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In July 1995, 40 Montana residents were identified with laboratory-confirmed Escherichia coli O157:H7 infection; 52 residents had bloody diarrhea without laboratory confirmation. The median age of those with laboratory-confirmed cases was 42 years (range, 4- 86); 58% were female. Thirteen patients were hospitalized, and 1 developed hemolytic-uremic syndrome. A case-control study showed that 19 (70%) of 27 patients but only 8 (17%) of 46 controls reported eating purchased (not home-grown) leaf lettuce before illness (matched odds ratio, 25.3; 95% confidence interval, 3.9-1065.6). Pulsed-field gel electrophoresis identified a common strain among 22 of 23 isolates tested. Implicated lettuce was traced to two sources: a local Montana farm and six farms in Washington State that shipped under the same label. This outbreak highlights the increasing importance of fresh produce as a vehicle in foodborne illness. Sanitary growing and handling procedures are necessary to prevent these infections. [ABSTRACT FROM AUTHOR]

Between 23 June and 15 July 1994, 21 cases (19 primary and 2 secondary) of Escherichia coli 0157:H7 infection were identified in the Bethel, Connecticut, area. Three pulsed-field gel electrophoresis (PFGE) patterns from 15 isolates (I, n = 13; II, n = 2; and III, n = 1) were observed. A casecontrol study that excluded secondary cases and patients with PFGE II and III patterns (n = 16) demonstrated that consumption of food from one supermarket was associated with illness (15/16 cases vs. 31/47 geographically matched controls, odds ratio [OR] undefined, lower 95% confidence interval OR = 1.45,P = .018). No one food was associated with illness. Inspection of the supermarket revealed deficiencies in hygiene and meat handling practices. The 2 cases with PFGE II ate raw beef and raw lamb from a second supermarket. These outbreaks demonstrate the value of PFGE in supporting epidemiologic investigations and the potential for outbreaks arising from retail outlets. [ABSTRACT FROM PUBLISHER]

Objective: Non-O157 Shiga toxin-producing Escherichia coli (STEC) have emerged as an important public health problem. Outbreaks attributed to non-O157 STEC rarely are reported. In 1999, follow-up of routine surveillance reports of children with hemolytic- uremic syndrome (HUS) identified a small cluster of 3 cases of HUS, all of whom had spent overlapping time in a Connecticut lake community in the week before onset of symptoms. We conducted an investigation to determine the magnitude and source of the outbreak and to determine risk factors associated with the transmission of illness., Methods: We conducted a cohort study and an environmental investigation. The study population included all people who were at the lake in a defined geographic area during July 16-25, 1999. This time and area were chosen on the basis of interviews with the 3 HUS case-patients. A case was defined as diarrhea (>/=3 loose stools/d for >/=3 days) in a person who was at the lake during July 16-25, 1999. Stool samples were requested from any lake resident with diarrheal illness. Stools were cultured for Salmonella, Shigella, Campylobacter, and E coli O157. Broth cultures of stools were tested for Shiga toxin. Case-patients were asked to submit a serum specimen for antibody testing to lipopolysaccharides of selected STEC. Environmental samples from sediment, drinking water, lake water, and ice were obtained and cultured for E coli and tested for Shiga toxin. An environmental evaluation of the lake was conducted to identify any septic, water supply system, or other environmental condition that could be related to the outbreak., Results: Information was obtained for 436 people from 165 (78%) households. Eleven (2.5%) people had illnesses that met the case definition, including the 3 children with HUS. The attack rate was highest among those who were younger than 10 years and who swam in the lake on July 17 or 18 (12%; relative risk [RR]: 7.3). Illness was associated with swimming (RR = 8.3) and with swallowing water while swimming (RR = 7.0) on these days. No person who swam only after July 18 developed illness. Clinical characteristics of case-patients included fever (27%), bloody diarrhea (27%), and severe abdominal cramping (73%). Only the 3 children with HUS required hospitalization. No bacterial pathogen was isolated from the stool of any case-patient. Among lake residents outside the study area, E coli O121:H19 was obtained from a Shiga toxin-producing isolate from a toddler who swam in the lake. Serum was obtained from 7 of 11 case-patients. Six of 7 case-patients had E coli O121 antibody titers that ranged from 1:320 to >1:20 480. E coli indicative of fecal contamination was identified from sediment and water samples taken from a storm drain that emptied into the beach area and from a stream bed located between 2 houses, but no Shiga toxin-producing strain was identified., Conclusions: Our findings are consistent with a transient local beach contamination in mid-July, probably with E coli O121:H19, which seems to be able to cause severe illness. Without HUS surveillance, this outbreak may have gone undetected by public health officials. This outbreak might have been detected sooner if Shiga toxin screening had been conducted routinely in HUS cases. Laboratory testing that relies solely on the inability of an isolate to ferment sorbitol will miss non-O157 STEC, such as E coli O121. Serologic testing can be used as an adjunct in the diagnosis of STEC infections. Lake-specific recommendations included education, frequent water sampling, and alternative means for toddlers to use lake facilities.

The frequency of Shiga toxin-producing Escherichia coli (STEC) serotypes associated with postdiarrheal hemolytic uremic syndrome (HUS) cases among children and adults in the United States and the proportion with IgM or IgG lipopolysaccharide antibodies to E. coli O157 were determined by use of a nationwide sample from January 1987 through December 1991. Among 83 patients, STEC were isolated from 30 (43%) of 70 whose stool cultures yielded bacterial growth (25 E. coli O157 isolates and 5 non-O157 STEC isolates). Fifty-three (80%) of 66 patients with serum samples had positive O157 lipopolysaccharide antibody titers. Of the 83 patients, 60 (72%) had evidence of STEC infection, including 6 of 8 adults whose illnesses also met criteria for thrombotic thrombocytopenic purpura. Data from a subset of patients suggest that E. coli O157 was the cause of > or = 80% of the STEC infections. All 3 women who were postpartum had evidence of E. coli O157 infection. STEC infection should be considered the likely cause for all persons with postdiarrheal HUS.

A cluster of gastrointestinal illnesses, including one case of hemolytic-uremic syndrome and one culture-confirmed Escherichia coli O157:H7 infection, followed a trailer park pool party. We interviewed a cohort of party attendees and park residents. A primary case was defined as the first gastrointestinal illness within a household between 5 July and 20 July in which the titer of IgG antibody to E. coli O157 (if determined) was elevated. Of 51 party attendees and trailer park residents, 18 developed a gastrointestinal illness, including 10 who met the definition of a primary case. Swimming in the pool significantly increased the risk of primary illness (relative risk = 6.3; 95% confidence interval = 1.8-18.9). No other exposure was significantly associated with primary illness, after pool exposure was controlled for. The implicated pool had little to no chlorine added during the period of 4-10 July. This outbreak provides new evidence of the importance of proper pool maintenance in controlling the spread of E. coli O157:H7.

Background: After an outbreak of gastroenteritis and fever among persons who attended a picnic in Illinois, chocolate milk served at the picnic was found to be contaminated with Listeria monocytogenes., Methods: In investigating this outbreak, we interviewed the people who attended the picnic about what they ate and their symptoms. Surveillance for invasive listeriosis was initiated in the states that receive milk from the implicated dairy. Stool and milk samples were cultured for L. monocytogenes. Serum samples were tested for IgG antibody to listeriolysin O., Results: Forty-five persons had symptoms that met the case definition for illness due to L. monocytogenes, and cultures of stool from 11 persons yielded the organism. Illness in the week after the picnic was associated with the consumption of chocolate milk. The most common symptoms were diarrhea (present in 79 percent of the cases) and fever (72 percent). Four persons were hospitalized. The median incubation period for infection was 20 hours (range, 9 to 32), and persons who became ill had elevated levels of antibody to listeriolysin O. Isolates from stool specimens from patients who became ill after the picnic, from sterile sites in three additional patients identified by surveillance, from the implicated chocolate milk, and from a tank drain at the dairy were all serotype 1/2b and were indistinguishable on multilocus enzyme electrophoresis, ribotyping, and DNA macrorestriction analysis., Conclusions: L. monocytogenes is a cause of gastroenteritis with fever, and sporadic cases of invasive listeriosis may be due to unrecognized outbreaks caused by contaminated food.

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.

In order to compare methods for subtyping Neisseria meningitidis serogroup B isolates, 96 isolates obtained from various locations in the United States and northwestern Europe were subtyped by five methods: monoclonal antibody (MAb)-based serotyping and serosubtyping, DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MEE), ribotyping, and PCR-restriction fragment length polymorphism of the internally transcribed spacer region of the rRNA operon (ITS PCR-RFLP). All N. meningitidis serogroup B isolates were typeable by PFGE, MEE, ribotyping, and ITS PCR-RFLP. Only 44.8% of the isolates were completely typeable (both serotype and serosubtype determination) by MAb-based serotyping and serosubtyping. 60.4% of the isolates could be serotyped but not serosubtyped, and 90.6% of the isolates could be either serotyped or serosubtyped. Simpson's discrimination indices of diversity for the methods were as follows: PFGE, 99.7%; MEE, 99.4%; ribotyping, 98.8%; MAb serotyping, 75.8%; MAb serotyping and/or serosubtyping 97.5%; and ITS PCR-RFLP, 84.2%. The high degree of diversity observed by PFGE, MEE, and ribotyping can be explained by the fact that isolates were collected from different geographic locations at various times. PFGE, MEE, and ribotyping showed greater discriminatory abilities than MAb-based serotyping and serosubtyping or ITS PCR-RFLP.

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.

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.

There is no standard immunoassay for evaluating immune responses to meningococcal vaccines. We developed an enzyme-linked immunosorbent assay to measure total levels of antibody to Neisseria meningitidis group A capsular polysaccharide. Five laboratories measured the antibody levels in six paired pre- and postvaccination serum samples by using the enzyme-linked immunosorbent assay. Methylated human serum albumin was used to bind native group A polysaccharide to microtiter plate surfaces. The between-laboratory coefficients of variation for pre- and postvaccination sera had ranges of 31 to 91 and 17 to 31, respectively. The mean laboratory coefficients of variation for pre- and postvaccination sera, respectively, were 17 and 11 (Molecular Biology Laboratory, Centers for Disease Control), 12 and 15 (Immunodiagnostic Methods Laboratory, Centers for Disease Control), 22 and 19 (Dana-Farber Cancer Institute), 38 and 38 (Bacterial Polysaccharide Laboratory, U.S. Food and Drug Administration), and 11 and 10 (Praxis Biologics, Inc.). Standardization of this enzyme-linked immunosorbent assay should allow interlaboratory comparison of meningococcal vaccine immunogenicity, thus providing a laboratory-based assessment tool for evaluating meningococcal vaccines.

We compared multilocus enzyme electrophoresis (MEE) and ribosomal DNA fingerprinting (ribotyping) for subtyping 44 strains of Neisseria meningitidis serogroup C that were isolated in Los Angeles County, California, between December 1985 and July 1986. The isolates were divided into six enzyme types (ETs) by MEE, but 36 of the isolates were clustered in one ET, 3. The same isolates were divided into 17 ribotypes by use of restriction endonucleases ClaI, EcoRI, and XhoI. Twenty of the 36 ET 3 isolates were divided into 17 ribotypes by use of restriction endonucleases ClaI, EcoRI, and XhoI. Twenty of the 36 ET 3 isolates were grouped in a single ribotype, J. The rate of infection with ribotype J strains was higher in the southern part of the study area than in the northern part. Isolates from each of eight pairs (each isolate pair was cultured from the same patient from the same or different sites) were found identical by MEE, but ribotyping revealed a difference in one pair. In this study, ribotyping showed a greater discriminating capacity than MEE for subtyping N. meningitidis serogroup C, but the epidemiologic relevance of this increased sensitivity needs further assessment.

On the basis of phenotypic characterization and DNA relatedness determinations, the genus Afipia gen. nov., which contains six species, is described. The type species is Afipia felis sp. nov. (the cat scratch disease bacillus). Afipia clevelandensis sp. nov., Afipia broomeae sp. nov., and three unnamed not associated with cat-borne disease. All but one strain (Afipia genospecies 3) were isolated from human wound and respiratory sources. All Afipia species are gram-negative, oxidase-positive, nonfermentative rods in the alpha-2 subgroup of the class Proteobacteria. They are motile by means of a single flagellum. They grow on buffered charcoal-yeast extract agar and nutrient broth, but rarely on MacConkey agar, at 25 and 30 degrees C. They are urease positive; but they are negative in reactions for hemolysis, indole production, H2S production (triple sugar iron agar), gelatin hydrolysis, esculin hydrolysis, and peptonization of litmus milk. They do not produce acid oxidatively from D-glucose, lactose, maltose, or sucrose. The major cell wall fatty acids are 11-methyloctadec-12-enoic (CBr19:1), cis-octadec-11-enoic (C18:1omega7c), and generally, 9,10-methylenehexadecanote and 11,12-methyleneoctadecanoate; and there are only trace amounts of hydroxy acids. The guanineplus-cytosine content is 61.5 to 69 mol%. A. felis is positive for nitrate reduction and is delayed positive for acid production from D-xylose, but it is catalase negative. A. clevelandensis is negative in all of these tests. A. broomeae is weakly positive for catalase production and acid production from D-xylose, but it is negative for nitrate reduction.

Active surveillance for invasive meningococcal disease was conducted during 1986 and 1987 in six areas of the United States with a total population of approximately 34 million persons. The incidence of meningococcal disease was 1.3:10(5). The highest incidence of disease among the surveillance areas was in Los Angeles County (1.65:10(5). Neisseria meningitidis serogroups B and C caused about equal amounts of disease, which reflects a recent increase in the incidence of group C disease. Group C caused more than half of the cases of meningococcal disease in Los Angeles and Tennessee but less than one-third of the cases in Missouri and Oklahoma. Multilocus enzyme electrophoresis demonstrated that a group of closely related isolates of N. meningitidis was prevalent in Los Angeles during the surveillance period and was associated with an increased incidence of meningococcal disease there.

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.

To investigate the microbiology and epidemiology of the 1,700 sporadic cases of listeriosis that occur annually in the United States, we developed a multilocus enzyme electrophoresis (MEE) typing system for Listeria monocytogenes. We studied 390 isolates by MEE. Eighty-two electrophoretic types (ETs) were defined. Two distinct clusters of ETs, ET group A (ETGA) and ET group B (ETGB), separated at a genetic distance of 0.440, were identified. Strains of ETGB were associated with perinatal listeriosis (P = 0.03). All strains of H antigen type a were in ETGA, while all strains of H antigen type b were in ETGB. Among 328 clinical isolates from cases of literiosis, 55 ETs of L. monocytogenes were defined. Thirty-four ETs were identified among 62 isolates from food products. The mean number of strains per ET (5.2) was significantly higher among clinical isolates than among food-borne isolates. Examination of isolates from outbreaks further documented the link between cases and contaminated food products. In one investigation, we found 11 different ETs, ruling out a single common source as a cause of that outbreak. By examining a large number of isolates collected over a specified time in diverse geographic locations in the United States, we have begun to establish a baseline for the study of the epidemiology of listeriosis by MEE.

Brazilian purpuric fever (BPF) is a recently described fatal pediatric disease caused by systemic infection with Haemophilus influenzae biogroup aegyptius. Previous studies have shown that all H. influenzae biogroup aegyptius strains isolated from BPF cases and case contacts share several unique phenotypic and genotypic characteristics that differentiate them from other H. influenzae biogroup aegyptius strains isolated from conjunctivitis cases in Brazil. One key characteristic of this BPF clone is reactivity in a BPF-specific monoclonal antibody enzyme-linked immunosorbent assay. We have purified and partially characterized a pilin, referred to as the 25-kilodalton (kDa) protein. Aggregates of this protein contain a heat-labile epitope which is recognized by a monoclonal antibody used in the BPF-specific enzyme-linked immunosorbent assay. The protein has a molecular weight of approximately 25,000, is insoluble in most detergents, and fractionates with outer membrane vesicles after LiCl extraction. Biochemical analysis of the 25-kDa protein shows it to have an amino acid composition similar but not identical to that of the H. influenzae type b pilin. The sequence of 20 N-terminal amino acids of the 25-kDa protein shows almost complete homology with the N terminus of the H. influenzae type b pilin and the types 1 and P pilins of Escherichia coli. Transmission electron microscopic analysis of the purified protein shows the presence of filamentous structures similar in morphology to those of H. influenzae pili. Reactivity between the 25-kDa protein and the BPF-specific monoclonal antibody is demonstrated by Western blotting (immunoblotting) and colloidal gold-enhanced immunoelectron microscopy. Hemadsorption analysis shows that expression of this protein is associated with increases in piliated cells and enhanced binding of these cells to human erythrocytes. These studies indicate that expression of the 25-kDa protein is a characteristic unique to the BPF clone and suggest that this protein plays a role in the pathogenesis of BPF.

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.

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 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.

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.

The cellular fatty acid composition of Legionella feeleii was determined by capillary gas-liquid chromatography, and the ubiquinone content was determined by reverse-phase high-pressure liquid chromatography. As in other Legionella species, this new species is characterized by relatively large amounts of branched-chain fatty acids and by major amounts of ubiquinones with more than 10 isoprene units in the side chain.

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.

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.

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.

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.

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.

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 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.

From December 1986 to March 1987 an outbreak of Listeria monocytogenes infection occurred in the Philadelphia metropolitan area. A patient-control study showed patients were more likely than controls to have had an ill family member and to have used antidiarrheal medication during the month before their illness. Diet histories showed patients were significantly more likely to have eaten ice cream or salami than were controls, and to have shopped at one grocery store chain. Subtyping of L. monocytogenes isolates of patients showed no predominant strain, and cultures of food products eaten by patients were negative except for Brie cheese eaten by one patient. With no predominant strain of L. monocytogenes in the patients, a common source for this outbreak is unlikely. Thus, the identified risk factors may have been associated with carriage of L. monocytogenes and a coinfecting organism may have precipitated disseminated disease. Possible cofactors should be considered in investigations of future outbreaks of listeriosis.

A new serogroup of Legionella pneumophila was isolated from bronchoscopic washings and an open-lung biopsy specimen of a patient from California with pneumonia. A serologically identical isolate was obtained from tap water of a hospital ward in the Netherlands, and a fatal case of pneumonia in a patient from Virginia was shown retrospectively to have been caused by this new organism. The type strain of what is now serogroup 9 of L. pneumophila is IN-23-G1-C2 (American Type Culture Collection no. 35289). Disease caused by L. pneumophila serogroup 9 is not apparently different from that caused by other L. pneumophila serogroups.

Six strains of a new species, Legionella sainthelensi, were isolated from freshwater in areas affected by the volcanic eruptions of Mt. St. Helens in the state of Washington. Strains of L. sainthelensi are culturally and biochemically similar to other legionellae. They grow on buffered charcoal yeast agar but not on media that lack cysteine. They are gram-negative, nonsporeforming, motile rods that are positive in reactions for catalase, oxidase, gelatin liquefaction, and beta-lactamase. They are negative in reactions for urease, hydrolysis of hippurate, reduction of nitrates, fermentation of glucose, and blue-white autofluorescence. Their cell wall fatty acid composition is qualitatively similar to those of other legionellae, with 50 to 62% branched-chain fatty acids. They contain the isobranched-chain 14- and 16-carbon acids and anteisobranched-chain 15- and 17-carbon acids and relatively large amounts of straight-chain 16-carbon acid. All strains of L. sainthelensi contain approximately equal amounts of ubiquinones Q9, Q10, Q11, and Q12, a pattern similar to those of Legionella bozemanii, Legionella dumoffi, and Legionella longbeachae. Serological cross-reactions were observed between L. sainthelensi, both serogroups of L. longbeachae, and Legionella oakridgensis. Three strains of L. sainthelensi were greater than 90% related by DNA hybridization. The type strain of L. sainthelensi, Mt. St. Helens 4, was 36% related to the type strain of L. longbeachae and 3 to 14% related to the other nine described Legionella species.

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.

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.

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.

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.

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.

We describe a new species of Legionella represented by 10 strains isolated from industrial cooling towers. Legionella oakridgensis differed genetically from the other seven species of Legionella in DNA hybridization studies and differed serologically in direct fluorescent-antibody tests. The new species, unlike all other species except L. jordanis, did not require added L-cysteine for growth in serial transfer on charcoal-yeast extract agar. L. oakridgensis, as well as three other species tested, required L-cysteine for primary isolation from animal tissues. L. oakridgensis was the only species of Legionella that failed to produce alkaline phosphatase at pH 8.5. In all other respects, it resembled other species of Legionella, including having a high content of branched-chain cellular fatty acids and being pathogenic for guinea pigs. These bacteria have not yet been associated with human disease, but they are potential causes of legionellosis.

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.

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.

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.

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.

Monoclonal antibodies were produced against two strains of Legionella pneumophila serogroup 1. A panel of nine monoclonal antibodies were selected for their unique specificities observed in indirect fluorescent antibody tests with 130 strains of L. pneumophila serogroup 1. One monoclonal antibody was reactive with an antigen possessed by 128 of 130 strains. Two major subgroups were identified and 13 different antigen patterns were observed among the 130 serogroup 1 strains.