Ellen W. Brown

Role of biofilms in the survival of Legionella pneumophila in a model potable-water system.

Legionellae can infect and multiply intracellularly in both human phagocytic cells and protozoa. Growth of legionellae in the absence of protozoa has been documented only on complex laboratory media. The hypothesis upon which this study was based was that biofilm matrices, known to provide a habitat and a gradient of nutrients, might allow the survival and multiplication of legionellae outside a host cell. This study determined whether Legionella pneumophila can colonize and grow in biofilms with and without an association with Hartmannella vermiformis. The laboratory model used a rotating disc reactor at a retention time of 6.7 h to grow biofilms on stainless steel coupons. The biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae and a Flavobacterium sp. The levels of L. pneumophila cells present in the biofilm were monitored for 15 d, with and without the presence of H. vermiformis, and it was found that, although unable to replicate in the absence of H. vermiformis, L. pneumophila was able to persist.

Hospital characteristics associated with colonization of water systems by Legionella and risk of nosocomial legionnaires' disease : A cohort study of 15 hospitals

OBJECTIVEnTo investigate an increase in reports of legionnaires disease by multiple hospitals in San Antonio, Texas, and to study risk factors for nosocomial transmission of legionnaires disease and determinants for Legionella colonization of hospital hot-water systems.nnnSETTINGnThe 16 largest hospitals in the cities of San Antonio, Temple, and Austin, Texas.nnnDESIGNnReview of laboratory databases to identify patients with legionnaires disease in the 3 years prior to the investigation and to determine the number of diagnostic tests for Legionella performed; measurement of hot-water temperature and chlorine concentration and culture of potable water for Legionella. Exact univariate calculations, Poisson regression, and linear regression were used to determine factors associated with water-system colonization and transmission of Legionella.nnnRESULTSnTwelve cases of nosocomial legionnaires disease were identified; eight of these occurred in 1996. The rise in cases occurred shortly after physicians started requesting Legionella urinary antigen tests. Hospitals that frequently used Legionella urinary antigen tests tended to detect more cases of legionnaires disease. Legionella was isolated from the water systems of 11 of 12 hospitals in San Antonio; the 12th had just experienced an outbreak of legionnaires disease and had implemented control measures. Nosocomial legionellosis cases probably occurred in 5 hospitals. The number of nosocomial legionnaires disease cases in each hospital correlated better with the proportion of water-system sites that tested positive for Legionella (P=.07) than with the concentration of Legionella bacteria in water samples (P=.23). Hospitals in municipalities where the water treatment plant used monochloramine as a residual disinfectant (n=4) and the hospital that had implemented control measures were Legionella-free. The hot-water systems of all other hospitals (n=11) were colonized with Legionella. These were all supplied with municipal drinking water that contained free chlorine as a residual disinfectant. In these contaminated hospitals, the proportion of sites testing positive was inversely correlated with free residual chlorine concentration (P=.01). In all hospitals, hot-water temperatures were too low to inhibit Legionella growth.nnnCONCLUSIONSnThe increase in reporting of nosocomial legionnaires disease was attributable to increased use of urinary antigen tests; prior cases may have gone unrecognized. Risk of legionnaires disease in hospital patients was better predicted by the proportion of water-system sites testing positive for Legionella than by the measured concentration of Legionella bacteria. Use of monochloramine by municipalities for residual drinking water disinfection may help prevent legionnaires disease.

More than 10 years of unrecognized nosocomial transmission of legionnaires' disease among transplant patients.

OBJECTIVEnTo investigate a cluster of cases of legionnaires disease among patients at a hospital.nnnSETTINGnA university hospital that is a regional transplant center.nnnDESIGNnRetrospective review of microbiology and serology data from the hospital laboratories and prospective surveillance via the radiology department; a case-control study and environmental sampling within the hospital and from nearby cooling towers.nnnRESULTSnDiagnosis of seven cases of legionnaires disease in the first 9 months of 1996 led to recognition of a nosocomial outbreak that may have begun as early as 1979. Review of charts from 1987 through September 1996 identified 25 culture-confirmed cases of nosocomial or possibly nosocomial legionnaires disease, including 18 in bone marrow and heart transplant patients. Twelve patients (48%) died. During the first 9 months of 1996, the attack rate was 6% among cardiac and bone marrow transplant patients. For cases that occurred before 1996, intubation was associated with increased risk for disease. High-dose corticosteroid medication was strongly associated with the risk for disease, but other immunosuppressive therapy or cancer chemotherapy was not. Several species and serogroups of Legionella were isolated from numerous sites in the hospitals potable water system. Six of seven available clinical isolates were identical and were indistinguishable from environmental isolates by pulsed-field gel electrophoresis. Initial infection control measures failed to interrupt nosocomial acquisition of infection. After extensive modifications to the water system, closely monitored repeated hyperchlorinations, and reduction of patient exposures to aerosols, transmission was interrupted. No cases have been identified since September 1996.nnnCONCLUSIONSnLegionella can colonize hospital potable water systems for long periods of time, resulting in an ongoing risk for patients, especially those who are immunocompromised. In this hospital, nosocomial transmission possibly occurred for more than 17 years and was interrupted in 1996, after a sudden increase in incidence led to its recognition. Hospitals specializing in the care of immunocompromised patients (eg, transplant centers) should prioritize surveillance for cases of legionnaires disease. Aggressive control measures can interrupt transmission of this disease successfully.

Legionella pneumophila associated with the protozoan Hartmannella vermiformis in a model multi-species biofilm has reduced susceptibility to disinfectants

Legionella pneumophila will infect biofilm-associated protozoa, and in this way might be protected from disinfectants in potable water systems. A base biofilm containing Pseudomonas aeruginosa, Klebsiella pneumoniae, and Flavobacterium spp. was grown on steel coupons in potable water prior to the addition of L. pneumophila and the protozoan H. vermiformis. After 7 d, coupons were removed and treated with 0.5u2009mgu2009l−1 free residual chlorine (FRC) or 0.5u2009mgu2009l−1 monochloramine (MCA) for 15, 60, or 180u2009min or 24u2009h. In a second experiment, only L. pneumophila and the base biofilm organisms were present but with an identical treatment protocol. Treatment of L. pneumophila for 180u2009min in a system without H. vermiformis resulted in log reductions of 2.07 and 2.11 for FRC and MCA, respectively. When H. vermiformis was present, however, the treatment resulted in log reductions of 0.67 and 0.81 for FRC and MCA, respectively. A similar pattern was observed for 15 and 60u2009min contact times. These results indicate that L. pneumophila was less susceptible to MCA or FRC when associated with biofilm-associated H. vermiformis in a model potable water biofilm.

Distribution of lag-1 Alleles and Sequence-Based Types among Legionella pneumophila Serogroup 1 Clinical and Environmental Isolates in the United States

ABSTRACT Approximately 84% of legionellosis cases are due to Legionella pneumophila serogroup 1. Moreover, a majority of L. pneumophila serogroup 1 clinical isolates react positively with monoclonal antibody 2 (MAb2) of the international standard panel. Over 94% of the legionellosis outbreaks investigated by the Centers for Disease Control and Prevention are due to this subset of L. pneumophila serogroup 1. To date, there is no complete explanation for the enhanced ability of these strains to cause disease. To better characterize these organisms, we subtyped 100 clinical L. pneumophila serogroup 1 isolates and 50 environmental L. pneumophila serogroup 1 isolates from the United States by (i) reactivity with MAb2, (ii) presence of a lag-1 gene required for the MAb2 epitope, and (iii) sequence-based typing analysis. Our results showed that the MAb2 epitope and lag-1 gene are overrepresented in clinical L. pneumophila serogroup 1 isolates. MAb2 recognized 75% of clinical isolates but only 6% of environmental isolates. Similarly, 75% of clinical isolates but only 8% of environmental isolates harbored lag-1. We identified three distinct lag-1 alleles, referred to as Philadelphia, Arizona, and Lens alleles, among 79 isolates carrying this gene. The Arizona allele is described for the first time in this study. We identified 59 different sequence types (STs), and 34 STs (58%) were unique to the United States. Our results support the hypothesis that a select group of STs may have an enhanced ability to cause legionellosis. Combining sequence typing and lag-1 analysis shows that STs tend to associate with a single lag-1 allele type, suggesting a hierarchy of virulence genotypes. Further analysis of ST and lag-1 profiles may identify genotypes of L. pneumophila serogroup 1 that warrant immediate intervention.

Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region

n Abstractn n Although the majority of cases of Legionnaires’ disease (LD) are caused by Legionella pneumophila, an increasing number of other Legionella species have been reported to cause human disease. There are no clinical presentations unique to LD and hence accurate laboratory tests are required for early diagnosis. Therefore, we designed a real-time PCR assay that targets the 23S-5S rRNA intergenic spacer region (23S-5S PCR) and allows for detection of all Legionella species and discrimination of L. pneumophila from other Legionella species. In total, 271 isolates representing 50 Legionella species were tested and the assay was validated using 39 culture-positive and 110 culture-negative patient specimens collected between 1989 and 2006. PCR-positive results were obtained with all 39 culture-positive samples (100% sensitivity). Specimens that tested positive according to 23S-5S PCR, but were culture-negative, were further analysed by DNA sequencing of the amplicon or the macrophage infectivity potentiator (mip) gene. In addition to L. pneumophila, Legionella longbeachae, Legionella cincinnatiensis and Legionella micdadei were identified in the specimens. The assay showed a 7-log dynamic range displaying a sensitivity of 7.5 CFU/mL or three genome equivalents per reaction. Sixty-one specimens containing viruses or bacteria other than Legionellae were negative according to 23S-5S PCR, demonstrating its specificity. Use of this assay should contribute to the earlier detection of respiratory disease caused by Legionella species, as well as to increased rates of detection.n n

Epidemiologic Investigation of a Restaurant-Associated Outbreak of Pontiac Fever

This case-control study investigated a cluster of respiratory illness among patrons of a restaurant. Of 173 patrons interviewed, 117 (68%) were ill. Symptoms included myalgias (93%), headache (87%), and fatigue (79%). The mean incubation period was 49 h and the mean duration of illness was 71 h. Patrons aged >15 years were more likely to have been ill than younger patrons (odds ratio [OR], 2.96; P=.002); 58% of persons who were ill sat near a large fountain, compared with 18% of respondents who were not ill (OR, 7.5; P=.005). Legionella anisa was cultured from water samples obtained from the fountain pool. Of 22 individuals who were ill, 11 (50%) had a > or =4-fold increase in the titer of antibody to that strain of L. anisa from acute-phase to convalescent-phase serum samples; 3 others (14%) had persistently elevated titers of > or =512; of a group of 20 individuals who had not been exposed to the restaurant, none had titers of >128. Pontiac fever should be considered as a diagnosis during acute outbreaks of influenza-like illness with a high attack rate and no other identified etiology.

Survey of Legionella Species Found in Thai Soil

Members of the Gram-negative genus Legionella are typically found in freshwater environments, with the exception of L. longbeachae, which is present in composts and potting mixes. When contaminated aerosols are inhaled, legionellosis may result, typically as either the more serious pneumonia Legionnaires disease or the less severe flu-like illness Pontiac fever. It is presumed that all species of the genus Legionella are capable of causing disease in humans. As a followup to a prior clinical study of legionellosis in rural Thailand, indigenous soil samples were collected proximal to cases homes and workplaces and tested for the presence of legionellae by culture. We obtained 115 isolates from 22/39 soil samples and used sequence-based methods to identify 12 known species of Legionella represented by 87 isolates.

Recurrence of Legionnaires Disease at a Hotel in the United States Virgin Islands over a 20-Year Period

We investigated 3 cases of legionnaires disease (LD) that developed in travelers who stayed at a hotel in the United States Virgin Islands where cases of LD occurred in 1981-1982 and in 1998. The temperature of the potable water at the hotel was in a range that could optimally support the growth of Legionella species, and the potable water was colonized with Legionella pneumophila in 1981-1982 and in 2002-2003.

Legionnaires’ Disease Outbreak in an Automobile Engine Manufacturing Plant

We investigated 4 cases of legionnaires disease (LD) reported among workers at an Ohio automotive plant in March 2001. A confirmed case of LD was defined as x-ray-confirmed pneumonia and a confirmatory laboratory test. A possible case of LD was defined as elevated titers of antibody and respiratory symptoms. Legionella pneumophila serogroup 1 (LP1) was isolated from 1 case patient. Legionella was isolated from 18 (9%) of 197 environmental samples; 3 isolates were LP1 but did not match the case isolate. We conducted a case-control study; 17 case patients with confirmed or possible LD and 86 control subjects (workers with low antibody titers and without symptoms) were enrolled. Visiting a specific cleaning line (odds ratio, [OR], 7.29; 95% confidence interval [CI], 2.31-23.00) and working in the cleaning region of the plant (OR, 3.22; 95% CI, 1.11-9.38) were associated with LD. LD can be transmitted in industrial settings in which aerosols are produced. Clinicians should consider LD when treating persons from these settings for pneumonia.