Legionellosis Outbreak Associated with Asphalt Paving Machine, Spain, 2009

TOC Summary: The source was untreated spring water in the machine’s water tank.

At the end of July 2009, the epidemiology surveillance system detected 2 cases of legionellosis in persons who had stayed in Alcoi, Spain, during their incubation periods. New cases appeared during the fi rst week of August, at which time an epidemic outbreak was declared and an epidemiologic investigation was started. Patients in the outbreak were questioned about clinical and personal aspects. Spatial-temporal analysis was used to identify the most likely areas of exposure for infection. L. pneumophila was isolated from environmental samples obtained in those areas. Because the usual facilities and municipal water systems associated with risk were not contaminated, other facilities not previously linked to legionellosis outbreaks were considered. To verify the common genetic origin of the outbreak and its environmental source, we performed an epidemiologic molecular analysis using sequence-based typing for clinical and environmental samples.

Legionellosis Cases
Cases of pneumonia reported in Alcoi during the incubation period were considered suspected cases of legionellosis unless this diagnosis could be ruled out. A confi rmed case of legionellosis was defi ned as a case of pneumonia with laboratory evidence of acute infection with L. pneumophila including 1) isolation of L. pneumophila serogroup 1 from respiratory secretions or lung tissue, 2) a >4-fold rise in antibody titers (from 128) against L. pneumophila serogroup1 by immunofl uorescence in paired acute-and convalescent-phase serum specimens, or 3) detection of L. pneumophila serogroup 1 antigen in urine.
Cases that conformed to the case defi nition and/or had positive results in at least 1 of the following tests were considered suspected cases: 1) high (>256) antibody titer against L. pneumophila serogroup 1 in convalescent-phase serum, 2) seroconversion to L. pneumophila serogroup 1 by indirect inmunofl uorescence in acute-and convalescentphase serum, or 3) direct staining of bacteria in respiratory secretions or lung tissue by direct fl uorescence using monoclonal or polyclonal antibodies against any Legionella spp. or serogroup, including serogroup 1.

Epidemiologic Investigation
A questionnaire was used to obtain personal information (age, gender, job, and free-time activities), clinical features (signs and symptoms, date of illness onset, date of hospitalization, previous hospitalizations, and medical history), predisposing and risk factors, place of residence, and recent urban mobility within Alcoi. Information about the patients' addresses and usual itineraries (roads and places visited) in Alcoi was used to delimit an area of infl uence, or a buffer, for the location of the likely source of infection. A 500-m radius was considered around home and city itineraries for each patient by using vectorial cartography (scale 1:10,000), ortophotography (scale 1:5,000), and ArcView software (www.esri.com). The buffer for each patient was thus represented by a polygonal area; intersecting areas yielded a common area representing a likely location for the source of the outbreak.

Environmental Investigation
Systematic environmental investigations are regularly performed in the municipal water distribution system, but when this outbreak was detected, an active search for L. pneumophila was made in patients' homes (bulk water and biofi lms from showerheads and taps) and the water distribution system (bulk water); results were negative. None of the other usual sources (e.g., public fountains, cooling towers, humidifi ers) were found to pose a risk. The absence of usual risk sources led us to consider other possible sources of aerosols, including moving devices used in street cleaning and asphalt repaving; the latter had been observed in the risk area during the epidemiologic inspection.

Clinical Sampling
Respiratory samples and, when available, corresponding cultures were obtained from 11 patients with a diagnosis of legionellosis made by positive urine test result at the Hospital Virgen de los Lirios. DNA was extracted from respiratory isolates by using an UltraClean BloodSpin Kit (Mobio Laboratories, Inc., Carlsbad, CA, USA). DNA from positive L. pneumophila cultures was extracted as described (23). Briefl y, bacterial colonies from pure cultures were resuspended in 200 μL of 20% Chelex 100 resin (Bio-Rad Laboratories, Richmond, CA, USA). DNA was then extracted during 3 freeze-thaw cycles (-75°C for 10 min and 94°C for 10 min), and cellular debris was removed by centrifuging at 10,000 × g for 1 min. The amount of genomic DNA was measured by spectrophotometry at 260 nm in triplicate, and DNA purity was checked by using the A260/A280 ratio. Purifi ed DNA was stored at -20°C until used. DNA extraction from respiratory samples (sputum, bronchoalveolar aspirated secretions, and lung biopsy tissue) was performed with a QIAamp DNA Mini Kit (QIAGEN, Valencia, CA, USA) and stored at -20°C.

Environmental Sampling
Water samples were fi ltered and treated with acid. Water and swab specimens were plated onto buffered charcoal yeast extract medium with and without supplemental antimicrobial drugs; standard plating techniques were used. Inoculated plates were incubated at 35°C and examined reg-ularly for colonies resembling Legionella spp. Suspected colonies were inoculated onto biplates containing buffered charcoal yeast extract medium with and without L-cysteine. Nine cultures that required L-cysteine for growth were subcultured and tested with specifi c antiserum to determine the Legionella spp. and serogroup.

PCR, Sequencing, and Allelic Profi le Assignment
For cultured isolates, the 7 loci in the European Working Group for Legionella Infections (EWGLI, www.ewgli. org) typing scheme were amplifi ed as detailed by Gaia et al. (17) and Ratzow et al. (18). DNA from respiratory samples was amplifi ed by using a seminested approach. For the fi rst PCR we used the same primers and conditions as for cultured isolates. For the second PCR we used the internal primers described by Coscollá et al. (22) and used 2 μL of the fi rst PCR products as template. Amplifi cation conditions and profi les were the same as for the fi rst PCR except for the annealing temperature (22). PCR products were purifi ed by using a High Pure PCR Product Purifi cation Kit (Roche Diagnostics, Mannheim, Germany). PCRs were conducted in a reaction volume of 50 μL containing 20 ng of genomic DNA, 1 U of Taq DNA polymerase, 200 μmol/L of each dNTP, 2.5 mmol/L of magnesium-free buffer, 2.5 mmol/L MgCl 2 , and 0.2 μmol/L of each pair of primers. We adopted the gene notation used in the fi rst L. pneumophila genome published (24); consequently, locus fl iC corresponds to locus fl aA used in the EWGLI typing scheme (17).
Purifi ed DNA was directly sequenced by the dideoxy method by using a BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit and was analyzed in an ABI PRISM 3700 sequencer (each from Applied Biosystems, Foster City, CA, USA). Sequencing of PCR products from respiratory samples differed only in the use of the same internal primers used in the second PCR of the seminested approach. Sequence chromatogram fi les were analyzed by using the Staden sequence analysis package (25).
Allelic profi les for the 7 sequenced genes were obtained from EWGLI and were aligned and compared with sequences derived in this study. Multiple sequence align-ments were obtained by using ClustalX (26) and further refi ned by visual inspection.

Molecular Phylogenetic Analysis
A phylogenetic reconstruction was obtained with the concatenated alignment of sequences from the 7 loci analyzed. Models of nucleotide substitution were assessed by using the maximum-likelihood approach implemented in jModeltest (27). Maximum-likelihood phylogenetic trees were obtained with PHYML 3.0 (28) by using the previously derived models of nucleotide substitution for each locus. Support for the nodes was evaluated by bootstrapping with 1,000 pseudoreplicates.

Epidemiologic Findings
Among patients with positive urine antigen test results, 11 cases of legionellosis were confi rmed and L. pneumophila was isolated from 4. All patients required hospitalization, and all except 1 recovered. (The patient who did not recover had severe signs and symptoms and subsequently died.) The main signs and symptoms were fever (100% incidence), pneumonia (100%), headache (27.3%), myalgia (27.3%), diarrhea and/or vomiting (18.2%), and confusion (45.5%). The average age was 70 years, range 49-88 years. More men than women were affected (male:female ratio = 4.5).
Confi rmed cases occurred from July 21 through September 17. According to the date of disease onset, the outbreak showed 3 epidemic waves: 2 cases in the second half of July, 8 cases in the fi rst half of August, and 1 case in the second half of August ( Figure 1).
No common indoor source of exposure was found, and the initial hypothesis was that the outbreak originated from environmental contamination of an unknown source capable of producing and dispersing large quantities of aerosols contaminated with L. pneumophila. The fi rst 2 patients lived in the northern part of the city, which suggested that the source could be located in that area. The spatial distribution of patients' buffers changed in August, thus indicating that the likely source of the outbreak had moved to the Santa Rosa quarter (Figure 2). The area of epidemic risk was modifi ed accordingly, and the search for putative environmental sources focused on that neighborhood.

Environmental Findings
L. pneumophila was not isolated from samples derived from the municipal water supply, traditional risk facilities, and patients' houses. The environmental investigation was extended to other potential sources, especially portable cleaning devices such as sweepers, hydrocleaners, and water tanks used to clean the streets, all of which used water from the municipal water supply. At that time, the Santa Rosa quarter was being repaved, and 1 of the machines used in the repaving process was a tank truck that carried water used by a large milling machine. The water in the tank was obtained from a natural spring untreated with chlorine or anything else. Because the average daytime temperature in Alcoi during July and August is 27°C, the water in the machine might have been warm enough for L. pneumophila growth. The milling machine had been working north of Alcoi around July 15 and in the Santa Rosa neighborhood from July 31 through August 20 ( Figure 2). This activity fi ts spatially and temporally with the incubation period of confi rmed cases. This machine was identifi ed and removed from service on August 21, thus was able to cause the last infection detected on August 23 ( Figure 2).

Microbiological Findings
L. pneumophila serogroup 1 was isolated from the water in the tank and from the atomizers in the milling machine ( Figure 3). L. pneumophila was also isolated from other machines used for street cleaning in the city, but these organisms were from serogroups other than serogroup1.
The milling machine and the water tank were immediately confi ned outside the city, sealed, cleaned, and disinfected. When the machine was put back into use, a new cleaning and maintenance protocol was implemented to prevent future contamination. The cleaning protocol consisted of treatment with chlorine (20 ppm) and removal of the atomizers and their replacement by gravity-based water distributors. Additionally, for operation in the city, use of a separate thermo-insulated water tank in which chlorine was continuously applied at 20 ppm was required, thus preventing any further growth of L. pneumophila. Additionally, water had to be obtained from the municipal system.

Molecular Characteristics
L. pneumophila sequence information was obtained from 7 uncultured sputum samples and from 4 cultures derived from the 11 patients studied. Complete allelic profi les, according to the EWGLI typing scheme, were obtained for 9 clinical samples in the study. All had the same profi le, which corresponded to sequence type (ST) 578 in the EWGLI database (Table 1). Partial allelic profi les for the other 2 samples were consistent with ST578. The 9 L. pneumophila isolates derived from putative environmental sources, including the milling machine and the water tank (   A phylogenetic tree was obtained from the concatenated alignment of the 2,984 bp ( Figure 4). The phylogenetic analysis showed that 4 environmental samples were identical to the clinical samples (represented by ST578 C/E in Figure 4). Their comparison with the remaining environmental samples showed 14-50 nt differences to strains 4159 and 4143/4160/7970 (represented by ST1 in Figure 4), respectively. The comparison of these to reference strains showed that outbreak samples were more closely related to the Corby strain (29), a virulent human isolate from which it differed by only 6 nt.

Discussion
During the epidemic outbreak of legionellosis in the summer of 2009 in Alcoi, Spain, 11 affected persons were identifi ed, and their L. pneumophila isolates shared the same sequence-based typing profi le (ST578). Spatialtemporal analysis of outbreak cases pointed to a milling machine used in street asphalt repaving and its water tank as the most likely source of infection. Molecular typing confi rmed that L. pneumophila isolated from the machine showed the same allelic profi le as the samples from the patients. When this machine was removed from service and cleaned, infections in this locality ceased.
The absence of apparent risk facilities in the area during the outbreak period and the changing spatial distribution of cases led us to consider alternative sources of con-tamination and spread. The heterogeneous spatial grouping led us to hypothesize that the transmission sources could be mobile. The analysis of the water tank and milling machine used in both neighborhoods where risk areas were identifi ed during the incubation period of confi rmed cases resulted in isolation of L. pneumophila. Molecular results confi rmed that 4 of the 9 environmental isolates obtained showed a sequence-based typing profi le identical to that of all clinical samples. This result highlights the fact that a device not previously considered to represent a risk for L. pneumophila infection, such as a street paving machine, can be associated with a legionellosis outbreak.
These kinds of machines are good candidates for spreading L. pneumophila infections, given their ability to generate aerosols. These machines are used in urban areas where contaminated aerosols can be inhaled by many citizens. They are continually moving, making their identifi cation as a source of infection more diffi cult because by the time an outbreak is detected and the causative L. pneumophila strains are characterized, these machines have usually moved to another location. Lack of suitable cleaning routines for these machines makes them excellent candidates for colonization with L. pneumophila. In this particular outbreak, use of untreated water from a natural spring contributed to the contamination of the tank and milling machine. These devices are frequently stored and operated in and from industrial areas where  nontreated water supplies are common, which increases risk for colonization by L. pneumophila. Whether they spread L. pneumophila more easily than other devices usually linked to such outbreaks, like cooling towers or spas, is unknown, but they should be considered risk devices for the reasons detailed above. During 1999-2005, ST578 has been found in clinical samples from patients with legionellosis in Alcoi (22) and has caused recurrent outbreaks and sporadic cases of community-acquired pneumonia. However, to our knowledge, no identical isolate has been found in the environment in this area (M. Coscollá et al., unpub. data). Finding ST578 in a sports club (Table 2), not related to the outbreak, indicates that it can occasionally be found in other risk facilities, which would help to explain its association with past clinical cases in Alcoi. Additionally, this profi le was reportedly found in an environmental strain detected in Mexico during an epidemiologic investigation of travelassociated legionellosis (EWGLI sequence-based typing database, www.ewgli.org).
Although culture isolates were available for only 4 of the 11 outbreak patients identifi ed, our use of a sequencebased typing approach to analyze clinical samples on the basis of direct extraction and sequencing of L. pneumophila from sputum samples increased the number of patients we were able to study (i.e., all those identifi ed in the outbreak). The effi ciency of this approach has been demonstrated to be higher than that of sequencing after isolating L. pneumophila from cultures (22). Moreover, the 100% match between the sequences directly obtained from respiratory samples and cultured isolates from the same patient corrob-orates the suitability of the direct sequencing approach for the identifi cation and molecular epidemiology studies of L. pneumophila (22). We think that this approach extends the usefulness of molecular epidemiologic tools in the study of L. pneumophila outbreaks, thus enabling more precise identifi cation of their sources.