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J Clin Microbiol. Jun 2003; 41(6): 2503–2508.
PMCID: PMC156525

A Hospital-Associated Outbreak of Legionnaires' Disease Caused by Legionella pneumophila Serogroup 1 Is Characterized by Stable Genetic Fingerprinting but Variable Monoclonal Antibody Patterns

Abstract

An outbreak of 18 pneumonia cases caused by Legionella pneumophila serogroup 1 occurred at a Swedish university hospital 1996 to 1999. Eight clinical isolates obtained by culture from the respiratory tract were compared to 20 environmental isolates from the hospital and to 21 epidemiologically unrelated isolates in Sweden, mostly from patients, by using pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism analysis (AFLP), and monoclonal antibody (MAb) typing. All patients and most environmental isolates from the outbreak hospital belonged to the same genotypic cluster in both PFGE and AFLP. This genotype was distinctly different from other strains, including a cluster from a second hospital in a different part of the country. The MAb subtype of the outbreak clone was Knoxville except for three isolates that were Oxford. A variation in the MAb reactivity pattern was also found in a second genotypic cluster. These changes in the MAb reactivity pattern were due to the absence or presence of the lag-1 gene coding for an O-acetyltransferase that is responsible for expression of the lipopolysaccharide epitope recognized by MAb 3/1 of the Dresden Panel. In all MAb 3/1-positive strains, the lag-1 gene was present on a genetic element that was bordered by a direct repeat that showed a high degree of sequence homology. Due to this homology, the lag-1 gene region seemed to be an unstable element in the chromosome. MAb patterns are thus a valuable adjunct to genotyping methods in defining subgroups inside a genotypic cluster of L. pneumophila sg 1.

Legionella pneumophila is increasingly recognized as an important pathogen causing hospital-associated pneumonia, and immunocompromised patients have an increased risk of acquiring legionellosis. It is important to relate patient strains to environmental isolates in order to initiate infection control programs. Subtyping with monoclonal antibodies (MAbs) directed against lipopolysaccharide epitopes on the surface of Legionella cells has been practiced for several years as a rapid procedure and, in recent years, as an adjunct to select Legionella strains for genotyping (16, 21, 33). These MAb panels have thus been useful for subtyping L. pneumophila serogroup 1 strains and also for differentiating strains expressing the virulence-associated epitope recognized by the MAb 3/1 in the Dresden Panel (corresponding to MAb 2 in the International Panel) and those that do not (4, 12, 14, 16, 24). MAbs may also be used for subgrouping non-L. pneumophila serogroup 1 strains (13). In general, MAb typing is a rapid technique that produces stable and reproducible typing patterns.

Several genotypic methods have also been developed and used for epidemiological investigations, in some cases together with MAb subtyping (10, 15, 17, 19, 26, 27, 28, 30, 32, 35). The European Working Group on Legionella Infections (EWGLI [www.ewgli.org]) studied the molecular methods that were currently in use and the collaboration group found two methods—macrorestriction followed by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism analysis (AFLP)—to be the two most useful (7). In later studies AFLP, described for Legionella in 1995 (32), was favored and found to be discriminatory, reproducible, and robust (8, 9).

In the present study, three different typing methods (MAb subtyping, PFGE, and AFLP) were applied on isolates of L. pneumophila sg 1 obtained from the patients and environment at the University Hospital of Uppsala during an outbreak. These were compared to other isolates obtained from unrelated cases in Sweden, including a nosocomial cluster from another Swedish university hospital. In addition, some isolates that showed identical genetic fingerprinting patterns but differed in the MAb subtype were analyzed for the lag-1 gene region, which codes for an O-acetyltransferase that is associated with the presence of the lipopolysaccharide epitope recognized by MAb 3/1 (14). By doing this, it was possible to demonstrate for the first time that MAb patterns might be unstable under environmental conditions. Such a phenomenon must be taken into consideration during epidemiological investigations.

(Preliminary results for part of the present study were presented at the 10th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Stockholm, Sweden, in May 2000 [2].)

MATERIALS AND METHODS

Bacterial isolates.

Table Table11 shows the origin of isolates used in the study. A hospital-associated outbreak caused by L. pneumophila serogroup 1 occurred in 18 patients from 1996 to 1999 at the University Hospital of Uppsala (hospital I). Serogroup 1 had not been found among patients and in the environment earlier despite the fact that an outbreak caused by L. pneumophila serogroups 4 and 10 had occurred in 1993 (unpublished data). Isolates were obtained from the respiratory tract of eight patients by culture on nonselective and selective BCYEα medium (6). Twenty environmental isolates of L. pneumophila sg 1 were cultured from five different buildings during the same period subsequent to filtration and acid treatment of hot water. A further 20 patient isolates and one environmental isolate from other parts of Sweden were included in the study. Most of these, except for five isolates from another hospital cluster (hospital II; Sahlgrenska University Hospital, Gothenburg, Sweden), were obtained from specimens sent to the Department of Clinical Microbiology at the Karolinska Hospital in Stockholm. Suspected legionellae were identified by cysteine requirement and serogrouping. All isolates were stored at −70°C. Bacteria were cultured on BCYEα for 3 days at 35°C, harvested, and suspended in phosphate-buffered saline at an optical density at 600 nm of 0.9 prior to serotyping and genotyping.

TABLE 1.
L. pneumophila serogroup 1 isolatesa

Serological tests.

The serogroup and MAb subtype of all isolates were confirmed by the use of the Dresden Panel of MAbs against L. pneumophila (13). This was done at two laboratories, in Dresden and in Stockholm, by methods described previously (13): either enzyme immunoassay or an immunofluorescent-antibody technique.

Macrorestriction with subsequent PFGE.

This method was applied in two laboratories, Stockholm and Uppsala. A modification of PFGE methods described previously was used (23, 25). Briefly, plugs were prepared containing legionella bacteria and lysozyme (Sigma-Aldrich). Restriction digestion of chromosomal DNA was performed with SfiI (Roche Diagnostics, Mannheim, Germany) overnight at 50°C. PFGE was run in a contour-clamped homogeneous electric field system (CHEF-DRII; Bio-Rad Laboratories, Hercules, Calif.) at 14°C and 6 V/cm, with an increasing switch time of 6.75 to 63.8 s for 22 h in Tris-borate-EDTA buffer. Each gel contained a lambda ladder PFG marker (New England BioLabs, Inc., Beverly, Calif.) and a control strain of L. pneumophila serogroup 1 (strain Corby, EUL 137 [7, 8]). Subsequent to ethidium bromide staining gels were scanned in a Geldoc instrument (Bio-Rad Laboratories).

AFLP.

The method was originally described for legionellae in 1995 (32). Our modification was similar to that used in the EWGLI harmonization study (8) (www.ewgli.org). AFLP was only run at one laboratory (Stockholm). Briefly, the harvested Legionella bacteria were suspended in phosphate-buffered saline at a concentration corresponding to a McFarland standard of 0.5, and DNA was prepared by using the the QIAamp tissue kit (Qiagen, Hilden, Germany). Each mix contained 1.5 μg of genomic DNA and 0.2 μg of each adapter-oligonucleotide (5′-CTC GTA GAC TGC GTA CAT GCA-3′ and 5′-TGT ACG CAG TCT AC-3′; PE Applied Biosystems, Warrington, United Kingdom), 20 U of PstI enzyme (Roche Diagnostics, Mannheim, Germany), and 1 U of T4-DNA ligase and ligation buffer (pH 7; Roche). Ligation was performed at 37°C for 3 h. PCR was performed by using Ready-to-Go PCR beads (Amersham Pharmacia Biotech, Inc., Uppsala, Sweden) and the selective primer 5′-GAC TGC GTA CAT GCA GG-3′ (PE Applied Biosystems). Amplification was performed in 34 cycles as follows: initial denaturation at 94°C for 4 min in the first cycle and for 1 min thereafter, annealing at 60°C for 1 min, and elongation at 72°C for 2.5 min. The amplified products were electrophoresed in a 1% agarose gel at 50 V for 16 h in Tris-borate-EDTA buffer, stained with ethidium bromide, and scanned in a Geldoc instrument (Bio-Rad). Each gel contained two ladders at each end (GeneRuler DNA ladder mix; MBI, Fermentas, United Kingdom) and the control strain EUL 137.

Data analysis.

The band profiles of the scanned gels were converted into a tagged information file format (TIFF). The files were analyzed visually and in a gel analysis software program (Molecular Analyst Software; Bio-Rad). Group analysis of bands was performed with the Dice coefficient and the unweighted pair group method by using arithmetic averages (UPGMA) for clustering. Further, a 2% band position tolerance and an optimization setting of 0.5% were used. AFLP isolates were considered to belong to the same genotype if the level of similarity between them was 95% or more. PFGE isolates were considered to belong to the same genotype if the difference between strains was <3 bands. Guidelines for the analysis of gels have been elaborated especially for PFGE (29, 31).

lag-1 gene detection.

Previous studies have suggested that the lag-1 gene might be mutated or deleted in MAb 3/1-negative strains (21, 34). Six isolates (i.e., isolates 3, 10, 21, 22, 59, and 60) belonging to two genotype clusters were selected in order to test whether changes in the lag-1 gene locus might have occurred in them. MAb 3/1-positive and -negative strains that could not be distinguished by using AFLP and PFGE were found within each cluster (Fig. (Fig.1).1). The design of the primers used for PCR and DNA sequence analysis of the lag-1 region were based on published sequences (21, 34) or sequences detected in the present study (Table (Table2).2). Several sets of primers were used to amplify fragments of ca. 500 to 2,000 nucleotides from the lag-1 gene locus of the investigated strains. These fragments were purified by using the QIAquick purification kit (Qiagen), and DNA sequences were determined by cycle sequencing on a DNA sequencer (model 377; Applied Biosystems, Weiterstadt, Germany) with appropriate primers. The nucleotide sequence was determined separately for each PCR fragment by using the sequence analysis package Lasergene (DNA-Star, Inc., Madison, Wis.). Finally, these sequences were assembled together to determine the DNA sequences of the whole lag-1 locus. Sequences were analyzed in more detail by using the BLAST search options of the National Center for Biotechnology Information program package (National Institutes of Health, Bethesda, Md. [1]) and the sequence analysis package Lasergene. The nucleotide sequence of the lag-1 region of strain 3 determined in the present study has been deposited in EMBL nucleotide sequence database under accession no. AJ504790.

FIG. 1.
(A) PFGE. Lanes 2 to 6 represent strains from cluster A (hospital I), two patient isolates (isolates 3 [lane 2] and 138 [lane 4], both Knoxville strains) and three environmental (isolates 10 [lane 3] and 139 [lane 5], both Oxford strains, and isolate ...
TABLE 2.
Oligonucleotides for amplification and sequencing of the lag-1 region

RESULTS

Genotypes.

Figure Figure1A1A (PFGE) and B (AFLP) show typical patterns obtained by both genotyping methods. The two main clusters are represented, as are, for comparison, five other isolates that are epidemiologically unrelated. About 10 to 15 distinct bands can be seen on most lanes.

The PFGE result is shown by the dendrogram in Fig. Fig.2.2. Similarly, the result of AFLP is shown in Fig. Fig.3.3. Both figures also show the results of MAb subtyping of the respective strain. Altogether, 10 PFGE types and 13 AFLP types were recognized. There was no real difference in clustering between the two laboratories running PFGE (data not shown). The typical pattern obtained at one laboratory could be recognized with the same method at the other.

FIG. 2.
Dendrogram of PFGE, with a Dice coefficient with 2% tolerance and UPGMA clustering of 50 isolates. The isolate number is shown, (though not in numerical ordrer), and the MAb subtype. Note the agreement of cluster A (31 isolates) in Fig. Fig.2 ...
FIG. 3.
Dendrogram of AFLP, with a Dice coefficient with 2% tolerance and UPGMA clustering of 50 isolates. See Fig. Fig.22 legend for more details.

The conformity in clustering between PFGE group A (31 isolates) with AFLP group 1 (27 isolates) should be noted. Thus, the groups A and 1 consist mainly of the Uppsala, hospital I, outbreak strain but differ in a few points. Similarly, there is agreement between PFGE group B with AFLP group 2. Isolates 21 to 25 in this cluster represent hospital II, whereas isolates 59 and 60 were obtained from patients who suffered from community-acquired pneumonia and who lived in other parts of Sweden. The similarity in band profiles can be seen in Fig. Fig.1.1. Both genotypic methods also included isolate 53 in cluster A/1, despite the fact that no epidemiological relationship could be found with the Uppsala region. Similarly, the non-hospital I isolates 50 and 55 were related to cluster A/1, although they were not quite identical in PFGE or AFLP analyses. Hospital I isolate 71 seemed to be related genotypically to the main group but not identical. However, surprisingly, hospital I isolate 40 was found in PFGE cluster A but not in AFLP cluster 1, differing by three bands.

MAb serotypes.

The MAb subtypes of the two main clusters, A/1 and B/2, were Knoxville or Oxford and Philadelphia or OLDA, respectively. Although all patients were infected with a Knoxville strain in the Uppsala outbreak, there was a variation inside genotype A/1 among the environmental isolates between Knoxville (Dresden Panel MAb 3/1 positive) and Oxford (Dresden Panel MAb 3/1 negative). Of the three Oxford isolates, one was sampled in 1996 and the other two were sampled in 1999 from the same building. Knoxville isolates of identical genotypes were also found there. The five OLDA isolates in the B/2 cluster did not react with MAb 3/1, whereas the two Philadelphia isolates did. MAb subtypes, including Knoxville from other patients, seemed to belong to quite different genotypes compared to the two main clusters. Altogether, six phenoms were recognized by using the Dresden MAb Panel. Figure Figure11 shows PFGE and AFLP patterns typical of the two main clusters. Note the lack of difference between the Knoxville and Oxford isolates belonging to the same genotype (cluster A/1), whether with PFGE or AFLP. The same was true for Philadelphia and OLDA isolates in cluster B/2.

Gene organization of the lag-1 region.

DNA fragments from each of the Legionella strains 3, 10, 21, 22, 59, and 60 were successfully amplified. The lag-1 gene was absent in the Oxford and OLDA isolates, which also lacked reactivity with Dresden MAb 3/1. The gene organization in isolate 3 is shown in Fig. Fig.4.4. It is clear from the figure that a fragment of 2,857 bp containing the lag-1 gene was absent in the genome of isolate 10, the MAb 3/1-negative strain. Due to the fact that a 797-bp direct repeat existed on both sides of the fragment, it is conceivable that the 2,857-bp fragment can be deleted or inserted easily. A similar genetic organization of the lag-1 region was found for the other MAb 3/1-positive strains investigated, i.e., isolates 21 and 59. Direct repeats on both sides of the fragment were present in both strains. These might be involved in a deletion of the lag-1 fragment, resulting in MAb 3/1-negative strains, i.e., isolates 10, 22, and 60.

FIG. 4.
Schematic representation of the gene organization in the lag-1 region in the MAb 3/1-positive patient strain 3. Open reading frames are represented as open boxes, with arrowheads indicating the orientation. The design of the open reading frame was based ...

DISCUSSION

Epidemiological investigations on outbreaks of L. pneumophila serogroup 1 infections have been studied earlier with MAbs directed against lipopolysaccharide epitopes (16, 21, 33). This method has been useful and rapid but is less discriminatory than recent genotyping methods. Among the latter, PFGE subsequent to macrorestriction has been shown to be an effective method for genotyping Legionellae (15, 19, 26, 30, 35). However, some laboratories may not have the requisite equipment. Furthermore, this method of genotyping may also be difficult to standardize (7). Although the reproducibility of PFGE subsequent to digestion with SfiI has been reported to be less satisfactory (35), this was not so in the present study. In our experience it is important to use lysozyme in addition to proteinase K in preparing DNA, which has not been the case in some studies (20, 35). The EWGLI harmonization group found that AFLP, although less discriminatory, was reliable and gave consistent results. It was also easier to standardize (7, 8, 9) and showed adequate epidemiological concordance. Another study in Germany confirmed the usefulness of AFLP in comparison with arbitrarily primed PCR and PFGE in the study of nosocomial outbreaks (17).

We found that isolates of L. pneumophila sg 1 from two university hospitals clustered in two groups, specific to each hospital, by using the two genotyping methods. Thus, PFGE genotype A agreed mainly with AFLP genotype 1 in defining the outbreak strain at hospital I, and PFGE genotype B agreed mainly with AFLP genotype 2 in defining the strain at hospital II. Both methods produced clusters that included epidemiologically unrelated isolates, namely, isolate 53 in the A/1 cluster (hospital I) and isolates 59 and 60 in the B/2 cluster (hospital II). Evidently, a certain genotype might be spread over a large geographical area, in which case it is not very discriminatory. The same phenomenon has recently been reported by others (5, 18). Some discrepant results were found with the two methods that in part may be explained by the criteria used to define genotypes in the present study. However, surprisingly, isolate 40 did differ in AFLP, but not in PFGE, from the main hospital I cluster by three bands.

Inside the two main genotype clusters there was a variation in the MAb subtype. Although most isolates from the outbreak hospital (hospital I) were Knoxville, a few environmental ones were Oxford. Similarly, in the second cluster (mainly hospital II) there was a variation between Philadelphia and OLDA among patient isolates. There was, however, no difference in the PFGE and AFLP patterns, even in a single band. Such serologic subtype variations have been reported earlier (11, 18, 30). The question whether these different phenoms represent an environmentally induced phenotypic expression or are caused by a true genetic event preceding the serologic change (14, 20, 34) could for the first time be answered in the present study. Thus, in all MAb 3/1-positive strains investigated the lag-1 gene was present on an unstable genetic element that was bordered by a direct repeat. It became apparent that in two different clusters the Oxford or OLDA isolates were genetically different compared to the Knoxville or Philadelphia isolates, respectively, in that they did not possess the lag-1 gene. This gene codes for an O-acetyltransferase of the lipopolysaccharide O-chain of L. pneumophila sg 1, and it is present in MAb 3/1-positive strains belonging to the MAb subtypes Knoxville, Benidorm, Philadelphia, Allentown, and France (16, 34). Thus, in two clusters there were isolates that seemed identical as genotypes but that differed as to the presence or absence of the lag-1gene.

There are two possible explanations for this phenomenon. First, there might have existed, coincidentally, two different strains in each water system that could not be distinguished by PFGE and AFLP. Second, the lag-1 region could have been inserted in the MAb 3/1-positive strains or deleted in the MAb 3/1-negative strains. The second assumption seems to be more likely because the DNA sequences of the up- and downstream regions of the lag-1 gene were 100% identical in each of the pairs of strains. These sequences, in turn, were different from those of six other MAb 3/1-positive strains, including strains Corby and Phildelphia-1, that were investigated for comparison. The fact that the sequences that border the lag-1 gene are identical in each of the sets of strains supports the assumption that the deletion or insertion is a recent event or occurs frequently. Experiments to determine the exact frequency are under way.

Insertion and excision of a large DNA fragment has been described by Lüneberg et al. (22). In this study the switching phenomenon was accompanied by the loss of virulence, as determined in cell culture assays and in a guinea pig model. In our study, the deletion or insertion has occurred in the environment and does not seem to be accompanied by reduced virulence, at least for the amebal hosts that must be present in the given water supplies. Altogether, five of the MAb 3/1-negative OLDA strains tested in the present study were clinical isolates. However, it should be noted that all clinical isolates in the large Uppsala cluster were MAb 3/1 positive, which gives some evidence in support of the concept of greater virulence and/or easier transmission of these strains because of increased hydrophobicity (14, 34).

There has been a general discussion about the usefulness of MAb subtyping: the same MAb subtypes can evidently be found among many different genotypes, which would make this method less discriminatory. However, as shown by Drenning et al. (5), different serotypes might have the same pulsotype in PFGE. Some genotypes may thus be widely spread, as also shown in a French study from the Paris area (18). In the present study (Fig. (Fig.1),1), a variation of MAb subtype was detected in two genotypic clusters by using both PFGE and AFLP. Since this variation is due to a genetic event related to the lag-1 gene, it is probably wise to use both genotyping and MAb subtyping methods in epidemiological investigations in order to define subpopulations inside a given genotypic cluster. The practical consequences of our observation was not epidemiologically important in our present investigation since the outbreak strains possessed identical MAb reactivity and were found in both patients and environment, making the hospital water supplies the most likely origin of infection.

The two outbreaks (1993 and 1996 to 1999) at the Uppsala University Hospital (hospital I) were associated with a special construction of the hot water system, in which ready-to-use water with a temperature of 45°C was produced. The system was subsequently reconditioned, and all buildings now have circulating hot water at 60°C. Over the years, continuous surveillance of buildings colonized by Legionella has been carried out, and the hot water system has been intermittently superheated in order to decrease the number of Legionella (unpublished data). It is interesting that L. pneumophila sg 1 had not been isolated from patients or environment before 1996, despite continuous surveillance for several years. The first outbreak at hospital I occurred in 1993 and was caused by L. pneumophila non-sg 1 (sg 4 and 10 [unpublished data]). Possibly, the new sg 1 strain began to colonize the water system because of a change in environmental conditions. The only change found in a retrospective analysis was the practice of intermittent superheating of fixtures and hot water plumbing. An increased heat resistance among virulent L. pneumophila sg 1strains compared to nonvirulent strains has been reported earlier (3). Also, virulence has been associated with the expression of the lipopolysaccharide epitope recognized by MAb 3/1 of the Dresden Panel (14).

In summary, MAb subtyping and two genotyping methods, PFGE and AFLP, were used in an investigation of a nosocomial outbreak of Legionnaires' disease. In general, both genotyping methods were equally discriminatory, a finding which is in agreement with previous studies (7, 17). MAb subtyping of L. pneumophila sg 1, although by itself less discriminatory, is a rapid and useful adjunct that increases the resolution of molecular typing methods. Genetic events, such as deletion or perhaps insertion of the lag-1 gene, may influence the reactivity of MAbs. Therefore, genotypic methods should always be used in outbreak investigations.

Acknowledgments

We thank Ulla Zettersten and Camilla Artaeus for skillful technical assistance with PFGE and AFLP, Sigrid Gäbler and Ines Wolf for monoclonal antibody typing, and Jutta Paasche for assistance with the lag-1 gene analysis. We also thank Leif Larsson at the Infections Control Unit, Department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden, for letting us use strains 21, 22, 23, 24, and 25 in our study.

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