Molecular epidemiology of Legionella pneumophila infection at a Canadian tertiary care institution

OBJECTIVE: To characterize the molecular epidemiology of LegioneLLa species infection at one Canadian tertiary care centre. DESIGN: Twenty-eight clinical isolates and 12 environmental isolates obtained over a six-year period were analyzed by resi.Jiction fragmentlenglh polymorphism (RFLP) of chromosomal DNA. Isolates included 15 from 12 patients wilh hospital acqu i1·ed illness and 13 from nine patients with community acquired infection. RESULTS: One nosocomial strain was LegioneLLa micdadei and one community strain was LegioneLLa pneumophila serotype 6: a ll olhers were L pneumophila serotype 1. RFLP typing revealed one clone for all cases of a 1985 single-ward outbreal< and five of six nonoutbreal< L pneumophila nosocomial cases. An RFLP patlern identical or highly related to that of the nosocomial clonal type was noted among nine of 12 L pneumophilaserotype l community isolates. The remaining three isolates had two related RFLP patterns distinct from the institutional stra in. The nosocomial and community strains were isolated from multiple institutional water samples in lhe institution. For lhe environmental isolates, monoclonal antibody typing was more discriminating than RFLP typing: seven monoclonal antibody subtypes were distinguished among 12 environmental isolates compi·ising lhree distinct RFLP patterns . CoNCLUSIONS: Despite multiple L pneumophila serotype l slrains isolated in the authors· institutional water. a single clone of L pneumophila produced most disease. Community acquired disease was caused by a wider Va.Jiety of slrains. (Pour resume. voir page 158)


T EGIONELLA PNEUMOPHILA I S AN IMPORTANT CAUSE OF
Lboth nosocomial and community acquired pneumonia worldwide (l).While Legionella species are ubiquitous in environmental water sources.relatively few strains.primarily L pneumophila serogroup 1. cau se human disease.Several studies have reported that endemic and epidemic nosocomial infection in an institution is usually attributable to a single strain among many contaminating the potable water system.suggesting that certain strains are uniquely viru lent (2-6) .Identification and characterization of these viru lent strains will contribute to our knowledge of the disease and could, potentially, have implications for management of contaminated institutional water sources.
The clinical microbiology laboratory at the Winnipeg Health Sciences Centre has cultured L pneumophila from clinical specimens since 1981.In 1985, an outbreak of L pneumophila occurred in renal transplant patients (7) and, subsequently, one to four cases per year of nosocomial L pneumophila h ave been identified (8).The institutional potable water is colonized with multiple different legionella strains.
Several approaches h ave been used to distinguish individual strains of L pneumophila in epidemio logical investigations.These methods include monoclonal antibody subtyping (3,9), multilocus enzyme electrophoresis (2,10), plasmid profile analysis (11), restriction fragment length polymorphism (RFLP) typing of whole DNA (12-14) and rRNA genes (15.16).Greater discriminatory power has been noted with two or more typing methods combined (5 , 12).The current study was undertalcen to describe the molecular epidemiology of L pneumophila.both nosocomial and community acquired.at the authors• institution using molecular typing and limited monoclonal antibody subtyping.

Institutional characteristics:
The Winnipeg Health Sciences Centre is an 1100 bed acute care hospital which includes a pediatric hospital and programs for renal transplantation, bone marrow transplantation and oncology .It is one of two tertiary care referral hospitals serving the populations of Manitoba and northern Ontario .An outbreak of L pneumophila occurred in renal transplant patients on one ward in 1985, and eA.'tensive isolation of Legionella species from potable water was documented at that time (7).Attempts to limit water colonization subsequent to that outbreak included intermittent shock chlorination and superheating.These interventions had limited utility in maintaining the water system free from Legionellaspecies .Further cases of L pneumophila in the hospitalized renal transplant population did not occur, however, after institution of trimethoprim/ sulfamethoxazole prophylaxis.
Cases of endemic nosocomial legionella pneumonia have continued to occur in the hospital population.with one to four cases identified each year (8) .These occur in all areas of the main hospital, but none in the women's and children's hospitals.Nosocomial cases ar•e generally.but not uniquely.identified in patients receiving high dose steroid therapy.A similar number of cases are admitted from the community or transferred from other institutions and diagnosed with L pneumophila pneumonia at admission each year.Microbiological methods: Clini cal specimens for L pneumophila were inoculated onto buffered charcoal yeast extract (BCYE) agar for isolation and identified using standard methods ( 1 7) .Serotyping was performed using commercially prepared antisera.Isolates from patients were stocked in skim milk at -70°C.All patient isolates identified between 1985 and 1991 were re-  mended by the supplier (Pharmacia Fine Chemicals, New York).Agarose gel electrophoresis and numerical analysis of RFLP patterns were carried out as previously described ( 19).The criteria for the identity of isolates were based on the relatedness of chromosomal DNA banding patterns.Isolates with more than 95% DNA banding similarity were considered to be identical strains.and those with 85 to 95% similarity were considered to be related strains.RFLP types were designated .lfor more than 95% DNA banding similarity and .2 for more than 85% and up to 95% s imilarity.Both identical and related strains were cons idered to be from the same clonal group.
For plasmid profile analysis, undigested total DNA was separated by agarose gel electrophoresis as described above .The molecular size of the plasmid was determined according to previously reported procedures (20).Monoclonal antibody typing was previously performed on L pneumophila isolated from patients during the 1985 outbreak and was routinely performed for environmental isolates.Methods have been previously described (7).Monoclonal antibody typing was not performed for other clinical isolates .
Definitions and data analysis: Pneumonia developing in a palicnl after 72 h of hospitalization was considered to be nosocomial infection: all others were community acquired.1\vo cases were transferred to the authors• institution from other hosp itals in the province and diagnosed with Legionnaire's disease on admission.
Both cases had been admitted to the peripheral hospital with pneumonia and, thus, were considered lobe community acqu ired.Comparisons of distinct strains identified by molecular typing were made of epidemic with endemic isolates for nosocomial cases, of nosocomial with community isolates, and of nosocomial clinical isolates with environmental isolates.

Bacte rial strains :
Twenty-eight isolates were available from 21 patients (Table 1).These included eight isolates from five patients in the 1985 outbreak, seven isolates from seven patients with hospital acquired infection isolated between 1985 and 1991.and 13 isolates from nine patients with community acquired infection admitled between 1986 and 1991.One nosocomial s lrai n was Legionella micdadei and one community acquired strain was L pneumophila serogroup 6.All other strains were L pneumophila serogrou p 1. RFLP and plasmid typing: RFLP typing identified one dominant clone with RFLP type 1.1, which included 16 of28 isolates (57%).and six isolates (21 %) characterized by a highly related RFLP type 1.2 (Table 1).The L micdadei and L pneumophila serogroup 6 strain had distinct RFLP types.The remaining four isolates had two highly related RFLP types 2. 1 and 2 .2(clonal group 2) that were distinct with respect to the major clone (Figure 1).In every case where multiple isolates were obtained from the same patient, the isolates were identical.Thirteen of 15 nosocomial acquired isolates from 10 patients.including all the epidemiologically linked outbreak strains, derived from a single strain characterized by the RFLP type 1.1 (Table 2).Three isolates from three patients of 13 community acquired isolates had RFLP patterns identical to lhis strain.A second strain.including six isolates identified in three of the 'None of the isolates hod plosmids.CCU Coronary core unit; SICU Surgical intensive core unit nine community acquired pneumonia patients.was highly related to the predominate nosocomial strain.
The single nosocomial isolate of L pneumopl1ila serotype 1 with RFLP type 2.1 d iffering from other nosocomial isolates was similar to three community isolates (with RFLP type 2.1 a nd RFLP type 2.2).The RFLP types for the two community acquired strains isolated from patients transferred from other facilities were 1.1 and 1.2.Fifteen isolates had no plasmid.and the remaining 13 had a similar large plasmid of size 21.6 MDa (Table 2).The presence of a plasmid was not unique to either nosocomial or community isolates.and was not cons istent for different isolates from the same patient.No environmental isolates had a plasmid.Mo noclo nal antibody typing: Strains from the 1985 outbreak were all monoclonal antibody type Philadelphia 1 (7).Among the environmental isolates.five monoclonal antibody types and two unlmowns were identified (Table 3).The monoclonal antibody typing appeared to be more discriminating than RFLP typing for identifying strain differences .RFLP type 1.1 included both Philadelphia l and Oxford 4032E monoc lonal types.and RFLP type 2.1 included both Bellingham and Olda or !leysham monoclonal types.

DISCUSSION
In 1985 an outbreak of L pneumopllila pneumonia occurred in renal transplant patients at our facility (7).The RFLP typing performed in this study is consistent with the previously reported monoclonal antibody typing showing that a s ingle strain was responsible for this outbreak.After this outbreak.sporadic cases of nosocomial Legionnaire's disease occurring throughout our institution have.with few exceptions.been of the same molecular type.The only exceptions are one nosocomial strain that occurred the same year as the outbreak and an L micdadei infection that occurred the next year.Thus.for our institution.a single strain of L pneuma-CAN J INFECT D1s VOL 5 No 4 JuLY 1 AuGusT 1994 pl1ila is responsible for most nosocomial Legionnaire's disease.
There was a wider molecular variety of strains causing legionellosis in patients admitted from the community.While a third of the community isolates was identical to the nosocomial strain.a related strain was identified as frequently among community isolates.but not in any nosocomial isolates.This strain was.however.present in the institutional water system.Finally.one L pneumopllila serogroup 6 and two other community strains were also isolated.One of these community isolates was similar to the single unique L pneumopl1ila serotype 1 nosocomial isolate and was identified in the institutional environmental isolates.RFLP typing, plasmid typing and.for environmental isolates.monoclonal antibody typing were compared.Strains either had no plasmids or had a single plasmid of 21.6 MDa. and strains with a plasmid were isolated from both community and nosocomial cases .No environmental isolates.however.had plasmids.The presence of a plasmid was not associated with any s ingle RFLP type.Thus.plasmid typing did not appear to be helpful in differentiating strains for epidemiological purposes.This is consistent with observations from other investigations (12.15.16).
Monoclonal antibody typing was not available for most clinical isolates.The monoclonal antibody typing of the environmental isolates.however.identified a greater number of unique strains than RFLP types.In particular.the single RFLP type identified in most nosocomial cases comprised two different monoclonal antibody types.and RFLP type 2 .1 also comprised two different monoclonal antibody types.Struelens el a! ( 21) also reported greater variation with monoclonal antibody typing.They suggested that phenotypic variation.in fact, compromised the utility of monoclonal antibody typing.
These observations of one predominant institutional strain causing disease are similar to those reported from other centres (2 , 12, 14,21).While mu ltiple strains of Legionella species are id entified in potable water sources, a restricted num ber of strains a re isolated from patients with n osocomial disease.Community strains included isolates s imilar to both nosocomial L pneLLmophila serogroup 1 strai n s, b u t L micdadei.isolated from one patient with nosocomial d isease, h as not been isolated from any patients wilh community acquired disease.Most of th e community strains were also isolated from the institu tional water samples.Thus these strains caused disease in the community and REFERENCES 1. Yu VL.Legionella pneumophila (Legionna ire's disease).
In were present in the institution , but did not contribute to in stitutional acquired disease.Th e organism or environ menta l determ inants of infection by a particular strain will need further clarification to explain these observations.