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.     

Molecular epidemiology of Legionella species by restriction endonuclease and alloenzyme analysis.

As part of an ongoing investigation into nosocomial Legionella infections at Stanford University Medical Center (SUMC), we applied the technique of restriction endonuclease analysis (REA) to determine strain differences among three species, including Legionella pneumophila, Legionella dumoffii, and Legionella micdadei. A total of 26 human and environmental water isolates from SUMC were selected for REA and compared with control strains that were not epidemiologically linked to SUMC. REA results were compared with results of alloenzyme typing, typing by monoclonal antibodies, and plasmid fingerprinting in all but L. micdadei strains. REA and alloenzyme typing showed that SUMC patient isolates were derived from distinct strains of three species. L. pneumophila strains from SUMC patients were genotypically identical to those isolated from potable water. REA was especially useful in proving that SUMC L. dumoffii patient isolates were derived from a single strain and that patients may have been exposed to a common source(s). REA typing correlated well with alloenzyme typing. These methods complement serologic typing of L. pneumophila and provide discriminating capability between strains of other Legionella species such as L. dumoffii, for which serologic types have not been identified. In addition, REA typing is somewhat easier to perform than alloenzyme typing and can be done in clinical laboratories.     

Diverse populations of Legionella pneumophila present in the water of geographically clustered institutions served by the same water reservoir.

We cultured potable water from seven institutions (six hospitals and one medical school) every 2 weeks for 6 months for Legionella pneumophila. All of the institutions were located close to each other and received water from the same freshwater source. Two institutions (the medical school and hospital F, a maternity hospital) never had L. pneumophila isolated from their potable water. The remaining five had 17 to 72% of their water samples positive for L. pneumophila. Most of the isolates were serogroup 1; however, in hospital B serogroup 5 accounted for 56% of the isolates. Oxford and OLDA monoclonal antibody subtypes of L. pneumophila serogroup 1 coexisted in four of the five institutions, while subtype France only was found in one institution. All 10 isolates from this institution lacked plasmids. The other four institutions had Legionella populations with plasmid profiles II, III, and VI. Two of these institutions also had isolates with no plasmids. The distribution of the plasmid types was significantly different for all institutions except C and D. The distribution of monoclonal antibody subtypes was significantly different for L. pneumophila isolates recovered from institutions C and D. There were no characteristics that distinguished the culture-positive institutions from the culture-negative areas. We conclude that diverse populations of L. pneumophila exist within these institutions despite their geographic proximity and identical potable water source.     

Multiple Types of Legionella pneumophila Serogroup 6 in a Hospital Heated-Water System Associated with Sporadic Infections

Five sporadic cases of nosocomial Legionnaires' disease were documented from 1989 to 1997 in a hospital in northern Italy. Two of them, which occurred in a 75-year-old man suffering from ischemic cardiopathy and in an 8-year-old girl suffering from acute leukemia, had fatal outcomes. Legionella pneumophila serogroup 6 was isolated from both patients and from hot-water samples taken at different sites in the hospital. These facts led us to consider the possibility that a single clone of L. pneumophila serogroup 6 had persisted in the hospital environment for 8 years and had caused sporadic infections. Comparison of clinical and environmental strains by monoclonal subtyping, macrorestriction analysis (MRA), and arbitrarily primed PCR (AP-PCR) showed that the strains were clustered into three different epidemiological types, of which only two types caused infection. An excellent correspondence between the MRA and AP-PCR results was observed, with both techniques having high discriminatory powers. However, it was not possible to differentiate the isolates by means of ribotyping and analysis of rrn operon polymorphism. Environmental strains that antigenically and chromosomally matched the infecting organism were present at the time of infection in hot-water samples taken from the ward where the patients had stayed. Interpretation of the temporal sequence of events on the basis of the typing results for clinical and environmental isolates enabled the identification of the ward where the patients became infected and the modes of transmission of Legionella infection. The long-term persistence in the hot-water system of different clones of L. pneumophila serogroup 6 indicates that repeated heat-based control measures were ineffective in eradicating the organism.     

Evaluation of different primers for DNA fingerprinting of Legionella pneumophila serogroup 1 strains by polymerase chain reaction

A DNA fingerprinting method for the characterization of Legionella pneumophila serogroup 1 strains was established. This method was based on the DNA extraction using Chelex 100 and subsequent PCR analysis using primers under conditions of low stringency. Sixteen single primers were tested for the typing of the 10 epidemiologically unrelated reference strains of L. pneumophila serogroup 1 as well as patient isolates and environmental strains isolated from the water system of a hospital where patients with legionellosis were treated. In addition, a combination of two primers (Lpm-1 and Lpm-2) originally established for the specific detection of Legionella strains was tested. The PCR results were compared with two further subtyping methods, i.e. monoclonal antibody analysis and pulsed-field gel electrophoresis. The type strains Philadelphia 1, Knoxville 1, Allentown 1, Benidorm 0303E, Bellingham 1, and France 5811 could be distinguished clearly in experiments using all of the primers. Depending on the primer used, Heysham 1 and Oxford 4032E showed different DNA profiles. The strains Olda and Camperdown 1 were nearly indistinguishable. In contrast, the analysis by PFGE and MAb subtyping revealed distinct types for all 10 reference strains. The discrimination of the patient isolates from two suspected cases of nosocomial legionellosis and environmental isolates was not possible with the 16 single primers used in the study. However, the PCR assay with the combination of Lpm-1 and Lpm-2 as well as the PFGE and MAb analysis were able to differentiate distinct types. The use of the sequence-specific primers under low-stringency annealing conditions allowed both simultaneous gene detection as well as epidemiological typing of Legionella strains.     

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

Arbitrarily primed PCR with three primers and pulsed-field gel electrophoresis were used to characterize a set of 75 clinical Legionella pneumophila serogroup 1 isolates, with no apparent epidemiological link, obtained from 24 hospitals in Paris, France, from 1987 to 1997. Unexpectedly, 25 clinical isolates from 15 hospitals had an identical profile (termed type A) by both methods. The same profile was subsequently found in 16 of 64 randomly selected environmental L. pneumophila serogroup 1 isolates from 15 different sites in the Paris area. There was no evidence of geographic clustering or a peak incidence of type A isolation. Type A has not been found in France outside the Paris area, suggesting that a particular type of L. pneumophila serogroup 1 is specifically present in the Paris water distribution network.     

Insertion sequences as highly resolutive genomic markers for sequence type 1 Legionella pneumophila Paris

The causative agent of legionellosis, Legionella pneumophila, colonizes all natural and human-made water networks, thus constituting the source of contaminated aerosols responsible for airborne human infections. Efficient control of infections, especially during epidemics, necessitates the fastest and most resolutive identification possible of the bacterial source for subsequent disinfection of reservoirs. We thus compared recognized typing approaches for Legionella with a method based on characterization of insertion sequence (IS) content. A total of 86 clinical or environmental isolates of L. pneumophila, including 84 Paris isolates, sampled from 25 clinical investigations in France between 2001 and 2007, were obtained from the Legionella National Reference Center. All strains were typed by monoclonal antibody subgrouping, sequence-based typing, pulsed-field gel electrophoresis, and restriction fragment length polymorphism based on the presence or absence of IS elements. We identified six different types of IS elements in L. pneumophila Paris and used them as genomic markers in hybridization experiments. One IS type, ISLpn11, revealed a high discriminatory power. Simpson's index of discrimination, calculated from the distribution of IS elements, was higher than that obtained with the other typing methods used for L. pneumophila Paris. Moreover, specific ISLpn11 copies were found only in strains isolated from particular cities. In more than half of the cases, each clinical isolate had an ISLpn11 profile that was recovered in at least one environmental isolate from the same geographical location, suggesting that our method could identify the infection source. Phylogenetic analysis suggests a clonal expansion for the L. pneumophila Paris strain.     

Comparison of ribotyping and restriction enzyme analysis using pulsed-field gel electrophoresis for distinguishing Legionella pneumophila isolates obtained during a nosocomial outbreak.

Because of the ubiquity of Legionella isolates in aquatic habitats, epidemiologic evaluation of Legionella pneumophila strains is important in the investigation and subsequent control of nosocomial outbreaks of legionellosis. In this study, ribotyping and restriction enzyme analysis by pulsed-field gel electrophoresis (PFGE) were used to compare isolates of L. pneumophila obtained from patients and the environment during a nosocomial outbreak with unrelated control strains. Restriction enzyme analysis by PFGE resolved 14 different patterns among the L. pneumophila serogroup 1 and L. pneumophila serogroup 6 isolates involved in the study. Two of the patterns were observed in the three L. pneumophila serogroup 6 isolates from patients with confirmed nosocomial infections and environmental isolates from the potable water supply, which was, therefore, believed to be the source of the patients' infections. Three more patterns that were not present in isolates from patients with legionellosis were seen in isolates from the hospital environment, demonstrating the presence of multiple strains in the hospital environment. In the outbreak, one distinct pattern occurred among the L. pneumophila serogroup 1 isolates from patients with nosocomial infections, suggesting a common source; however, the source could not be determined. By comparison, ribotyping generated five patterns. However, some control strains of both L. pneumophila serogroups 1 and 6 possessed the same ribotypes as were present in the outbreak isolates. Both techniques were used successfully to subtype the isolates obtained during the investigation of the outbreak. Furthermore, restriction enzyme analysis by PFGE was useful for subdividing ribotypes and for distinguishing strains involved in the outbreak from epidemiologically unrelated strains.     

A hospital-associated outbreak of Legionnaires' disease caused by Legionella pneumophila serogroups 4 and 10 with a common genetic fingerprinting pattern

An outbreak of eight cases of pneumonia caused by Legionella pneumophila non-serogroup 1 (non-sg 1) occurred at a Swedish university hospital in 1993. Including previous and subsequent sporadic cases, the total number of culture-positive patients was 13. Twelve available non-sg1 isolates from patients were compared to 50 environmental water isolates using a monoclonal antibody test for serogrouping and amplified fragment length polymorphism analysis (AFLP). Of the 12 hospital-associated Legionella non-sg 1 patient isolates, 4 were serogrouped as sg 4, 7 as sg 10, and one as sg 6. Using AFLP fingerprinting all serogroup (sg) 4 and 10 isolates were genetically related except for minor variations. Furthermore, sg 4 isolates were identical in AFLP to sg 10 isolates. Patient isolates were also identical to isolates found in the water system of several hospital buildings, but quite unrelated to isolates obtained in a subsequent outbreak at the same hospital caused by L. pneumophila sg 1. Serogroup variations in outbreaks may occur despite a common molecular fingerprinting pattern. Evidently, the L. pneumophila sg 4 and 10 strains were closely related genetically, which raises the question whether this variation in phenotype is due to a genetic event or to a variable phenotypic expression. Genetic fingerprinting should be used in conjunction with serogrouping in epidemiological investigations.     

A hospital-associated outbreak of Legionnaires' disease caused by Legionella pneumophila serogroup 1 is characterized by stable genetic fingerprinting but variable monoclonal antibody patterns

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.     

Evaluation of arbitrarily primed polymerase chain reaction analysis for typing Legionella pneumophila

Objective: To evaluate the performance of arbitrarily primed polymerase chain reaction (AP-PCR) analysis in epidemiologic typing of Legionella pneumophila.
Methods: Sixty-two isolates of L. pneumophila of serogroups 1, 3, 6 and 10, including epidemiologically related and unrelated isolates, were analyzed by AP-PCR using the primer BG2. Twenty-six of the serogroup 1 isolates were typed by pulsed-field gel electrophoresis (PFGE).
Results: AP-PCR analysis showed 98% typeability and complete reproducibility. A majority of unrelated isolates of each serogroup could be distinguished (discrimination index: 92%). Clinical isolates showed AP-PCR patterns indistinguishable from those of the isolates of the related environmental source. PFGE and AP-PCR results were in agreement for 88% of isolates.
Conclusions: Single-primer AP-PCR analysis can be used as a simple and reproducible screening method for typing L. pneumophila strains of different serogroups.     

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.     

Characterization of a tandem repeat polymorphism in Legionella pneumophila and its use for genotyping

We have analyzed the variability of minisatellite sequences (also called variable-number tandem repeats [VNTRs]) in the genome of Legionella pneumophila. Based upon the genome sequence of the Philadelphia-1 strain (serogroup 1), 25 minisatellites were selected and their polymorphisms were analyzed by PCR with the DNA of serogroup 1 to 14 reference strains. For 22 markers, a PCR product of the expected size was found with the DNA of the Philadelphia-1 strain. Most of these markers did not amplify the DNA of other Legionella species or other bacteria used as controls. A polymorphism was observed for seven markers among the L. pneumophila strains tested. To check whether these markers could be used to compare strains of L. pneumophila, we analyzed two groups of isolates from clinical and environmental samples which had been independently genotyped by other methods. The results showed that, for the isolates in these two sets of samples, VNTR typing is as informative as pulsed-field gel electrophoresis for comparison of strains. Sequencing of one minisatellite from 14 reference strains was performed. Comparison of the sequences allowed a classification and confirmed the existence of subspecies of L. pneumophila. We also tested the usefulness of one very polymorphic marker as a tool for the rapid screening of colonies grown from water samples. This allowed the rapid identification of the L. pneumophila colonies and gave a first hint as to the presence of several strains in a single sample.     

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.     

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.     

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.     

Taxonomic investigation of Legionella pneumophila using monoclonal antibodies.

A panel of 19 monoclonal antibodies was used to produce patterns of immunofluorescent staining of 468 isolates of Legionella pneumophila. Twelve monoclonal antibodies were selected that divided L. pneumophila into 17 phenons which, in the majority of cases, conform to serogroup divisions. These phenons are more easily defined than the present serogroups, and isolates can be placed in them with little ambiguity. The standardized set of monoclonal antibodies was also used to define the subgroups of serogroup 1.     

Restriction fragment length polymorphism of rRNA genes for molecular typing of members of the family Legionellaceae.

Typing of Legionella pneumophila remains important in the investigation of outbreaks of Legionnaires' disease and in the control of organisms contaminating hospital water. We found that the discriminatory power of a nonradioactive ribotyping method could be improved by combining results obtained with four restriction enzymes (HindIII, NciI, ClaI, and PstI). Fifty-eight clinical and environmental L. pneumophila strains including geographically unrelated as well as epidemiologically connected isolates were investigated. Epidemiologically related strains had the same ribotypes independent of the combinations of enzymes used. Some strains belonging to the same serogroup were assigned to different ribotypes, and some ribotypes contained members of different serogroups, indicating, as others have found, that serogroup and genotype are not always related. The discriminatory power of the method was estimated by calculating an index of discrimination (ID) for individual enzymes and combinations thereof. The combined result with all four enzymes was highly discriminatory (ID = 0.97), but results for three enzymes also yielded ID values acceptable for epidemiological purposes. In addition, the testing of 27 type strains and 6 clinical isolates representing Legionella species other than L. pneumophila indicated that ribotyping might be of value for species identification within this genus, as previously suggested.     

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 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.