Molecular typing of Legionella pneumophila serogroup 1 isolates from patients and the nosocomial environment by arbitrarily primed PCR and pulsed-field gel electrophoresis.

The ubiquity of Legionella pneumophila in aquatic habitats means that epidemiological evaluation is important for the investigation and control of nosocomial outbreaks of legionellosis. Pulsed-field gel electrophoresis (PFGE) of chromosomal DNA following digestion with SfiI is considered to be one of the most discriminative methods for detecting DNA polymorphisms amongst L. pneumophila serogroup 1 (Lp1) isolates. This paper describes an arbitrarily primed PCR (AP-PCR) method with three different primers (20-mers) for detecting DNA polymorphisms of Lp1 isolates. The AP-PCR assay was compared with PFGE analysis. Both experimental methods were found to have good discriminatory power (discrimination index of 98% and 94.3%, respectively) with 27 unrelated isolates from different geographical areas collected between 1987 and 1997. Furthermore, when the AP-PCR was used in the epidemiological investigation of nosocomial cases of infection, convergent results with the three primers allowed an epidemiological link to be established between isolates from patients and their environment. The AP-PCR method, which is rapid and easy to perform, gave results at least as discriminatory as those obtained with the PFGE method and is proposed for use in the molecular typing of Lp1 outbreaks.     

Molecular typing of nosocomial strains of Legionella pneumophila by arbitrarily primed PCR.

Arbitrarily primed PCR with two different primers was compared with ribotyping and monoclonal antibody analysis for typing Legionella strains. Applied to 11 epidemiologically unrelated strains, arbitrarily primed PCR resulted in an index of discrimination of 100% with both primers. It was found able to identify an epidemic clone of Legionella pneumophila serogroup 1 that was isolated from both patients and a hot water circuit of the same hospital.     

Molecular epidemiology of Legionella pneumophila serogroup 1

The DNA of patient and environmental isolates of Legionella pneumophila serogroup 1 was analyzed by restriction endonuclease cleavage. The electrophoretic patterns of the DNA digests of isolates from a group of patients with Legionnaires disease acquired in a hospital were indistinguishable from one another and were identical to the DNA pattern of a strain isolated from the hot water supply of the hospital. On the other hand, they were easily differentiated from strains isolated from patients and hot water supplies in other hospitals in the same city. The homogeneity of populations of L. pneumophila serogroup 1 colonizing plumbing systems was also investigated by DNA restriction endonuclease analysis in three hospitals. We distinguished two subtypes in one hospital; the two other hospitals had homogeneous populations. Restriction endonuclease digest analysis of L. pneumophila serogroup 1 DNA enables subtyping and appears to be a useful method for examining the epidemiology of outbreaks of Legionnaires disease.     

New serogroup of Legionella pneumophila, serogroup 8.

A strain of Legionella pneumophila (designated Concord 3) was isolated from a postmortem lung specimen of a patient with pneumonia. Direct fluorescent-antibody and immunoelectrophoresis tests, using absorbed and unabsorbed reagents prepared to this and other strains of legionellae, indicated that the Concord 3 strain represents a new serogroup of L. pneumophila, serogroup 8.     

Molecular comparison of Legionella pneumophila serogroup 1 isolated in Tunisia

Legionella pneumophila is a common cause of hospital and community-acquired pneumonia, being transmitted by inhalation of aqueous aerosols. Most outbreaks are linked to contaminated hot water systems and cooling towers. Our study was about the molecular typing of 35 strains of L. pneumophila including four clinical isolates and 31 environmental strains isolated from the distribution systems of 14 hotels. Among the clinical strains, two have the same pattern, however, all were different from the studied environmental strains. For the 31 environmental strains, ten patterns were obtained. Among which, a same pulsotype was found for four strains isolated from four different establishments. In addition, two different pulsotypes were found for strains isolated from the same establishment. The pulsed-field gel electrophoresis showed the existence of various patterns. Although cases of legionellosis were declared in these hotels, there are no epidemiological links between the clinical and environmental strains.     

Plasmid profiles of clinical and environmental isolates of Legionella pneumophila serogroup 1

Clinical and environmental Legionella pneumophila serogroup 1 isolates from a single water source in Columbus, Ohio, exhibited five different plasmid profiles. The multiplicity of plasmid profiles observed within a single geographic area and a skewed distribution of isolates bearing these plasmid profiles within this area suggest that plasmid analyses will be useful in the study of the epidemiology of Legionnaires disease and the environmental distribution of legionellae.     

Subtyping of Legionella pneumophila serogroup 1 isolates by monoclonal antibody and plasmid techniques.

A group of environmental and clinical Legionella pneumophila serogroup 1 isolates was subtyped by monoclonal antibody dot immunoblotting and plasmid analysis. Monoclonal antibody analysis defined seven subtypes within three major groups. Plasmid analysis (including restriction endonuclease digestion) revealed 10 subtypes. By combining plasmid and monoclonal techniques, all 16 strains were shown to be distinct. Plasmid profiles and monoclonal antibody reactivities of selected strains were stable despite serial passage (greater than 100 times). No plasmid-associated antigen was defined by this panel of monoclonal antibodies. The observed dissociation of plasmid profiles and monoclonal antibody reactivity patterns suggests that accurate epidemiologic typing of L. pneumophila serogroup 1 strains will require use of both techniques.     

Identification of protein antigens of Legionella pneumophila serogroup 1.

Growth of Legionella pneumophila serogroup 1 in yeast extract broth was standardized, and protein profiles of detergent-solubilized cells were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Over 30 protein bands were identified, 6 of which were more prominent both in Coomassie brilliant blue-stained profiles and in fluorograms with intrinsically radiolabeled bacteria. These major proteins were 22,000 dalton (22K), 24K, 43K, 63K, 76K, and 78K. We found that the 24K and 63K major proteins were antigenic, as demonstrated both by radioimmunoprecipitation (RIP) of [35S]methionine-labeled organisms and by immunoblotting with rabbit hyperimmune sera. In addition, both techniques detected antigens migrating at 58K, 72K, 76K, and 78K. The major 22K and 43K major proteins and antigens migrating at 25.5K, 29K, and 46K were only detected by radioimmunoprecipitation, whereas antigens of 19K, 48K, 53K, and 68K were detected only by immunoblotting. Cell-surface localization of the proteins was determined by a modified radioimmunoprecipitation assay designed to react specifically with surface antigens and by the use of hyperimmune sera that had been extensively preabsorbed with intact cells to deplete the sera of antibodies directed against surface components. The 22K, 24K, 43K, 63K, and 78K major proteins and several minor proteins were found to be located on the surface of L. pneumophila cells.     

Crossed immunoelectrophoretic analysis of Legionella pneumophila serogroup 1 antigens.

By crossed immunoelectrophoresis, 85 different antigens were demonstrated in sonicated preparations of Legionella pneumophila serogroup 1 (Lp1). The precipitin patterns of 82 anodic-migrating antigens were numbered and were designated the Lp1 reference system. Eleven antigens were stable to boiling, and seven of these were shown to be surface antigens. One heat-stable surface antigen (antigen no. 61) was highly reactive with limulus amoebocyte lysates and formed a precipitin resembling lipopolysaccharide. Serum from an isolation confirmed case of Lp1 infection and serogroup-specific rabbit antiserum reacted specifically with antigen no. 61, which was designated the serogroup-specific antigen. Normal human and rabbit sera commonly had antibodies to antigen no. 66 of the Lp1 reference system. This antigen is antigenically related to the "common antigen" of Pseudomonas aeruginosa.     

Antigenic and genetic variation in Legionella pneumophila serogroup 6.

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.     

Legionella pneumophila serogroup 4 isolated from joint tissue

We report the isolation of Legionella pneumophila serogroup 4 from synovial tissue obtained from an 80-year-old female with chronic swelling of her right metacarpophalangeal joint. Synovial tissue infections caused by L. pneumophila are rare. Interestingly, this isolate was recovered from chocolate agar after 5 days of incubation.     

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.     

Isolation of Legionella pneumophila serogroup 14 from a human source.

A strain of Legionella pneumophila serogroup 14 was isolated during a retrospective study, after death from the sputum of a patient who had had acute leukaemia and pneumonia. This is the third strain of that serogroup to be isolated from a human source. This event emphasises the importance of performing culture as well as serological tests, so as to detect cases of legionellosis caused by strains which rarely cause fatal clinical illness.     

Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila

A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of > 0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.     

Molecular typing of Legionella pneumophila serogroup 1 by pulsed-field gel electrophoresis with SfiI and comparison of this method with restriction fragment-length polymorphism analysis.

A panel of 131 well-characterised Legionella pneumophila serogroup 1 strains, previously used to evaluate a restriction fragment-length polymorphisms (RFLP) typing scheme, was examined by pulsed-field gel electrophoresis (PFGE) with the restriction endonuclease SfiI. The data obtained show that PFGE with SfiI is a highly discriminatory method yielding an index of discrimination (IOD) of 0.992 and 0.975, with 100% and 90% similarity thresholds respectively, compared with an IOD of 0.909 for the RFLP typing method. Reproducibility of PFGE profiles within gels was excellent and it was possible to compare the profiles visually. However, the reproducibility of the technique between gels was poor and visual comparison of the patterns was extremely difficult. Computer-aided analysis assisted the assessment of inter-gel reproducibility. Of 11 duplicates examined only four pairs showed 100% similarity, although 9 of 11 showed > or =90% similarity. In an attempt to determine if the PFGE banding patterns were sufficiently unambiguous to allow the method to be used as a definitive typing method, 20 coded strains were examined. At a 90% similarity level, 16 of these were placed in the correct PFGE type and four were not allocated to a type. Partial digestion of DNA by SfiI was noted despite careful control of DNA and enzyme concentrations, suggesting that an alternative enzyme might give more reproducible results.     

Thirteenth serogroup of Legionella pneumophila isolated from patients with pneumonia.

A Legionella-like organism (strain 82A3105; ATCC 43736) was isolated from a lung aspirate taken from a patient with pneumonia. Results of physiologic, gas-liquid chromatographic, genetic, and serologic tests showed that strain 82A3105 and four additional clinical isolates belong to a new Legionella pneumophila serogroup, serogroup 13.     

Legionella pneumophila serogroup 12 isolated from human and environmental sources.

A Legionella-like organism (strain 570-CO-H [= ATCC 43290]) isolated from the lung tissue of a patient with pneumonia was shown by growth, as well as physiological, serological, and genetic characteristics, to belong to a new Legionella pneumophila serogroup, serogroup 12. Two additional strains were detected with antiserum specific for strain 570-CO-H. These strains were isolated from environmental sources.     

Legionella pneumophila serogroup 14 isolated from patients with fatal pneumonia.

Two Legionella-like organisms, one isolated from postmortem lung tissue and the other from a bronchial aspirate, were shown by growth, physiologic, and genetic characteristics to belong to the species Legionella pneumophila. Subsequent serologic testing indicated that both strains belonged to a new serogroup, serogroup 14.     

Genetic typing in a cluster of Legionella pneumophila infections.

Legionella pneumophila strains isolated from six patients, three air-conditioning- and cooling tower-derived strains, and three hot water supply-derived strains were analyzed by three genetic typing methods. The results of the whole-cell DNA restriction endonuclease analysis and the restriction patterns based on genes coding for rRNA correlated with each other and demonstrated that the patient isolates were indistinguishable from the air-conditioning- and cooling tower-derived isolates but differed markedly from the hot water supply-derived isolates. The patient and air-conditioning- and cooling tower-derived strains contained plasmids of the same molecular weight; the hot water supply-derived strains were plasmidless. These results indicated that the cooling tower or the air-conditioning system was the environmental source for the examined cluster of Legionnaires disease strains.     

A method for typing strains of Legionella pneumophila serogroup 1 by analysis of restriction fragment length polymorphisms

A restriction fragment length polymorphism (RFLP) typing method for Legionella pneumophila serogroup 1 was developed. The method depended upon the use of cloned EcoR1 fragments from L. pneumophila (Knoxville-1) probing Nci1 restriction fragments of chromosomal DNA. Examination of strains of L. pneumophila which were apparently unrelated showed that inter-strain RFLPs were common, and these formed the basis of the typing scheme. The technique was found to be highly reproducible and discriminatory. When the RFLP data were compared to that obtained by monoclonal antibody (MAb) subgrouping both methods of strain differentiation gave consistent results. The isolates examined by either method were also sub-divided by the alternative technique. The analysis of RFLPs by cloned probes should be of considerable epidemiological value.