Paleoepidemiologic investigation of Legionnaires disease at Wadsworth Veterans Administration Hospital by using three typing methods for comparison of legionellae from clinical and environmental sources.

Multilocus enzyme electrophoresis, monoclonal antibody typing for Legionella pneumophila serogroup 1, and plasmid analysis were used to type 89 L. pneumophila strains isolated from nosocomial cases of Legionnaires disease at the Veterans Administration Wadsworth Medical Center (VAWMC) and from the hospital environment. Twelve L. pneumophila clinical isolates, obtained from patients at non-VAWMC hospitals, were also typed by the same methods to determine typing specificity. Seventy-nine percent of 33 VAWMC L. pneumophila serogroup 1 clinical isolates and 70% of 23 environmental isolates were found in only one of the five monoclonal subgroups. Similar clustering was found for the other two typing methods, with excellent correlation between all methods. Enzyme electrophoretic typing divided the isolates into the greatest number of distinct groups, resulting in the identification of 10 different L. pneumophila types and 5 types not belonging to L. pneumophila, which probably constitute an undescribed Legionella species; 7 clinical and 34 environmental VAWMC isolates and 2 non-VAWMC clinical isolates were found to be members of the new species. Twelve different plasmid patterns were found; 95% of VAWMC clinical isolates contained plasmids. Major VAWMC epidemic-bacterial types were common in the hospital potable-water distribution system and cooling towers. Strains of L. pneumophila which persisted after disinfection of contaminated environmental sites were of a different type from the prechlorination strains. All three typing methods were useful in the epidemiologic analysis of the VAWMC outbreak.     

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 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.     

Nosocomial Legionella pneumophila serogroup 5 outbreak associated with persistent colonization of a hospital water system

An outbreak of infections caused by Legionella pneumophila serogroup 5 was detected in a university hospital, and nosocomial reservoirs of the legionella epidemic were examined. Clinical isolates from two patients who had been affected by the L. pneumophila serogroup 5 outbreak, and from another patient with a legionella infection caused by the same serogroup 3 years later, were compared to L. pneumophila serogroup 5 isolates from the hospital water supply by two molecular methods, amplified fragment length polymorphism (AFLP) analysis and random amplified polymorphic DNA analysis (RAPD). Genotyping confirmed the epidemiological linkage of the first two patients, and linked their infections with the hospital water supply. The third clinical strain, which was also linked to the hospital water, was very similar to the epidemic strain. Even though the water distribution system was sanitized (superheat and flush sanitation), the epidemic strain was shown to be persisting in the hospital water outlets several years after its initial discovery.     

Cefamandole-susceptible strains of Legionella pneumophila serogroup 1: implications for diagnosis and utility as an epidemiological marker.

The standard selective Legionella medium that contains cefamandole failed to grow legionella pneumophila serogroup 1, subtype Bellingham, from a sputum sample from a patient with nosocomial Legionnaires' disease; the isolate did grow on a similar selective medium that substitutes vancomycin for cefamandole. Two Bellingham isolates from this patient's hospital environment also failed to grow when tested on the cefamandole medium. We tested 106 additional L. pneumophila serogroup 1 isolates that belonged to nine different monoclonal antibody subtypes and demonstrated that susceptibility to cefamandole was rare (10%) and limited to the Bellingham subtype. The diagnosis of Legionnaires' disease may be missed unless the culture protocol includes both a nonselective medium and a selective medium that does not contain cefamandole. In vitro susceptibility to cefamandole also provided an epidemiologic marker that linked a water source for a patient to nosocomial Legionnaires' disease.     

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.     

Epidemiologic investigation by macrorestriction analysis and by using monoclonal antibodies of nosocomial pneumonia caused by Legionella pneumophila serogroup 10.

A 67-year-old woman was hospitalized with an acute pneumonia of the left lower lobe. Legionella pneumophila serogroup 10 was cultured from two sputum specimens taken on days 18 and 20 and was also detected by direct immunofluorescence assay by using a commercially available species-specific monoclonal antibody as well as serogroup 10-specific monoclonal antibodies. Antigenuria was detected in enzyme-linked immunosorbent assays by using serogroup 10-specific polyclonal and monoclonal antibodies. In the indirect immunofluorescence test rising antibody titers against serogroups 1, 4, 5, 8, 9, 10, 14, and 15 were found in serum, with the highest titers found against serogroups 8, 9, and 10. L. pneumophila serogroups 10 and 6 and a strain that reacted with serogroup 4 and 14 antisera were cultured from both central and peripheral hot water systems of the hospital. Macrorestriction analyses of the genomic DNAs by pulsed-field gel electrophoresis showed that the isolate from the patient was identical to the serogroup 10 strains from the hospital hot water system. In contrast, the genomic DNAs of 16 unrelated L. pneumophila serogroup 10 strains showed 12 different restriction patterns. Monoclonal antibody subtyping revealed only minor differences in L. pneumophila serogroup 10 strains isolated from different sources. In conclusion, macrorestriction analysis is a valuable tool for studying the molecular epidemiology of L. pneumophila serogroup 10.     

Potable water as a cause of sporadic cases of community-acquired legionnaires' disease.

BACKGROUND. The environmental sources of sporadic, community-acquired legionnaires' disease are largely unknown, and culturing of water sources after identification of a case is currently not recommended. We conducted a prospective study of sporadic cases of community-acquired legionnaires' disease to determine whether the environmental reservoirs could be identified. METHODS. We cultured samples of potable water obtained from sources to which each of 20 patients with culture-confirmed, community-acquired legionnaires' disease had been exposed during the two weeks before the onset of symptoms. Monoclonal-antibody subtyping and restriction-endonuclease analysis were performed on the legionella isolates recovered from both the patients and the associated environmental cultures. RESULTS. For 8 of the 20 patients, isolates of Legionella pneumophila with identical subtypes were identified in cultures from both the patient and the potable water to which the patient had been exposed. The environmental reservoirs linked to the infections were the water supplies of two private residences, two nursing homes, two hospital outpatient clinics, and an industrial plant. CONCLUSIONS. Potable-water supplies that harbor L. pneumophila are an important source of community-acquired legionnaires' disease. Future studies should include attempts to identify the environmental sources of this infection.     

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.     

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 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.     

Production of monoclonal antibodies to Legionella pneumophila serogroups 1 and 6

To better define the surface antigens of Legionella pneumophila for clinical and experimental purposes, we have produced monoclonal antibodies to L. pneumophila serogroups 1 and 6. Two hybridomas were produced in serogroup 1. One antibody, LP-I-17, recognized a serogroup-common antigen. The second antibody, LP-I-81, was specific for serogroup 1. This antibody was able to agglutinate bacterial cells belonging to the serogroup 1 reference strains. Philadelphia and Knoxville. Microagglutination assays of environmental and clinical isolates revealed a subgroup of serogroup 1 environmental isolates which were not agglutinated by LP-I-81. This subset of isolates was segregated to certain buildings in the medical complex. Immunodiffusion studies showed identity between the LP-I-81 antigen and the serogroup-specific antigen of serogroup 1 organisms. This antigen could be absorbed out of the serogroup 1 organism extract with LP-I-81-coated Staphylococcus aureus, leaving the serogroup-common antigens. Three hybridomas were produced to serogroup 6. All three produced antibodies which were serogroup 6 specific and agglutinated serogroup 6 bacteria.     

Prevalence of Legionella pneumophila serogroup 1 in water distribution systems in Izmir province of Turkey

Legionella pneumophila serogroup 1 occurrence has been investigated in 168 hot water samples from 24 hotels, situated in 6 counties in Izmir province of Turkey, from 15 June to 30 September of the year 2000. Sampling was carried out at 15-day intervals and seven samples were taken from each of the hotels' hot water reservoirs and hot water networks. The samples were (1 L) concentrated using polycarbonate filters (mesh size 0.22 microm). Isolation was achieved using selective medium, GVPC agar. The samples were concentrated by membrane filtration, divided into three portions and cultured without pretreatment, after acid treatment, and after heat treatment, on GVPC agar. One hundred and ten isolates were identified as L.pneumophila sg 1 using the Legionella Latex Test (Oxoid). Arbitrarily primed PCR (AP PCR) was employed to assess the clonal relationship between Legionella pneumophila sg 1 isolates from the hot water samples of the hotels. Three genotypes of L. pneumophila sg 1 isolates were identified. With a high prevalence of type A, 22 hotels were found to be colonized with L. pneumophila serogroup 1, while only 2 were free from the bacteria.     

Molecular typing of nosocomial isolates of Legionella pneumophila serogroup 3.

In Paris, France, an outbreak of pneumonia due to Legionella pneumophila serogroup 3 was observed in Necker (four cases) and Pitié (six cases) hospitals. Neither the 10 clinical isolates nor 5 tap water isolates from Necker Hospital harbored plasmids. Clinical and environmental serogroup 3 isolates and serogroup 3 reference strain Bloomington 2 were analyzed by chromosomal probe fingerprinting. rRNA, 16S and 23S from Escherichia coli and a randomly cloned 15-kilobase-pair nucleotide sequence from L. pneumophila serogroup 3 were used as probes. All strains tested showed a single pattern after HindIII digestion of DNA and hybridization with the 32P-end-labeled rRNA probe, whereas three patterns were obtained after hybridization with the 32P-labeled 15-kilobase-pair DNA probe. One pattern was given by all clinical and tap water isolates from Necker Hospital, another one was given by all clinical isolates from Pitié Hospital, and a last one was given by reference strain Bloomington 2. Thus, molecular analysis showed that the two hospital outbreaks of legionellosis were unrelated and could link the outbreak in Necker Hospital to contaminated tap water.     

Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies.

A panel of monoclonal antibodies to Legionella pneumophila serogroup 1 and a subclassification scheme were developed in a collaborative project among three laboratories. The seven most useful monoclonal antibodies were selected from three previously developed panels on the basis of indirect fluorescent antibody patterns with 83 strains of L. pneumophila serogroup 1 that were obtained from widely distributed geographic locations. The isolates were divided into 10 major subgroups on the basis of reactivity patterns that can be readily reproduced in any laboratory and are not subject to major inconsistencies of interpretation of staining intensity. A standard protocol for the indirect fluorescent antibody procedure was also developed.     

Determination of antigenic relationships among legionellae and non-legionellae by direct fluorescent-antibody and immunodiffusion tests.

Six isolates, five from water samples and one from a human tracheal swab taken at autopsy, reacted strongly with working dilutions of Legionella fluorescent-antibody conjugates. Of these, two isolates of Pseudomonas fluorescens (EB and CDC93), one isolate of the Flavobacterium-Xanthomonas group (CDC65), and one isolate of P. alcaligenes (CDC11) reacted with Legionella pneumophila serogroup 1 conjugate. P. alcaligenes ABB 50 reacted with an L. pneumophila serogroup 3 conjugate and of P. maltophilia reacted with the L. micdadei conjugate. Antisera and labeled conjugates were prepared for these new cross-reacting isolates, and their relationships to the legionellae were examined by direct fluorescent-antibody and immunodiffusion tests. A nonreciprocal cross-reaction existed between L. micdadei and P. maltophilia and also between serogroups 3 of L. pneumophila and P. alcaligenes ABB50. Of the four isolates that reacted with serogroup 1 of L. pneumophila, P. fluorescens CDC93 had the strongest relationship, and the other three had only minor relationships. Although cross-reactivity among non-legionellae and legionellae has not been a major problem, these findings are relevant to the interpretation of direct fluorescent-antibody tests for detecting these bacteria.     

Comparison of arbitrarily primed polymerase chain reaction, ribotyping, and monoclonal antibody analysis for subtyping Legionella pneumophila serogroup 1.

Arbitrarily primed polymerase chain reaction (AP-PCR) was used to characterize Legionella pneumophila serogroup 1. Cells from a single colony could be subtyped by AP-PCR within a few hours. The discrimination between strains of L. pneumophila serogroup 1 by AP-PCR was equivalent to that by monoclonal antibody analysis and ribotyping. Four strains representing the monoclonal antibody pattern most frequently associated with outbreaks all yielded unique amplicon patterns by AP-PCR.     

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.     

The role of arbitrarily primed PCR in identifying the source of an outbreak of Legionnaires' disease.

An outbreak of community-acquired Legionnaires' disease (LD) occurred in Providence, R.I., in fall 1993. To find the outbreak source, exposures of 17 case patients were compared to those of 33 matched controls. Case patients were more likely than controls to have visited a section of downtown (area A) during the 2 weeks before illness (11 [65%] versus 9 [27%]; matched odds ratio, 6.5; P = 0.01). Water samples were cultured from 27 aerosol-producing devices within area A. Legionella pneumophila serogroup 1 isolates underwent monoclonal antibody (MAb) subtyping and arbitrarily primed PCR (AP-PCR). All four L. pneumophila serogroup 1 isolates available from case patients who visited area A had identical MAb and AP-PCR patterns. Among 14 environmental isolates, 5 had MAb patterns that matched the case patient isolates, but only 1 had a matching AP-PCR pattern. This investigation implicates a cooling tower in area A as the outbreak source and illustrates the usefulness of AP-PCR for identifying sources of LD outbreaks.     

Microbiological investigations on a nosocomial case of Legionella pneumophila pneumonia associated with water birth and review of neonatal cases

A case of Legionella pneumophila 1 pneumonia, confirmed by positive serology and urinary antigen, occurred in a 7-day old neonate after water birth in hospital. As respiratory samples were not available for culture, further microbiological investigations were performed in neonate and environment, in order to recognize the source of infection. The hospital water supply was contaminated by L. pneumophila 1 strains (300-2000 cfu/L) of two monoclonal subtypes of Pontiac subgroup. L. spiritensis (10-225 cfu/L) was isolated from cold tap water of the patient's home. PCR from tap and humidifiers water of the patient's home was positive for Legionella spp, but not for L. pneumophila. Because L. pneumophila 1, responsible of child infection, was only isolated from the hospital pool water for waterbirthing, we conclude that the infant acquired the nosocomial legionellosis by prolonged delivery in contaminated water, perhaps by aspiration. Infection control measures for waterbirthing are highly recommended. A review of neonatal case of legionellosis is also presented. As this rare infection may have a high fatality rate if unrecognized, pediatricians should be aware of the possibility of the legionellosis in newborns.