Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bernander, S. Articles by Lundholm, M. Search for Related Content PubMed PubMed Citation Articles by Bernander, S. Articles by Lundholm, M.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, June 2003, p. 2503-2508, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2503-2508.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Sverker Bernander,^1* Kerstin Jacobson,¹ Jürgen Herbert Helbig,² Paul Christian Lück,² and Monica Lundholm^3,

Department of Clinical Microbiology, Karolinska Hospital, and Microbiology and Tumor Biology Center, MTC, Karolinska Institute, Stockholm,¹ Department of Clinical Microbiology, Uppsala University Hospital, Uppsala, Sweden,³ Institut Medizinische Mikrobiologie und Hygiene, Medizinische Fakultät TU Dresden, Dresden, Germany²

Received 15 October 2002/ Returned for modification 19 November 2002/ Accepted 7 March 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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].)

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial isolates. Table 1 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 BCYE 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 BCYE 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.

View this table: [in this window] [in a new window]

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. 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 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.

View larger version (79K): [in this window] [in a new window]

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 83 [lane 6], a Knoxville strain). The patient isolates in lanes 7 (Philadelphia, isolate 59) and 8 (OLDA, isolate 60) show a genotypic similarity with other strains from cluster B (hospital II) in lanes 9 (Philadelphia, isolate 21) and 10 (OLDA, isolate 22) but are epidemiologically unrelated. The strains in lanes 11 to 13 (Benidorm, isolates 28 and 56, and Philadelphia, isolate 54) have been isolated from travelers' pneumonia and are completely unrelated epidemiologically. (B) AFLP. The same series of strains as in Fig. 1 is shown. Note the similarities between isolates in cluster 1(lanes 2 to 6) and between the isolates in lanes 7 to 10.

View this table: [in this window] [in a new window]

TABLE 2. Oligonucleotides for amplification and sequencing of the lag-1 region

Top Abstract Introduction Materials and Methods Results Discussion References

 
Genotypes. Figure 1A (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. 2. Similarly, the result of AFLP is shown in Fig. 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.

View larger version (28K): [in this window] [in a new window]

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. 2 with cluster 1 (27 isolates) in Fig. 3. Similarly, cluster B (7 isolates) agrees with cluster 2 (7 isolates). Patient isolates are marked by "#" symbols. Also, note the variation between the serotypes Oxford/OLDA and Knoxville/Philadelphia inside the same genotypic clusters.

View larger version (25K): [in this window] [in a new window]

FIG. 3. Dendrogram of AFLP, with a Dice coefficient with 2% tolerance and UPGMA clustering of 50 isolates. See Fig. 2 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. 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 1 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. 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.

View larger version (9K): [in this window] [in a new window]

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 on published sequences (21). The closed box indicates the 2,857-bp fragment that was deleted in the MAb 3/1-negative strain 10, which has the same genotype as strain 3. Two repeats of 797 bp that showed an identity of 99.6% are also shown.

Top Abstract Introduction Materials and Methods Results Discussion References

 
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. 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.

 
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.

 
* Corresponding author. Mailing address: Department of Clinical Microbiology, Karolinska Hospital, SE-171 76 Stockholm, Sweden. Phone: 46-8-51773539. Fax: 46-8-308099. E-mail: sverker.bernander{at}ks.se.

Deceased.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
2
2. Bernander, S., K. Jacobson, U. Zettersten, J. H. Helbig, and M. Lundholm. 2000. PFGE, AFLP, and MAb subtyping of Legionella pneumophila serogroup 1. Clin. Microbiol. Infect. 6(Suppl. 1):223.
3
3. Colbourne, J. S., and P. J. Dennis. 1989. The ecology and survival of Legionella pneumophila. J. Institution Water Environ. Management 3:345-350.
4
4. Dournon, E., W. F. Bibb, P. Rajagopalan, N. Desplaces, and R. M. McKinney. 1988. Monoclonal antibody reactivity as a virulence marker for Legionella pneumophila serogroup 1 strains. J. Infect. Dis. 157:496-501.[Medline]
5
5. Drenning, S. D., J. E. Stout, J. R. Joly, and V. L. Yu. 2001. Unexpected similarity of pulsed-field gel electrophoresis patterns of unrelated clinical isolates of Legionella pneumophila, serogroup 1. J. Infect. Dis. 183:628-632.[CrossRef][Medline]
6
6. Edelstein, P. H. 1981. Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens. J. Clin. Microbiol. 14:298-303.[Abstract/Free Full Text]
7
7. Fry, N. K., S. Alexiou-Daniel, J. Bangsborg, S. Bernander, M. Castellani Pastoris, J. Etienne, B. Forsblom, V. Gaia, J. H. Helbig, D. Lindsay, P. C. Lueck, C. Pelaz, S. A. Uldum, and T. G. Harrison. 1999. A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study. Clin. Microbiol. Infect. 5:462-477.[Medline]
8
8. Fry, N. K., J. M. Bangsborg, A. Bergmans, S. Bernander, J. Etienne, L. Franzin, V. Gaia, P. Hasenberger, B. Baladron-Jimenez, D. Jonas, D. Lindsay, S. Mentula, A. Papoutsi, M. Struelens, S. A. Uldum, P. Visca, W. Wannet, and T. G. Harrison. 2002. Designation of the European Working Group on Legionella Infection (EWGLI) amplified fragment length polymorphism types of Legionella pneumophila serogroup 1 and results of intercentre proficiency testing using a standard protocol. Eur. J. Clin. Microbiol. Infect. Dis. 21:722-728.[CrossRef][Medline]
9
9. Fry, N. K., J. M. Bangsborg, S. Bernander, J. Etienne, B. Forsblom, V. Gaia, P. Hasenberger, D. Lindsay, A. Papoutsi, C. Pelaz, M. J. Struelens, S. A. Uldum, P. Visca, and T. G. Harrison. 2000. Assessment of intercentre reproducibility and epidemiological concordance of Legionella pneumophila serogroup 1 genotyping by amplified fragment length polymorphism analysis. Eur. J. Clin. Microbiol. Infect. Dis. 19:773-780.[CrossRef][Medline]
10
10. Gomez-Lus, P., B. S. Fields, R. F. Benson, W. T. Martin, S. P. O'Connor, and C. M. Black. 1993. Comparison of arbitrarily primed polymerase chain reaction, ribotyping, and monoclonal antibody analysis for subtyping Legionella pneumophila serogroup 1. J. Clin. Microbiol. 31:1940-1942.[Abstract/Free Full Text]
11
11. Harrison, T. G., N. A. Saunders, A. Haththotuwa, G. Hallas, R. J. Birtles, and A. G. Taylor. 1990. Phenotypic variation amongst genotypically homogeneous Legionella pneumophila serogroup 1 isolates: implications for the investigation of oubreaks of Legionnaires' disease. Epidemiol. Infect. 104:171-180.[Medline]
12
12. Helbig, J. H., S. Bernander, M. Castellani-Pastoris, K. Etienne, V. Gaia, S. Lauwers, D. Lindsay, P. C. Lück, T. Marques, S. Mentula, M. F. Peeters, C. Pelaz, M. Struelens, S. A. Uldum, G. Wewalka, and T. G. Harrison. 2002. Pan-European study on culture-proven Legionnaires' disease: distribution of Legionella pneumophila serogroups and monoclonal subgroups. Eur. J. Clin. Microbiol. Infect. Dis. 21:710-716.[CrossRef][Medline]
13
13. Helbig, J. H., J. B. Kurtz, M. Castellani-Pastoris, C. Pelaz, and P. C. Lück. 1997. Antigenic lipopolysaccharide components of Legionella pneumophila recognized by monoclonal antibodies: possibilities and limitations for division of the species into serogroups. J. Clin. Microbiol. 35:2841-2845.[Abstract]
14
14. Helbig, J. H., P. C. Lück, Y. A. Knirel, W. Witzleb, and U. Zähringer. 1995. Molecular characterization of a virulence-associated epitope on the lipopolysaccharide of Legionella pneumophila serogroup 1. Epidemiol. Infect. 115:71-78.
15
15. Johnson, W. M., K. Bernard, T. J. Marrie, and S. D. Tyler. 1994. Discriminatory genomic fingerprinting of Legionella pneumophila by pulsed-field electophoresis. J. Clin. Microbiol. 32:2620-2621.[Abstract/Free Full Text]
16
16. Joly, J. R., R. M. McKinney, J. O. Tobin, W. F. Bibb, I. D. Watkins, and D. Ramsay. 1986. Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies. J. Clin. Microbiol. 23:768-771.[Abstract/Free Full Text]
17
17. Jonas, D., H.-G. W. Meyer, P. Matthes, D. Hartung, B. Jahn, F. D. Daschner, and B. Jansen. 2000. Comparative evaluation of three different genotyping methods for investigation of nosocomial outbreaks of Legionnaires' disease in hospitals. J. Clin. Microbiol. 38:2284-2291.[Abstract/Free Full Text]
18
18. Lawrence, C., M. Reyrolle, S. Dubrou, F. Forey, B. Decludt, B. C. Goulvestre, P. Masiota-Bernard, J. Etienne, and C. Nauciel. 1999. Single clonal origin of a high proportion of Legionella pneumophila serogroup 1 isolates from patients and the environment in the area of Paris, France, over a 10-year period. J. Clin. Microbiol. 37:2652-2655.[Abstract/Free Full Text]
19
19. Lawrence, C., E. Ronco, S. Dubrou, R. Leclercq, C. Nauciel, and P. Masiota-Bernard. 1999. Molecular typing of Legionella pneumophila serogroup 1 isolates from patients and the environment by arbitrarily primed PCR and pulsed-field gel electrophoresis. J. Med. Microbiol. 48:327-333.[Abstract/Free Full Text]
20
20. Lück, P. C., R. J. Birtles, and J. H. Helbig. 1995. Correlation of MAb subgroups with genotype in closely related Legionella pneumophila serogroup 1 strains from a cooling tower. J. Med. Microbiol. 43:50-54.[Abstract/Free Full Text]
21
21. Lück, P. C., T. Freier, C. Steudel, Y. A. Knirel, E. Lüneberg, U. Zähringer, and J. H. Helbig. 2001. A point mutation in the active site of Legionella pneumophila O-acetyltransferase resulted in modified lipopolysaccharide but does not influence virulence. Int. J. Med. Microbiol. 291:345-352.[CrossRef][Medline]
22
22. Lüneberg, E., B. Mayer, N. Daryab, O. Kooistra, U. Zahringer, M. Rohde, J. Swanson, and M. Frosch. 2001. Chromosomal insertion and excision of a 30-kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila. Mol. Microbiol. 39:1259-1271.[CrossRef][Medline]
23
23. Maslow, J. N., A. M. Slutsky, and R. D. Arbeit. 1993. Application of pulsed-field gel electrophoresis to molecular epidemiology, p 563-572. In D. H. Persing, T. F. Smith, F. C. Tenover, and T. J. White (ed.), Diagnostic molecular microbiology: principles and applications. American Society for Microbiology, Washington, D.C.
24
24. Plouffe, J. F., M. F. Para, W. E. Maher, B. Hackman, and L. Webster. 1983. Subtypes of Legionella pneumophila serogroup 1 associated with different attack rates. Lancet ii:649-650.
25
25. Poddar, S. K. 1991. A modified method of genomic DNA preparation in agarose insert for pulsed field gel electrophoresis. Electrophoresis 12:674-675.[CrossRef][Medline]
26
26. Pruckler, J. M., L. A Mermel, R. F. Benson, C. Giorgio, P. K. Cassiday, R. F. Breiman, C. G. Whitney, and B. S. Fields. 1995. Comparison of Legionella pneumophila isolates by arbitrarily primed PCR and pulsed-field gel electrophoresis: analysis from seven epidemic investigations. J. Clin. Microbiol. 33:2872-2875.[Abstract]
27
27. Saunders, N. A., T. G. Harrison, A. Haththotuwa, N. Kachwalla, and A. G. Taylor. 1990. A method for typing strains of Legionella pneumophila serogroup 1 by analysis of restriction fragment length polymorphisms. J. Med. Microbiol. 31:45-55.[Abstract/Free Full Text]
28
28. Stout, J. E., J. Joly, M. Para, J. Plouffe, C. Ciesielski, M. J. Blaser, and V. L. Yu. 1988. Comparison of molecular methods for subtyping patient and epidemiologically linked environmental isolates of Legionella pneumophila. J. Infect. Dis. 157:486-495.[Medline]
29
29. Struelens, M. J., et al. 1996. Consensus guidelines for appropriate use and evaluation of microbial epidemiologic typing systems. Clin. Microbiol. Infect. 2:2-11.[Medline]
30
30. Struelens, M. J., N. Maes, F. Rost, A. Deplano, F. Jacobs, C. Liesnard, N. Bornstein, F. Grimont, S. Lauwers, M. P. McIntyre, and E. Serruys. 1992. Genotypic and phenotypic methods for the investigation of a nosocomial Legionella pneumophila outbreak and the efficacy of control measures. J. Infect. Dis. 166:22-30.[Medline]
31
31. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.[Medline]
32
32. Valsangiacomo, C., F. Baggi, V. Gaia, T. Balmelli, R. Peduzzi, and J.-C. Piffaretti. 1995. Use of amplified fragment length polymorphism in molecular typing of Legionella pneumophila and application to epidemiological studies. J. Clin. Microbiol. 33:1716-1719.[Abstract]
33
33. Watkins, I. D., J. O'H Tobin, P. J. Dennis, W. Brown, R. Newnham, and J. B. Kurtz. 1985. Legionella pneumophila serogroup 1 subgrouping by monoclonal antibodies: an epidemiological tool. J. Hyg. 95:211-216.
34
34. Zou, H. Z., Y. A. Knirel, J. H. Helbig, U. Zähringer, and C. S. Mintz. 1999. Molecular cloning and characterization of a locus responsible for O acetylation of the O polysaccharide of Legionella pneumophila serogroup 1 lipopolysaccharide. J. Bacteriol. 181:4137-4141.[Abstract/Free Full Text]
35
35. Zoysa, A. S., and T. G. Harrison. 1999. 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. J. Med. Microbiol. 48:269-278.[Abstract/Free Full Text]

---

Journal of Clinical Microbiology, June 2003, p. 2503-2508, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2503-2508.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Kozak, N. A., Benson, R. F., Brown, E., Alexander, N. T., Taylor, T. H. Jr., Shelton, B. G., Fields, B. S. (2009). Distribution of lag-1 Alleles and Sequence-Based Types among Legionella pneumophila Serogroup 1 Clinical and Environmental Isolates in the United States. J. Clin. Microbiol. 47: 2525-2535 [Abstract] [Full Text]  
• Thurmer, A., Helbig, J. H., Jacobs, E., Luck, P. C. (2009). PCR-based 'serotyping' of Legionella pneumophila. J Med Microbiol 58: 588-595 [Abstract] [Full Text]  
• Ratzow, S., Gaia, V., Helbig, J. H., Fry, N. K., Luck, P. C. (2007). Addition of neuA, the Gene Encoding N-Acylneuraminate Cytidylyl Transferase, Increases the Discriminatory Ability of the Consensus Sequence-Based Scheme for Typing Legionella pneumophila Serogroup 1 Strains. J. Clin. Microbiol. 45: 1965-1968 [Abstract] [Full Text]  
• Fernandez-Moreira, E., Helbig, J. H., Swanson, M. S. (2006). Membrane Vesicles Shed by Legionella pneumophila Inhibit Fusion of Phagosomes with Lysosomes.. Infect. Immun. 74: 3285-3295 [Abstract] [Full Text]  
• Scaturro, M., Losardo, M., De Ponte, G., Ricci, M. L. (2005). Comparison of Three Molecular Methods Used for Subtyping of Legionella pneumophila Strains Isolated during an Epidemic of Legionellosis in Rome. J. Clin. Microbiol. 43: 5348-5350 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bernander, S. Articles by Lundholm, M. Search for Related Content PubMed PubMed Citation Articles by Bernander, S. Articles by Lundholm, M.