct groups. Group 1 contained L. longbeachae, L. bozemanii, L. dumoffi, and L. gormanii; group 2 contained only L. micdadei; and group 3 contained only L. pneumophila. The identities of the quinones were established by UV spectroscopy and mass spectrometry.

A strain of Legionella longbeachae (Tucker 1) that was isolated from the postmortem lung tissue of a pneumonia patient was serologically distinct from four other strains of L. longbeachae. The recognition of a second serogroup of L. longbeachae represents the first reported instance of serogroup diversity within a species of Legionella other than L. pneumophila. The disease caused by the Tucker 1 strain does not seem to be readily distinguishable from that of pneumonia caused by other legionellae.

The genetic structure of populations of Legionella pneumophila was defined by an analysis of electrophoretically demonstrable allelic variation at structural genes encoding 22 enzymes in 292 isolates from clinical and environmental sources. Nineteen of the loci were polymorphic, and 62 distinctive electrophoretic types (ETs), representing multilocus genotypes, were identified. Principal coordinates and clustering analyses demonstrated that isolates received as L. pneumophila were a heterogeneous array of genotypes that included two previously undescribed species. For 50 ETs of L. pneumophila (strict sense), mean genetic diversity per locus was 0.312, and diversity was equivalent in ETs represented by isolates recovered from clinical sources and those collected from environmental sources. Cluster analysis revealed four major groups or lineages of ETs in L. pneumophila. Genetic diversity among ETs of the same serotype was, on average, 93% of that in the total sample of ETs. Isolates marked by particular patterns of reactivity to a panel of nine monoclonal antibodies were also genetically heterogeneous, mean diversity within patterns being about 75% of the total. Both Pontiac fever and the pneumonic form of legionellosis may be caused by isolates of the same ET. The genetic structure of L. pneumophila is clonal, and many clones apparently are worldwide in distribution. The fact that L. pneumophila is only 60% as variable as Escherichia coli raises the possibility that isolates recovered from clinical cases and man-made environments are a restricted subset of all clones in the species as a whole.

Legionella pneumophila subsp. pneumophila serogroup 6 is second in importance only to L. pneumophila serogroup 1 as a cause of legionellosis. Monoclonal antibody (MAb) reactivity and multilocus enzyme electrophoretic analyses were used to subtype serogroup 6 isolates as a potential aid for epidemiologic and virulence studies. Forty-eight serogroup 6 isolates submitted to the Centers for Disease Control from 1980 to 1985 were examined by these methods. The isolates were divided into two groups based on differential reactivity with two MAbs. Thirty-two of the isolates were of a single electrophoretic type (ET) and were reactive with both MAbs. The remaining 16 isolates were distributed among 10 ETs and were reactive with one or both MAbs. The mean genetic diversity for serogroup 6, as determined from the degree of variability at 20 enzyme loci, was found to be essentially the same as that for L. pneumophila subsp. pneumophila as a whole. The ETs of serogroup 6 isolates were unique but closely related genetically to the ETs of L. pneumophila subsp. pneumophila serogroups 1 to 5, 7, and 8. The range of serogroup 6 subtypes distinguished by MAbs and enzyme electrophoresis suggests that the combination of these two methods can be useful as a typing system.

Previous DNA relatedness and enzyme electrophoretic mobility studies indicated heterogeneity among strains of Legionella pneumophila serogroups 1, 4, 5, and Lansing 3 (a new, as yet unnumbered serogroup). In this study 60 L. pneumophila strains were studied by DNA hybridization (hydroxyapatite method) to assess their genomic relatedness. These strains were also studied biochemically and serologically to determine whether they formed one or more phenotypic groups. DNA relatedness studies identified three groups. DNA group 1 contained the type strain Philadelphia 1 and strains from serogroups 1 through 14 of L. pneumophila. The average relatedness of DNA group 1 strains was 88% at 60 degrees C with 1.1% divergence in related sequences and 85% at 75 degrees C. DNA group 2 contained strain Los Angeles 1, the reference strain of serogroup 4, and strains of serogroups 1, 4, 5, and Lansing 3, an unnumbered serogroup. Average relatedness of DNA group 2 strains was 84% at 60 degrees C with 0.7% divergence and 87% at 75 degrees C. Reciprocal relatedness of DNA groups 1 and 2 was approximately 67% at 60 degrees C with 6.0% divergence and 48% at 75 degrees C. DNA group 3 strains were in serogroup 5. They were 98% related at 60 degrees C with 0.5% divergence and 97% related at 75 degrees C. Reciprocal relatedness of DNA group 3 and DNA group 1 was approximately 74% at 60 degrees C with 5.3% divergence and 43% at 75 degrees C, and reciprocal relatedness of DNA groups 3 and 2 was 66% at 60 degrees C with 5.7% divergence and 55% at 75 degrees C. The DNA groups could not be separated biochemically or serologically or by cell wall fatty acid and isoprenoid quinone composition. Three subspecies of L. pneumophila are proposed to accommodate the three DNA groups: L. pneumophila subsp. pneumophila subsp. nov. for DNA group 1, L. pneumophila subsp. fraseri subsp. nov. for DNA group 2, and pneumophila subsp. pascullei subsp. nov. for DNA group 3.

Brazilian purpuric fever (BPF) is a recently recognized fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. BPF is usually preceded by purulent conjunctivitis that has resolved before the onset of fever. Both the conjunctivitis and BPF are caused by Haemophilus influenzae biogroup aegyptius (formerly called H. aegyptius). Isolates from 15 BPF cases, mainly from blood or hemorrhagic cerebrospinal fluid, case-associated isolates from 42 persons in towns where BPF cases occurred, and control strains from 32 persons in towns without BPF cases were characterized biochemically, genetically, and epidemiologically. Results indicated that a single clone was responsible for all BPF cases identified in six Brazilian towns from 1984 through 1986. All of 15 (100%) case strains were the same clone as was 1 of 32 (3%) control strains (P = less than 10(-8). Isolates of the clone were preferentially intrarelated by DNA hybridization (99% relatedness, hydroxyapatite method at 60 and 75 degrees C) and were separable from other H. influenzae biogroup aegyptius strains (approximately 90% relatedness at 60 degrees C and 82% relatedness at 75 degrees C). All isolates of the BPF clone and no other strains contained a 24-megadalton plasmid of restriction endonuclease type 3031, were of a single multilocus enzyme mobility type, were of a single sodium dodecyl sulfate-polyacrylamide gel electrophoresis type, and were in one of two ribosomal DNA restriction patterns. All BPF clone isolates reacted with monoclonal antibodies produced from a case strain; only 3 of 62 (5%) other strains reacted with this monoclonal antibody. Ninety percent of BPF clone strains and 27% of other strains were relatively resistant to sulfamethoxazole-trimethoprim.

Autopsy specimens of lung tissues from 15 patients that contracted legionellosis during the 1976 Philadelphia outbreak of Legionnaires disease were examined for the presence of Legionella organisms and soluble antigens by indirect fluorescent-antibody (IFA) testing and by an enzyme-linked immunosorbent assay (ELISA) with both polyclonal and monoclonal antibodies. In all 15 cases, at least one specimen was positive for Legionella pneumophila serogroup 1 (Lp-1) antigens by a polyclonal antibody ELISA system. Of the 15 cases tested for Lp-1, 9 were positive by a polyclonal antibody IFA test. Nine mouse monoclonal antibodies to Lp-1 gave essentially the same reactivity pattern with extracts from lung tissue homogenates as that obtained with a Philadelphia 1 culture extract by using a monoclonal antibody ELISA system. The same monoclonal antibody panel gave similar results when used in the IFA system with tissue homogenates. Monoclonal antibodies can be used as epidemiological marker systems with both IFA and ELISA testing. This study provides evidence that the 1976 common source outbreak in Philadelphia was probably caused by a single Lp-1 strain. ELISA testing of the soluble antigen of Lp-1 from lung tissue homogenate supernatants was more sensitive than IFA testing of the homogenates and should be extremely useful as either a primary test or as an adjunct to fluorescent antibody testing for legionellosis.

Pseudomonas aeruginosa PAO1 possessed a carbamate kinase (CKase) distinct from carbamoylphosphate synthetase as well as from a constitutive acetate kinase which also catalyzes the phosphorylation of ADP by carbamoylphosphate. CKase was purified to homogeneity. Polyacrylamide gel electrophoresis of cross-linked CKase in the presence of sodium dodecyl sulfate showed that the enzyme consists of two subunits with identical molecular weights (37,000). The optimal pH of enzyme activity is 7.0. The double-reciprocal plot for carbamoylphosphate was linear at 2 mM ADP, yielding an apparent Km of 5 mM. However, at 0.25 mM ADP, the plot was concave upward, and a Hill plot of the data yielded a coefficient of 1.4. This apparent cooperativity at low ADP concentrations might serve to reduce the extent of catabolism of carbamoylphosphate under growth conditions yielding high energy charge. Experiments on the regulation of synthesis under various growth conditions showed a response to three regulatory signals: CKase was induced to high levels by anaerobiosis, induced to moderate levels by arginine, and repressed by ammonia. Thus, CKase expression is regulated in a manner that allows the enzyme to function as a provider of ammonia under aerobic conditions and of ATP under anaerobic conditions. ATP was an effective inhibitor of CKase activity; this inhibition provides the cell with an effective mechanism for avoiding a futile cycle resulting from the simultaneous operation of CKase and carbamoylphosphate synthetase when cells are grown in the presence of exogenous arginine.