Vectorial role of Acanthamoeba in Legionella propagation in water for human use.

Legionella spp. is the causative agent of Legionnaires' disease and is transmitted through aerosols emanating from man-made water systems. Legionella resistance to water treatments has been related to its association with environmental amoebae such as Acanthamoeba. Due to the high presence of this protozoon in Spain and the high rate of notification of Legionnaires' disease of this country, the aims of this work were to study the coexistence of these bacteria and protozoa in water as well as their interaction. The usefulness of Acanthamoeba co-culture for the isolation of environmental Legionella was also studied. For this purpose, 70 water samples were collected in 2011 from three Drinking Water Treatment Plants, three Wastewater Treatment Plants and five Natural Pools in Spain. Acanthamoeba was found by PCR in 87.1% (61/70) samples and, by culture in 85.7% (60/70) samples. Legionella was detected by PCR in 58.6% (41/70) of water samples, in 5.7% (4/70) by agar culture and 75.7% (53/70) by Acanthamoeba co-culture. From the 54 Acanthamoeba water isolates, Legionella was detected in 43 of them independently of Acanthamoeba's genotype (T3, T4 and T11). Legionella feeleii, Legionella birminghamiensis, Legionella gresilensis/berliardensis, Legionella fairfieldensis, Legionella drozanski and Legionella falloni were identified. In conclusion, our results showed that environmental Acanthamoeba is infected by Legionella to a high percentage, and due to its ubiquity, high resistance and its pathogenic potential per se, new methods for its elimination should be studied. Also, the high effectivity of Acanthamoeba co-culture for Legionella detection has been shown.

Analysis of Bordetella pertussis clinical isolates circulating in European countries during the period 1998-2012.

Despite more than 50 years of vaccination, pertussis is still an endemic disease, with regular epidemic outbreaks. With the exception of Poland, European countries have replaced whole-cell vaccines (WCVs) by acellular vaccines (ACVs) in the 1990s. Worldwide, antigenic divergence in vaccine antigens has been found between vaccine strains and circulating strains. In this work, 466 Bordetella pertussis isolates collected in the period 1998-2012 from 13 European countries were characterised by multi-locus antigen sequence typing (MAST) of the pertussis toxin promoter (ptxP) and of the genes coding for proteins used in the ACVs: pertussis toxin (Ptx), pertactin (Prn), type 2 fimbriae (Fim2) and type 3 fimbriae (Fim3). Isolates were further characterised by fimbrial serotyping, multi-locus variable-number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE). The results showed a very similar B. pertussis population for 12 countries using ACVs, while Poland, which uses a WCV, was quite distinct, suggesting that ACVs and WCVs select for different B. pertussis populations. This study forms a baseline for future studies on the effect of vaccination programmes on B. pertussis populations.

Persistence of chlorine-sensitive Legionella pneumophila in hyperchlorinated installations.

AIMS: To study the persistence of Legionella over time in different disinfected facilities and analysing whether failures in bacterial eradication could be the result of a decrease in the susceptibility of the persistent strains to subsequent treatments. METHODS AND RESULTS: A long-term environmental surveillance was carried out in three different facilities associated with cases of Legionnaires' disease (a hospital, a fishing boat and a hotel). Despite several hyperchlorination episodes, amplified fragment length polymorphism, pulsed field gel electrophoresis-SfiI and arbitrarily primed polymerase chain reaction methods demonstrated that a specific clone of L. pneumophila serogroup 1 was able to survive for 17, 5 and 10 years in the hospital, fishing boat and the hotel, respectively. Isolates from different years from the same facility showed similar minimal inhibitory concentration and minimal bactericidal concentration values against eight different disinfectants. CONCLUSIONS: Hyperchlorination over long periods of time did not prevent the persistence of L. pneumophila. The lack of effectiveness did not appear to be the result of a decreased susceptibility of Legionella to chlorine. Hyperchlorination did not modify in vitro susceptibility of Legionella to other disinfectants to which the bacteria had not previously been exposed. SIGNIFICANCE AND IMPACT OF THE STUDY: Persistent Legionella in treated installations remain sensitive to disinfectants; hence, new strategies of treatment, different from hyperchlorination, should be developed to achieve bacterial eradication.

Effectiveness of disinfectants used in cooling towers against Legionella pneumophila.

BACKGROUND: Legionella persists in man-made aquatic installations despite preventive treatments. More information about disinfectants could improve the effectiveness of treatments. This study tests the susceptibility of Legionella pneumophila serogroup (sg) 1 against 8 disinfectants used in cooling tower treatments. METHODS: We determined the minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and bactericidal effect of sodium hypochlorite (A), hydrogen peroxide with silver nitrate (B), didecyldimethylammonium chloride (C), benzalkonium chloride (D), tributyltetradecylphosphonium chloride (E), tetrahydroxymethylphosphonium sulfide (F), 2,2-dibromonitropropionamide (G) and chloromethylisothiazolone (H) against 28 L. pneumophila sg 1 isolates. RESULTS: MIC and MBC values were equivalent. Bacteria are less susceptible to disinfectants F, B, D and A than to H, E, C and G. All disinfectants induced a bactericidal effect. The effect rate is dose dependent for G, H, F and B; the effect is fast for the rest of disinfectants at any concentration. The bactericidal activity of disinfectants A, G and F depends on the susceptibility test used. CONCLUSIONS: All disinfectants have bactericidal activity against L. pneumophila sg 1 at concentrations used in cooling tower treatments. Results depend on the assay for some products.

A large Legionnaires' disease outbreak in Pamplona, Spain: early detection, rapid control and no case fatality.

An outbreak of Legionnaire's disease was detected in Pamplona, Spain, on 1 June 2006. Patients with pneumonia were tested to detect Legionella pneumophila antigen in urine (Binax Now; Binax Inc., Scarborough, ME, USA), and all 146 confirmed cases were interviewed. The outbreak was related to district 2 (22 012 inhabitants), where 45% of the cases lived and 50% had visited; 5% lived in neighbouring districts. The highest incidence was found in the resident population of district 2 (3/1000 inhabitants), section 2 (14/1000). All 31 cooling towers of district 2 were analysed. L. pneumophila antigen (Binax Now) was detected in four towers, which were closed on 2 June. Only the strain isolated in a tower situated in section 2 of district 2 matched all five clinical isolates, as assessed by mAb and two genotyping methods, AFLP and PFGE. Eight days after closing the towers, new cases ceased appearing. Early detection and rapid coordinated medical and environmental actions permitted immediate control of the outbreak and probably contributed to the null case fatality.

Identification and serotyping of atypical Legionella pneumophila strains isolated from human and environmental sources.

AIMS: To validate identification methods for Legionella pneumophila strains that cannot be serotyped into the known serogroups and to characterize their antigenic diversity. METHODS AND RESULTS: Fifty L. pneumophila strains that could not be serogrouped, but which had been confirmed as L. pneumophila by mip gene sequencing, were further identified phenotypically. We used (i) MONOFLUO anti-Legionella Staining Reagent (Bio-Rad) (50/50), (ii) an in-house prepared immunoblot assay for the detection of L. pneumophila- specific Mip protein epitope (50/50), (iii) fatty acid analysis using the Microbial Identifications System (MIDI) (47/50) and (iv) Oxoid agglutination tests (44/50). The serological diversity was further characterized by testing with five serogroup-cross-reactive monoclonal antibodies, resulting in nine phenons. CONCLUSIONS: The division of L. pneumophila into 15 serogroups does not reflect the serogroup heterogeneity. Results of these tests indicate that there are more serogroups. SIGNIFICANCE AND IMPACT OF THE STUDY: MONOFLUO anti-Legionella Staining Reagent is the only commercially available tool for identifying atypical strains of L. pneumophila. If necessary for epidemiological purposes, the antigenic heterogeneity of these strains can be analysed by monoclonal antibodies.

Legionnaires' disease clusters associated with travel to Spain during the period January 2001 to July 2003.

Epidemiological surveillance and control of travel associated cases of legionnaires' disease are necessary tasks for public health and collaboration between countries is necessary to do this. Within the framework of the European Surveillance Scheme for Travel Associated Legionnaires' Disease (EWGLINET), European Guidelines for Control and Prevention of Travel Associated Legionnaires' Disease have been produced . This has established the reporting and response criteria when cases or clusters appear. In this paper the analysis of the information corresponding to the 46 reported clusters related to Spain is presented. Data corresponds to the period January 2001 to July 2003.

Pan-European study on culture-proven Legionnaires' disease: distribution of Legionella pneumophila serogroups and monoclonal subgroups.

This pan-European study included unrelated strains of Legionella pneumophila obtained from 1335 cases of Legionnaires' disease. The isolates were serotyped into the serogroups 1 to 15 by monoclonal antibodies (MAb) and/or rabbit antisera. Additionally, MAb subgrouping was undertaken for isolates belonging to serogroups 1, 4, and 5. Monoclonal types of serogroup 1 were subdivided as having, or not having, the virulence-associated epitope recognized by the MAb 3/1 (Dresden Panel). This epitope is not present on strains belonging to any other serogroups. Taking all Legionella incidents together, MAb 3/1-positive cases were most frequent (66.8%); 11.7% of the isolates belonged to MAb 3/1-negative serogroup 1 subgroups and 21.5% to other serogroups, with serogroups 3 and 6 predominating. Among all serotypes discriminated in this study, monoclonal subtype Philadelphia was the most frequent. If categories of infection were considered, the proportion of MAb 3/1-negative strains differed significantly ( P<0.0005) between community-acquired cases (139/510; 27.3%) and travel-associated (42/295; 14.2%) or hospital-acquired infections (176/329; 53.5%). Moreover, taking distribution in different European areas into account, the proportion of MAb 3/1-negative strains was significantly higher in the Scandinavian region than in the Mediterranean countries or the UK for both community-acquired (48.7% vs. 18.6% or 12.0%; P<0.0005) and nosocomial cases (87.7% vs. 32.6% or 52.6%; P

An outbreak of Legionnaires' disease in an inner city district: importance of the first 24 hours in the investigation.

OBJECTIVE: To present the main results of the investigation of an outbreak of Legionnaire's disease that occurred in an inner city district of Barcelona between 15 October and 15 November 2000. METHODS: Epidemiological surveys of patients and environmental investigations were initiated on the day the first five cases were notified. Water samples and smears from cooling tower trays were taken for microbiological analysis. Maps of the distribution of cases and possible contamination foci were elaborated. Incidences were calculated for each census tract. RESULTS: A total of 54 patients related to the outbreak were identified, with a case fatality rate of 5.5%. Incidence rate in the area closest to the cooling tower (6.40/1000) was significantly higher than that of the rest of the neighbourhood (2.23/1.000, RR 2.87, 95%CI 1.37-6.12, P = 0.0035). Cultures positive for Legionella pneumophila serogroup 1, subtypes Pontiac, Philadelphia or Allentown, were obtained from eight patients. On the 39th day of the investigation it was found that the strain isolated in one of the cooling towers coincided with the serogroup, subtype and molecular profiles identified in clinical samples. CONCLUSIONS: Rapid coordination of clinicians, microbiologists, epidemiologists and environmentalists permitted the source of infection and the affected cases to be correlated within a few days.

[The role of the clinical microbiology laboratory during the outbreak of Legionella spp. in the municipality of Alcoy: the effectiveness of the different diagnosis methods]. / El laboratorio de microbiología clínica en el brote de Legionella spp. en la comarca de Alcoy: rentabilidad de las diferentes técnicas diagnósticas.

BACKGROUND: The effectiveness of the different laboratory test methods to diagnose Legionella spp. in clinical specimens varies according to the epidemiological context. In this study, the usefulness of the laboratory methods used for an outbreak that occurred in the municipality of Alcoy (Alicante, Spain) are evaluated. MATERIALS AND METHODS: 222 community-acquired cases of infection caused by Legionella pneumophila serogroup 1, subtype Pontiac-Knoxville, genotypes I and II were studied, that had been diagnosed by the Microbiology laboratory from January 1999 to December 2000, corresponding to patients residing in the municipality of Alcoy (Alicante). The methods used were direct antigen detection in respiratory specimens by immunoflurescence, direct antigen detection in urine, cultures and serology. RESULTS: The detection of the antigen in urine diagnosed 201 cases (90,5%). Direct immunofluorescence provided a high number of false positives (n=24). A culture was essential to confirm the etiology of the outbreak (25 sputum) strains from 22 patients). Serology complemented the other methods and helped to retrospectively diagnose 21 patients (9%) when the other tests were not carried out or when they provided negative results. CONCLUSIONS: A rapid diagnosis is essential to evaluate the patients and to control epidemical outbreaks, and the detection of the urinary antigen is very useful, but should be complemented with other methods. The culture of the respiratory specimens and the subsequent typing of the strains means that the etiology can be established with certainty and this helps to determine the source(s) of the infection. Serology complemented the diagnosis in 9% of the cases.

A small outbreak of Legionnaires' disease in a cargo ship under repair.

It was reported that two mechanics working on a cargo ship under repair in the port of Barcelona had died after having fever. An investigation was made into the possibility of any additional cases and the presence of Legionella pneumophila in the ship they were repairing and in their hotel. The contaminated water system was treated with sodium hypochlorite. Both patients died after having been repeatedly diagnosed as having influenza. The two cases occurred among those who had been working with the pump of the ship's water system, while no cases were observed among the other workers (p = 0.02). Various serogroups of L. pneumophila were isolated from the ship's water pump and distribution system. However, organism of serogroup 1, subgroup Pontiac (Knoxville) were identified with identical deoxyribonucleic acid (DNA) patterns in the lung tissue of one patient and in the cooling water circuit valve of the ship's water pump. The first postintervention control water samples showed no further growth of legionella, but serogroups 4 and 8 were identified 8 months later. This legionellosis outbreak, although small, was highly lethal, probably due to the high levels of bacteria to which the patients were exposed and also because of the failure of correct diagnosis. International recommendations on prevention and control of legionellosis, which include ships under repair, are required.

Assessment of intercentre reproducibility and epidemiological concordance of Legionella pneumophila serogroup 1 genotyping by amplified fragment length polymorphism analysis.

The aims of this work were to assess (i) the intercentre reproducibility and epidemiological concordance of amplified fragment length polymorphism analysis for epidemiological typing of Legionella pneumophila serogroup 1, and (ii) the suitability of the method for standardisation and implementation by members of the European Working Group on Legionella Infections. Fifty coded isolates comprising two panels of well-characterised strains, a "reproducibility" panel (n=20) and an "epidemiologically related" panel (n=30), were sent to 13 centres in 12 European countries. Analysis was undertaken in each centre following a previously determined standard protocol. Results were analysed by the participants, using gel analysis software where available, and submitted to the coordinating centre. The coordinating centre reanalysed all results visually and selected data-sets with gel analysis software. Data analysis by participants yielded reproducibility (R) values of 0.20-1.00 and epidemiological concordance (E) values of 0.11-1.00, with 6 to 34 types. Following visual analysis by the coordinating centre, R=0.78-1.00, and E=0.67-1.00, with 10-20 types. Analysis of three data-sets by the coordinating centre using gel analysis software yielded R=1.00 and E=1.00, with 12, 13 or 14 types. This method can be used as a simple, rapid screening tool for epidemiological typing of isolates of Legionella pneumophila serogroup 1. Results demonstrate that the method can be highly reproducible (R=1.00) and epidemiologically concordant (E=1.00), with good discrimination. The electropherograms generated are amenable to computer-aided analysis, but strict adherence to a previously defined laboratory protocol is required. Following designation of representative type strains and patterns, this method will be adopted by the European Working Group on Legionella Infections as the first internationally standardised typing method for use in the investigation of travel-associated Legionella infections.

In vitro activities of gemifloxacin versus five quinolones and two macrolides against 271 Spanish isolates of Legionella pneumophila: influence of charcoal on susceptibility test results.

The MICs at which 90% of isolates are inhibited for gemifloxacin, trovafloxacin, and grepafloxacin were low (/=6 dilutions) when they were determined by the agar dilution method. This was due to the charcoal in the agar dilution medium, as shown by the progressive decrease in the MICs when the charcoal concentrations decreased. As free drug is the active fraction, charcoal binding should be considered.

Nosocomial Legionnaire's disease in a children's hospital.

BACKGROUND: Only a few cases of nosocomial Legionella sp. infection have been reported in children. We report the clinical and epidemiologic data of five nosocomial legionellosis cases that occurred in the Pediatric Nephrology Service between August, 1994, and December, 1998, and the control measures adopted. METHODS: The Hospital Materno-Infantil Vall d'Hebron, Barcelona, is a 407-bed tertiary care hospital. The pediatric kidney transplant unit has three isolated beds in the same ward within the Pediatric Nephrology Service. Diagnostic workup to establish Legionella pneumophila infection included culture, fluorescent antibody and serologic studies. Macrorestriction analysis of genomic DNA was used as epidemiologic markers of the isolated strains. RESULTS: In May, 1996, a case of L. pneumophila serogroup 6 pneumonia was identified in a 19-year-old youth who had received a kidney transplant 16 days earlier. Retrospective and prospective analysis of legionellosis cases diagnosed at our center up to August, 1994, yielded four additional cases. Four patients had had a kidney transplant and were receiving immunosuppressive therapy, and the fifth had been diagnosed with systemic lupus erythematosus with renal involvement. L. pneumophila serogroup 6 was isolated in bronchial secretions in four cases; in the fifth patient the diagnosis was made by serology. L. pneumophila serogroup 6 was isolated from potable water of the hospital. Molecular epidemiologic methods revealed the identity of the environmental and clinical isolates. Showering was implicated as the most feasible means of exposure to contaminated water. CONCLUSIONS: Nosocomial legionellosis, albeit rare in children, should be considered in the differential diagnosis of pneumonias, particularly in immunosuppressed children, because the fatality rate may be high without early diagnosis and treatment.

Detection of specific IgM antibody in the investigation of an outbreak of pneumonia due to Legionella pneumophila serogroup 1.

OBJECTIVE: Management of outbreaks of pneumonia due to Legionella pneumophila serogroup 1 (SG1) infection requires rapid and accurate diagnostic tests. Current serologic approaches, based on detection of seroconversion for total antibody, do not fulfil this requirement. METHODS: A diagnostic test based on detection of IgM antibody to L. pneumophila SG1 by indirect immunofluorescence was developed and used to evaluate serum samples from patients involved in a community outbreak of L. pneumophila SG1 pneumonia that occurred in Spain. RESULTS: Testing of samples from serologically proven, sporadic cases of pneumonia due to L. pneumophila SG1 (14), cases of atypical pneumonia due to other infectious agents (16) and healthy controls (100) supported the sensitivity and specificity of the assay. On samples from the outbreak, the IgM assay recognized five of six cases with isolation of L. pneumophila SG1 from respiratory secretions or lung tissue and more than 70% of cases with confirmed or presumptive diagnosis as determined by the current serologic criteria. In addition, the IgM assay was positive in 23-70% of patients who fulfilled the clinical and epidemiologic criteria of case definition but did not display diagnostically significant serologic results or who lacked a detectable antibody response in the routine assay. Among cases confirmed by the current criteria, detection of specific IgM was occasionally achieved before the conventional serology gave significant results. CONCLUSION: Incorporation of IgM antibody detection in the current diagnostic criteria for L. pneumophila SG1 infection may help to improve the management of outbreaks of pneumonia due to this agent.

Treatment of a Legionella pneumophila-colonized water distribution system using copper-silver ionization and continuous chlorination.

The detection in April 1997 of a case of nosocomial legionellosis in our hospital led to the discovery that both our hot- and cold-water circuits were heavily colonized with Legionella pneumophila. Conventional methods for eradication of the organisms were unsuccessful, so a copper-silver (Cu-Ag) ionization system and a continuous chlorination system were installed. Five months later, the number of colonized sites decreased from an initial 58.3% to 16.7%.

Antigenic lipopolysaccharide components of Legionella pneumophila recognized by monoclonal antibodies: possibilities and limitations for division of the species into serogroups.

Legionella pneumophila accounts for the majority of cases of Legionnaires' disease. By using rabbit antisera, the species has been divided into 14 numbered and 1 unnumbered serogroups. To recognize the antigenic diversity of the lipopolysaccharide (LPS) responsible for this classification, the Dresden Legionella LPS MAb panel, containing 98 monoclonal antibodies (MAbs), was created. Each serogroup reference strain possesses at least one specific epitope not found on any other reference strain and therefore designated the serogroup-specific epitope. When the appropriate MAbs were used for serotyping of 1,064 human and environmental isolates, 1,045 (98%) could be placed into the known serogroups. In most cases (97%), this was in agreement with the polyclonal typing. Of the 29 isolates that showed strong cross-reactivities with the rabbit antiserum panel, 11 could be typed easily by MAbs; for the remaining 18, however, only serogroup-cross-reactive epitopes could be determined. Below the serogroup level, monoclonal subtypes were found for 11 serogroups. Altogether, the Dresden Legionella LPS MAb panel was able to divide the 1,064 isolates tested into 64 phenons, indicating its usefulness for both serogrouping and subgrouping of L. pneumophila strains. In order to compare the identities of patient and environmental isolates, testing their reactivity with MAbs should be the first step, especially if large numbers of colonies are to be typed. Only in cases of identical patterns are the more time consuming and expensive genetic fingerprints necessary. Moreover, the MAbs can also be used for specific antigen detection in respiratory specimens on the serogroup or subgroup level.

Outbreak of legionnaire s disease in two groups of tourists staying at camp sites in France.

On 11 June 1996, three suspected cases of legionnaires disease in a group of 42 Dutch tourists were reported to the local public health authority by Millau hospital in south west France. The group (group 1) had been touring with caravans and staying at d

An international investigation of an outbreak of Legionnaires disease among UK and French tourists.

Five cases of legionnaires disease and one death were associated with four members of a tour group from the United Kingdom (UK) and one French tourist who all visited Spain in the spring of 1993. The UK group stayed at four hotels, one of which was also used by the French tourist. Phenotypic and genotypic comparison of isolates of Legionella pneumophila obtained from one of the UK cases and the French patient demonstrated that they were indistinguishable from each other and from environmental isolates obtained from the water supply of the hotel at which all five cases had stayed. A cohort study of the UK tour group was carried out to determine the extent of the outbreak and showed that three further members of the group had respiratory illness but were serologically negative to legionella infection. International participation in this investigation has highlighted the value of a European surveillance scheme and the benefit of microbiological collaboration between legionella reference laboratories in Europe.

[Characterization of clinical and environmental isolates of Legionella associated with outbreaks and study of the infection sources]. / Caracterización de aislados clínicos y ambientales de Legionella asociados a brotes y estudio de las fuentes de infección.

BACKGROUND: The study of isolates of Legionella related with six outbreaks of community-acquired legionellosis and seven nosocomial outbreaks is presented. METHODS: The isolates were serogrouped by indirect immunofluorescence testing and those belonging to serogroup (SG) 1 L. pneumophila strains were subtyped using the international panel of monoclonal antibodies. RESULTS: SG 1 L. pneumophila appeared to be the etiologic agent of all the community outbreaks analyzed and the main one of the nosocomial cases. However, in the latter situation, cases produced by SG3, SG4, SG6, SG8, SG8, 10 y SG4,8,10 were also found. Among subgroup 1 Legionella, the Pontiac, Philadelphia 1 or Allentown 1 subtypes were responsible for four out of the six community and three of the seven nosocomial outbreaks. The Pontiac, Knoxville 1 subtype was the cause of a community-outbreak and of another nosocomial outbreak, and the Pontiac, Benidorm 030E caused a community and another hospital outbreak, also being the main agent found in another hospital. The remaining subtypes appeared in small numbers in some hospitals, where cases due to different isolates coexisted. Likewise, nosocomial cases produced by SGs other than SG1 (Sg 4, 6 and 8) were found in one hospital (with SG 8 also being found in environmental samples) and Sg 4, 8, 10 together with Sg 3 in another (both SGs were also found in different hospital facilities). The sources of infection found were air conditioning systems in two cases and the lavatory water system in the remaining cases, with no colonization being found in a series of natural sources (spring water, wells, water tanks, watering systems) also studied in several of the outbreaks. CONCLUSIONS: These results with suggest that although building installations are colonized by a large variety of species, SGs and subtypes of Legionella, only some (mainly SG 1 L. pneumophila, Pontiac subtype) actually produces outbreaks. This is specially relevant in community outbreaks, while in hospital outbreaks other species or SGs may produce infection in isolated cases or outbreaks due to the presence of highly susceptible hosts.