Immunoaffinity purification, stabilization and comparative characterization of listeriolysin O from Listeria monocytogenes serotypes 1/2a and 4b.

We developed a simple and highly effective procedure for stabilizing the haemolytic activity of listeriolysin O (LLO) from Listeria monocytogenes after immunoaffinity purification. The haemolytic activity of LLO was stabilized by eluting it directly into tubes containing an alkaline buffer (5 mM lysine, 140 mM KCl, 50% ethylene glycol, pH 11.5). The purified LLO retained 100% of its haemolytic activity after 6 weeks of storage at -20 degrees C. LLO purified from a strain of L. monocytogenes serotype 1/2a (ATCC 43249) and LLO purified from a strain of L. monocytogenes serotype 4b (F 2365) isolated from a Mexican-style cheese, showed no significant differences in pH and temperature stability. When incubated in buffers at pH values from 4 to 12 at 4 degrees C and 25 degrees C, LLO from serotypes 1/2a and 4b retained maximal haemolytic activity at pH 8 after 4 h of incubation. LLO from both serotypes lost their haemolytic activity after incubation at 50 degrees C for 25 min.

Analysis of Listeria monocytogenes by multilocus enzyme electrophoresis and application of the method to epidemiologic investigations.

We examined 310 strains of Listeria monocytogenes by multilocus enzyme electrophoresis. Fifty-six electrophoretic types (ETs) of the organism were defined: 10 for serovar 4b strains, 11 for serovar 1/2b strains, and 30 for serovar 1/2a strains. Strains of serovars 1/2c, 3a, and 3b, and a non-typable strain were distributed among the remaining five ETs. The mean genetic diversity of the species was 0.41. Principal coordinate analysis revealed a sharp division among ETs which divided the species into two major clusters. ETs containing serovar 1/2a strains were in one cluster while all ETs containing serovar 4b, 1/2b, and 3b strains were in the second cluster. Except for two ETs that contained strains from both serovar 1/2b and serovar 3b, no ET contained strains from more than one serovar. Multilocus enzyme electrophoresis facilitated the analysis of epidemiologic data. In three separate epidemiologic investigations electrophoretic typing confirmed a common source as a cause of an outbreak; in a fourth investigation a single common source as a cause of an outbreak was effectively ruled out. Electrophoretic typing was also useful in documenting potential links between Listeria contaminated foods and persons with listeriosis who consumed those foods.

Multilocus enzyme electrophoresis for characterization of Listeria monocytogenes isolates: results of an international comparative study.

Multilocus enzyme electrophoresis (MEE) is a standard technique that is used to elucidate the epidemiology of a variety of bacterial species. Recently, the method has been employed by several laboratories for investigations of clinical and foodborne isolates of Listeria monocytogenes. To assess the sensitivity and reproducibility of MEE in characterising L. monocytogenes isolates for epidemiological purposes and, ultimately, to agree on a standard protocol, seven laboratories participated in a blinded study of 80 strains. The strain collection included both epidemiologically related and unrelated isolates. Each laboratory used its own protocol for MEE. The number of enzymes that were assayed by the laboratories ranged from 8 to 23, and the total number of identified electrophoretic types (ETs) varied between 14 and 25. Of the II pairs of duplicate strains, the number of pairs recognised as identical by the seven laboratories ranged from 3 to 10 (median = 8). From 10 to 18 (median = 15) of the 22 groups of epidemiological related strains were recognised as homogeneous by the different laboratories. The discriminatory power of the method, calculated using Simpson's index of diversity for 69 strains (80 strains minus the 11 duplicates), ranged from 0.827 to 0.925. This relatively low discriminatory power is a consequence of a somewhat low genetic diversity of L. monocytogenes compared to other bacterial species. Efforts should be pursued to standardise the method in order to improve the intra- and inter-laboratory reproducibility.

[Brazilian purpuric fever. Fast characterization of invasive strains of Haemophilus aegyptius]. / Febre purpúrica brasileira. Caracterização rápida das cepas invasoras de Haemophilus aegyptius.

Strains of H. aegyptius isolated during outbreak of Brazilian Purpuric Fever (BPF) in Brazil were characterized antigenically by slide agglutination test utilizing antiserum produced with a H. aegyptius strain isolated from blood culture from a patient with BPF. By means of this method, it were identified H. aegyptius strains responsible for outbreaks of conjunctivitis with identical antigenic characteristics to strains isolated from BPF. The sensitivity and specificity of slide seroagglutination test was 97.7% and 89.6% respectively; therefore this assay was efficient to be used as a screening method in the studies of purulent conjunctivitis for detecting high risk populations for BPF, and to implement measures that will increase the efficiency of epidemiologic surveillance.

Use of absorbed antisera for demonstration of antigenic variation among strains of Legionella pneumophila serogroup 1.

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.

Retrospective examination of lung tissue specimens for the presence of Legionella organisms: comparison of an indirect fluorescent-antibody system with direct fluorescent-antibody testing.

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.

Isoprenoid quinones of the genus Legionella.

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.

Carbamate kinase from Pseudomonas aeruginosa: purification, characterization, physiological role, and regulation.

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.

Use of monoclonal antibodies in an epidemiological marker system: a retrospective study of lung specimens from the 1976 outbreak of Legionnaires disease in Philadelphia by indirect fluorescent-antibody and enzyme-linked immunosorbent assay methods.

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.

Monoclonal antibody reactivity as a virulence marker for Legionella pneumophila serogroup 1 strains.

Using a panel of nine monoclonal antibodies, we subgrouped 85 environmental and 129 clinical Legionella pneumophila serogroup 1 isolates from Paris, France. Patients were unlikely to be epidemiologically linked either with each other or with the 44 sampled environmental sites (14 air conditioning systems and 30 buildings) that were selected at random in the Paris area. According to their monoclonal antibody patterns, isolates belonged to 14 subgroups. Monoclonal antibody 2 recognized 121 (93.8%) of 129 clinical isolates and 30 (35.3%) of 85 environmental isolates (P less than 10(-9)). Of the eight patients infected with L. pneumophila not recognized with monoclonal antibody 2, seven were immunocompromised; only 46.3% of the 121 patients infected with L. pneumophila reactive with monoclonal antibody 2 were immunocompromised (P = .02). We conclude that monoclonal antibody 2 can be used as a marker for the more virulent strains of L. pneumophila serogroup 1.

Isolation and characterization of a seventh serogroup of Legionella pneumophila.

An environmental isolate (Chicago 8) and a clinical isolate (Dallas 5) of Legionella pneumophila were shown to have similar serological characteristics; however, these characteristics were distinct from those of L. pneumophila serogroups 1 through 6. Chicago 8, ATCC 33823, was designated as the reference strain for L. pneumophila serogroup 7. The use of Mongolian gerbils for the isolation of L. pneumophila from the environment is described. Even though guinea pigs are the animals of choice in such studies, the isolation of Chicago 8 illustrates that the use of gerbils may be a viable option when cost is a major consideration in study design.

Determination of antigenic relationships among legionellae and non-legionellae by direct fluorescent-antibody and immunodiffusion tests.

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.

Detection of soluble Legionella pneumophila antigens in serum and urine specimens by enzyme-linked immunosorbent assay with monoclonal and polyclonal antibodies.

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.

A rapid dot immunoassay for detecting the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius with a "flow through" device.

Brazilian purpuric fever (BPF) is a highly fatal pediatric disease that may follow an episode of purulent conjunctivitis caused by a virulent clone of Haemophilus influenzae biogroup aegyptius (Hae). Oral rifampin prophylaxis, by eliminating carriage of the BPF clone in children with conjunctivitis, may prevent onset of the systemic disease. A test to detect the BPF clone directly from eye swabs could identify those in need of prophylaxis. This is a preliminary report of a rapid dot immunoassay performed on a "flow-through" cartridge that was developed for use under field conditions. The test is based upon recognition of a unique epitope of the 25-kDa pilin protein on the surface of BPF clone cells by a monoclonal antibody. With 36 laboratory-maintained cultures of Hae (15 clone isolates and 21 others), sensitivity of the assay was 67% and specificity was 95%. When fimbrial-enriched (25-kDa+) phenotypes of five false-negative clone strains were prepared for use as test antigens, sensitivity rose to 100%. Evaluation of the immunoassay under field conditions is necessary to prove its efficacy.

The epidemiology of listeriosis in the United States--1986. Listeriosis Study Group.

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.

Multilocus enzyme analysis of Legionella dumoffii.

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.

Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies.

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.