Discriminatory genomic fingerprinting of Legionella pneumophila by pulsed-field electrophoresis.

Eight strains of Legionella pneumophila were used to optimize cleavage of DNA with BssHII, SalI, or SpeI and separation by pulsed-field electrophoresis. Isolates from a community outbreak involving a contaminated hot tub were genomically identical. Cleavage patterns were distinctly different for unrelated environmental and nosocomial strains from a single hospital.     

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 study of Taylorella equigenitalis strains by field inversion gel electrophoresis of genomic restriction endonuclease fragments.

Contagious equine metritis (CEM), a sexually transmitted bacterial disease, was first described in thoroughbred horses. It also occurs in nonthoroughbred horses, in which it produces isolated, apparently unrelated outbreaks. Thirty-two strains of Taylorella equigenitalis, the causative agent of CEM, from all over the world were characterized by field inversion gel electrophoresis of fragments of genomic DNA obtained by digestion with low-cleavage-frequency restriction enzymes. This resulted in a division into five clearly distinct groups. Strains from thoroughbred horses from all continents belonged to one group. Strains from nonthoroughbred horses from various countries were different from strains from thoroughbred horses; four groups could be determined. Two groups contained both streptomycin-resistant and streptomycin-susceptible strains. The data indicate that CEM in nonthoroughbreds did not originate from the thoroughbred population; also, the reverse was not demonstrated. Thus, extensive international transportation directives regarding the testing of nonthoroughbred horses for CEM may need reconsideration.     

Species-specific detection of Legionella pneumophila in water by DNA amplification and hybridization.

A sensitive detection system specific for Legionella pneumophila in water was developed. This system is based on amplification of a chromosomal DNA sequence from L. pneumophila by the polymerase chain reaction, followed by detection of the amplified product by hybridization of a radiolabeled oligodeoxynucleotide. After 35 cycles of amplification, a water sample which had been seeded with 35 CFU of L. pneumophila contained sufficient amplified DNA to be detected on dot blots. Bacteria of other genera tested did not generate positive signals under these conditions. Application of this technique to environmental water samples may help identify the natural reservoirs of nosocomial and community-acquired L. pneumophila infections.     

Evaluation of a real-time PCR hybridization assay for rapid detection of Legionella pneumophila in hospital and environmental water samples

A real-time LightCycler assay for Legionella pneumophila was evaluated with 120 water samples potentially contaminated with PCR inhibitors. Results were obtained within five hours, with a detection limit equivalent to 800 cells/L. However, 11 of 22 culture-positive samples containing < 100 CFU/L were also positive by LightCycler assay, indicating the presence of significant numbers of non-viable cells. Following extraction, amplification inhibitors remained in four culture-positive samples, but only one contained > 800 CFU/L. The assay seemed suitable for rapidly screening large sample numbers for heavy contamination with L. pneumophila , but conventional culture should continue to be used to detect low contamination levels.     

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.     

Immunohistological detection of Legionella pneumophila in lung sections.

Immunohistology was used for the detection of Legionella pneumophila serogroup I in necropsy tissue. Study of pneumonic lung from the recent Stafford outbreak has shown that this technique has a high sensitivity. A retrospective postmortem examination showed that L pneumophila serogroup 1 was an unusual cause of pneumonia in Oxfordshire during the study period. L pneumophila serogroup 1 can be successfully subgrouped, using a panel of monoclonal antibodies on formalin fixed paraffin embedded sections. Immunohistological methods have a potentially useful role in the diagnosis of Legionellosis at postmortem examination and in the epidemiological investigation of individual cases and outbreaks.     

Evaluation of Legionella V-TesT for the detection of Legionella pneumophila antigen in urine samples

We evaluated a new immunochromatographic assay (Legionella V-TesT, Coris BioConcept, Gembloux, Belgium) for its ability to detect Legionella pneumophila serogroup 1 antigen in urine. Test devices were read at various time points to determine the optimum incubation time regarding performance. The results were compared with those obtained with the BinaxNOW urinary antigen test. The sensitivity and specificity were 82.2% and 98.6%, respectively, for the Legionella V-TesT and 83.9% and 100%, respectively, for the BinaxNOW urinary antigen test after 15 min of incubation. When tests were examined after 60 min, the sensitivity for both tests increased to 91.5%.     

An ELISA test for the detection of antibodies to Legionella pneumophila.

An enzyme-linked immunosorbent assay (ELISA) test has been developed to detect antibodies to Legionella pneumophila serogroup 1. There is good correlation between indirect fluorescent antibody (IFA) and ELISA titres but ELISA is more sensitive.     

Epidemiological fingerprinting of Enterobacter cloacae by small-fragment restriction endonuclease analysis and pulsed-field gel electrophoresis of genomic restriction fragments.

A cluster of infections caused by Enterobacter cloacae was observed among preterm neonates in a neonatal intensive care unit (NICU) of a pediatric hospital in Osnabrück, Germany. The presence of similar antimicrobial susceptibility patterns among the bacterial isolates prompted an investigation to determine whether a limited spread of a single strain existed. All 12 E. cloacae isolates from the NICU and 50 nonrelated strains were fingerprinted by small-fragment restriction endonuclease analysis (SF-REA) of EcoRI DNA digests. Selected isolates were further characterized by pulsed-field gel electrophoresis (PFGE) of NotI- or XbaI-generated genomic restriction fragments. Epidemiologically unrelated strains were clearly discriminated by both methods. Results achieved by SF-REA and PFGE revealed that of the 12 isolates from the NICU, 11 belonged to the same genotypic cluster. Since all reagents and equipment for both techniques are commercially available, DNA fingerprinting by SF-REA or PFGE is proposed as a useful tool in the microbiology laboratory for investigating the epidemiological relatedness of E. cloacae strains of clinical and environmental origin.     

Direct immunofluorescent detection of Legionella pneumophila in respiratory specimens.

Respiratory secretions from patients with clinically suspected Legionnaires pneumonia were examined by direc immunofluorescent tests at the Medical Center Hospital of Vermont and at the Center for Disease Control. No fluorescent bacteria were found by either laboratory in eight specimens from eight patients who were seronegative. Twenty specimens were obtained from seven patients who had serologically confirmed Legionnaires disease. Four of the seven cases were identified at the Medical Center Hospital of Vermont, and six of the seven were identified at the Center for Disease Control. Of 20 specimens, 8 were positive at the Center for Disease Control (six or more bacilli per slide), and 7 specimens were suspicious (one to five bacilli per slide); at the Medical Center Hospital of Vermont, 4 of 20 specimens were positive, and 2 were suspicious. The inclusion of a rhodamine-conjugated counterstain at the Center for Disease Control facilitated the examination and may have improved the sensitivity. Smears from transtracheal aspirates, bronchoscopic aspirates, transcutaneous lung aspirates, pleural fluids, and tracheal aspirate-expectorated sputum produced positive results. Several specimens contained fluorescing bacilli when stained for serogroup 2 as well as serogroup 1, perhaps reflecting the presence of cross-reacting antigens in vivo.     

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.     

Legionella pneumophila growth restriction in permissive macrophages cocultured with nonpermissive lipopolysaccharide-activated macrophages.

Macrophages can be activated by lipopolysaccharides (LPS) from gram-negative bacteria to evince a number of biological activities, including increased resistance to intracellular infection by opportunistic bacteria. In the present study, intraperitoneal injection of LPS into A/J mice activated peritoneal macrophages so that they resisted subsequent in vitro infection with Legionella pneumophila. Coculture of these macrophages with those from nontreated A/J mice converted the entire population of cells from permissive to nonpermissive. This effect did not appear to be mediated by soluble factors released from the LPS-treated macrophages, since the levels of interleukins-1 and -6 and tumor necrosis factor alpha produced by the macrophages were not found to be markedly elevated at the time when the macrophages from the LPS-treated mice were most effective in converting normal macrophages to nonpermissiveness. Furthermore, macrophages from mice injected intraperitoneally with either interferon or tumor necrosis factor alpha did not evince nonpermissiveness and also did not have the ability to convert normal spleen cells to nonpermissiveness. Polymyxin B, a known inactivator of LPS activity, did not inhibit the macrophages from the LPS-treated mice from inducing this resistance. It seemed unlikely that free LPS released from the macrophages mediated this effect. The results of this study thus showed that macrophages activated by LPS in vivo can evince nonpermissiveness for Legionella growth in vitro and also can induce macrophages from normal, permissive mice to become nonpermissive for Legionella growth in vitro.     

Kinetic-dependent enzyme-linked immunosorbent assay for detection of antibodies to Legionella pneumophila.

A semiautomated, kinetic-dependent, enzyme-linked immunosorbent assay (K-ELISA) was adapted to detect serum antibodies to Legionella pneumophila. In a comparative study, 158 human serum samples (79 pairs) were tested by K-ELISA and the standard indirect immunofluorescence assay for determination of antibody levels to L. pneumophila serogroup 1. K-ELISA determinations were made by using a serogroup-specific antigen or a preparation (unfractionated antigen) which contained both common antigen and serogroup-specific reactivity. There was good correlation between the immunofluorescence assay and the K-ELISA by using either antigen, although greater correlation was achieved with the unfractionated antigen (coefficients of correlation, 0.894 with unfractionated antigen and 0.841 with serogroup-specific antigen). These results indicate that the K-ELISA is a reliable alternative to the immunofluorescence assay for serologically diagnosing legionellosis.     

Cyclic AMP inhibition of lipopolysaccharide-induced restriction of Legionella pneumophila growth in macrophage cultures.

The mechanism of the effects of lipopolysaccharide (LPS) on macrophages in terms of replication of intracellular facultative bacteria is unclear. It was found in the present study that the anti-Legionella pneumophila activity induced by LPS in macrophages from susceptible A/J mice was reversed in vitro by dibutyryl cyclic AMP (DcAMP). A 24-h pretreatment of murine thioglycolate-elicited macrophages with LPS resulted in an enhanced ability of these cells to inhibit the intracellular growth of L. pneumophila. This anti-L. pneumophila activity of macrophages induced by LPS was inhibited when DcAMP (10(-3) to 10(-5) M) was present during preincubation with LPS. The addition of DcAMP to the cultures was more effective before LPS treatment than after treatment. The effect of DcAMP was dose dependent. The secretion and production of acid phosphatase by LPS-activated macrophages were also inhibited by the addition of DcAMP before LPS treatment. Furthermore, the anti-L. pneumophila activity of macrophages induced by LPS could also be reversed in vitro by treatment with prostaglandin E2, colchicine, isoproterenol, theophylline, or hydrocortisone, all of which are known to increase the intracellular levels of cyclic AMP in various tissues. These observations indicate that the anti-L. pneumophila activity induced by LPS treatment can be modified by mechanisms involving cyclic nucleotide metabolism.     

Molecular typing of Legionella pneumophila serogroup 1 isolates from patients and the nosocomial environment by arbitrarily primed PCR and pulsed-field gel electrophoresis.

The ubiquity of Legionella pneumophila in aquatic habitats means that epidemiological evaluation is important for the investigation and control of nosocomial outbreaks of legionellosis. Pulsed-field gel electrophoresis (PFGE) of chromosomal DNA following digestion with SfiI is considered to be one of the most discriminative methods for detecting DNA polymorphisms amongst L. pneumophila serogroup 1 (Lp1) isolates. This paper describes an arbitrarily primed PCR (AP-PCR) method with three different primers (20-mers) for detecting DNA polymorphisms of Lp1 isolates. The AP-PCR assay was compared with PFGE analysis. Both experimental methods were found to have good discriminatory power (discrimination index of 98% and 94.3%, respectively) with 27 unrelated isolates from different geographical areas collected between 1987 and 1997. Furthermore, when the AP-PCR was used in the epidemiological investigation of nosocomial cases of infection, convergent results with the three primers allowed an epidemiological link to be established between isolates from patients and their environment. The AP-PCR method, which is rapid and easy to perform, gave results at least as discriminatory as those obtained with the PFGE method and is proposed for use in the molecular typing of Lp1 outbreaks.     

Evaluation of commercial amplification kit for detection of Legionella pneumophila in clinical specimens.

A commercial kit (EnviroAmp) designed to detect the DNA of Legionella species in environmental water samples using PCR and reverse dot hybridization was applied to clinical specimens. Results correlated well with culture for bronchoalveolar lavages. In addition, this test was easy to perform and showed good sensitivity.     

New methods for the isolation of Legionella pneumophila.

Some new methods for the isolation of Legionella pneumophila are described which have been successful in recovering this organism from 6/10 patients with clinical evidence of Legionnaires' disease. The increased sensitivity of these methods combined with speedier isolation of the organisms than has hitherto been possible will hopefully lead to eventual isolation of this organism as a routine procedure in diagnostic microbiology laboratories.