Search for Legionella pneumophila in Domestic Water System in Benin Metropolis

: Legionella species, a Gram negative bacterium is the causative agent of Legionnaires’ disease, a potentially fatal pneumonic syndrome of widely recognized public health importance. The aim of the study was to determine the presence of Legionella pneumophila in domestic water (borehole) in Benin metropolis by cultural method. One hundred and ninety-eight (198) water samples from the eight-two facilities (grouped into three: public apartments/hotels, private apartments and eateries) were cultured on BCYE made selective with the addition of legionella supplement IV and growth supplement after concentration and heat treatment at 50 0 C for 30 minutes and incubated at 37 0 C. Isolates were identified by doing gram stain, oxidase, catalase and hippurate test, final identification was done by using PCR, sequence and blast search using National Center for Biotechnology Information (NCBI). The results obtained showed that Legionella pneumophila or Legionella species was not isolated though other bacteria (such as Burkholderia bacterium MSMB7 (32%), Pseudomonas antarctica (28%), Cupriavidus gilaridii (14%), Microbacterium paraoxydans (14%), Bacillus thuringensis (4%), Bacillus cereus (2%), Acinetobacter johnsonii (6%)) were detected. In conclusion zero percent (0%) prevalence of Legionella species in the water systems investigated though other bacteria with pathogenic potential were recovered. This finding suggests that water systems in Benin metropolis may not present vehicles for the transmission of diseases associated with Legionella species.

Legionellaceae are fastidious, opportunistic Gram negative bacteria that reside in aquatic environment with wide spread distribution (Pel-Yi et al., 2000). In their natural environment, Legionellaceae are intracellular parasite of free living protozoa. Legionella is transmitted to humans by inhalation of contaminated aerosols.
Common sources are air conditioning system, cooling towers, dental devices, river, streams, taps and showers (David et al., 2007;Alli et al., 2011;Maura et al., 2014). Legionellosis can appear in two distinct clinical presentation: Legionnaries' disease (LD), a mild to fatal pneumonia with 3.4% fatality rate (Anonymous, 2000) and Pontiac fever, an acute selflimited influenza-like illness. Transmission between human beings has never been observed to this date (Nathalie et al., 2010). Over 50% species of Legionella have been recognized and at least 24 of these have been associated with human infection. One species of Legionella, L. pneumophila is the etiological agent of about 90% of legionellosis case and serogroup1 (Sg1) account for the most frequent cause of infection (Doleans et al., 2004;Amemura-Maekawa et al., 2010).
Domestic system are complex environment in which concentration of Legionella can fluctuate considerably depending upon water temperature, biocide level and presence of natural host (protozoa) for legionellae to parasitize. Domestic water in Benin are 99% from borehole which are usually 80 to 120 meters deep or more (Ikhile, 2016). Culturing the domestic water is the first step to assess the risk for Legionella in Benin metropolis. This approach is well adopted in the national guide-lines for European countries, France, Italy and in other regional guidelines and recommendations (Italy: Ministero della Salute, 2000; France: Ministere de l'Emploi et de la Solidarite, 2002). Legionnaries' disease has not been reported in Benin and environmental culture of domestic water for the isolation of Legionella has never been performed hence no documented epidemiological data to determine the presence of the bacterium. The objectives of this study was to determine the presence of Legionella in domestic water and air conditioner water system in Benin metropolis.

MATERIALS AND METHODS
Study area: Benin City is situated in the southern part of Edo state, Nigeria. It is the capital of Edo state. Its geographical coordination are 6 0 20ˈ 0 " north and 5 0 38' 0" east. It is estimated to have a population of 1,147,187 people (Okhakhu, 2016). Its major attractive sites includes Oba's palace, Central hospital, banks, police station, legislative building, Oba Akenzua cultural center, Okada house, museum (where some of the importance statues of ancient times are preserved). The past Benin City was known for the artisans who did excellent bronze and ivory casting.

Study facility:
The study was conducted between September, 2015 and March, 2016 in eighty-two facilities grouped into three namely: private apartments, public apartments (hotels) and eateries all in Benin metropolis. Water samples were collected after obtaining verbal consent from the management of the facilities and ethical clearance from the Edo state Ministry of Health.
Sample collection: A total of one hundred and ninetyeight (198) water samples were aseptically collected from taps, showers and air conditioner (AC). Sterile swab sticks were first used to scrape the mouth of facet (shower with the shower head removed) and AC pipes and placed in 15mls sterile plastic centrifuge tube containing 3mls of water obtained from same point. A liter of water sample was collected into specimen container containing 1% sodium thiosulphate and stored at room temperature before processing. One hundred milliliter (100ml) of water from same source were stored at 2-4 o C for physico-chemical analysis.
Sample processing: Water samples were processed in Central Hospital Benin Medical Microbiology laboratory for isolation of Legionella as described by Alli et al., (2011). Briefly 100ml of each collected water sample was placed in a centrifuge tube and centrifuged at 3000g for 20minutes. The supernatant was carefully discarded leaving about 3mls of water with the sediment. The sediment was mixed (vortex) to dislodge the sediment bacteria. 1ml of the concentrated water sample was placed at 50 o C in a water bath for 30min (to reduce the no of nonlegionella bacteria), the heated water sample was placed on the bench for 10min in order to attain room temperature.
Culture method: Prepared concentrated sample were cultured on buffered charcoal yeast extract (BCYE) according to PHE, 2015. Briefly, an aliquot of 100µl of prepared sample was inoculated on duplicate plate of BCYE agar base made selective by adding legionella selective supplement (glycine, vancomycin, anisomycin and polymycin B sulphate) and enrich with legionella growth supplement. A set of plates were placed in a candle jar and other without candle jar at 90% humidity incubated at 36 0 C. Plates were examined at periodic interval starting from the third day of incubation for the typical ground glass colony and those without growth were re-incubated and reexamined later. Plate without growth after day fourteen (14) were discarded. Each presumptive colony was first Gram stained and those that were Gram negative rods were tested for catalase, oxidase and hippurate. Subcultures were done on α-BCYE-GVP with L-cysteine and non-selective medium BCYE agar without L-cysteine and incubated as describe above.
DNA extraction of isolate: The isolated bacteria had their DNA extracted using ZR fungal/ bacterial DNA Mini Prep TM . The manufacturer's DNA extraction protocol was used with elution volume of 25µl, and was kept at -20 o C until PCR amplification was performed.
PCR, Sequence and Blast of isolates: DNA extract obtained from isolates had their 16S target region amplified using Dream Taq TM DNA polymerase according to manufacturer's procedure with universe primer 16S-27F 5 1 -AGAGTTTGATCMFGGCTCACG-3 1 and 16S-1492R 5 1 -CGGTTACCTTGTTAGGACTT-3 1 in an ABI PRISM TM 3500xL Genetic Analyzer and CLC Main-Workbench 7.5.1 blast search was done using NBCI as describe by Stephen et al., 1997. Water quality analysis: Calcium, zinc, magnesium, iron and copper analysis were done adopting Akpan-Idiok et al., 2012 method using Buck 210 Atomic Absorption Spectrophotometer.
Statistical analysis: All statistical analysis were done using statistical package for social science (SPSS). Values were recorded as mean and standard deviation. Comparison of mean was by one-way analysis of variance (ANOVA).

RESULTS AND DISCUSSION
A search for Legionella species in domestic water in Benin metropolis in 82 facilities grouped into three (public apartments/hotels (18.3%), private apartments (70.7%) and eateries (11%)) was done. Out of the total facilities examined 36.6% yielded growth while 70.7% yielded no growth as presented in Table1.    Table 2 shows gram reaction and biochemical test performed on isolates. Suspected colonies were gram stained and those that were Gram negative rods were sub-cultured on BCYE (with and without growth supplement) and biochemically tested (catalase, oxidase and hippurate; a test suggestive of Legionella) in line with Alli et al., 2011 study. All suspected isolates grew on BCYE (with and without growth supplement) and were hippurate negative. C. gilaridii and P. antarctica were both oxidase and catalase positive while B. bacterium was oxidase positive but catalase negative and A. johnsonii was oxidase negative but catalase positive. The positive control organism (L.pneumophila) gave typical biochemical reaction (i.e. oxidase, catalase and hippurate positive) and growth only on BCYE (with growth supplement). The Gram positive isolates were not subjected to biochemical test. The used of BCYE with antibiotic to search for Legionella was in line with Alli et al., 2011 in which BCYE was used in detecting and isolating of legionellae from the environment although legionellae was not isolated in our study. Table 3 shows identified bacteria from molecular level. Results shows that Legionella has a zero prevalence. This is in contrast to result reported by Alli et al., 2011 in which Legionella spp was isolated from wells and streams. Other bacteria isolated are P. antarctica (28%), B. bacterium (32%), M. paraoxydan and C.gilaridii (14% each), B. cereus (2%), B.thuringensis (4%) and A. johnsonii (6%).    One question that puzzles the mind is; 'why was Legionella not isolated?' The main source of domestic water in the facilities examined is from the deep ground (borehole) which is 80m to 120m or more, at this depth the metals concentration (such as Cu, Zn and DO with no significant level: p >0.05) ( Table 4) may play important role in the survival of this bacterium (Legionella species) such as: (1) water percolation causes filtration of the studied organism and particles thereby accounting for the non-isolation of legionellae.

Table1: System examined in this study
(2) in this study we observed a low level DO (0.06ppm) as against a higher level reported by Wadowsky et al., 1985 in which Legionella require 0.3-9.6ppm (DO) to survive. The low DO observed may have caused non-isolation of Legionella in this study. (3) the high level of copper (0.37±0.07ppm to 0.39±0.07ppm) and zinc (0.58±0.10ppm to 0.58±0.11ppm) observed in this study may have greater effect on Legionella as studies have shown that copper greater than 50ppb and zinc level above 200ppb or below 100ppb may inhibit or lower Legionella concentration (Borella et al., 2004;2005). The effect of pH on Legionella could not be ascertain in this study as pH of the medium was not recorded. Also observed in this study is the non-isolation of Legionella from the air conditioner water samples examined, this is in agreement with zero percent (0%) reported by Alli et al., 2011 in Osogbo, Osun State, Nigeria, as against a study carried out in Germany in which 3.3% of the air conditioner water systems examined were positive for Legionella (Dermitzel et al., 1992). The non-isolation of Legionella from air conditioner water in this study could be attributed to air conditioner being an artificial man-made that is devoid of amoeba, which is supportive of legionellae growth (Alli et al., 2011). The temperature required (above 49 0 C or below 21 0 C depending on heat load, ambient temperature) by the air conditioner to effectively chill a room may not be conducive for Legionella to survive. The erratic power failure in Benin metropolis may also contribute to the nonisolation of the bacterium. When this occur air conditioner stops functioning causing the AC outlets to dry up and any Legionella present within the outlets is reduced due to the death of the bacterium. In this study, bacteria isolated were Burkholderia bacterium MSMB7 with 32% prevalence, Pseudomonas antarctica (28%), Cupriavidus gilaridii (14%), Microbacterium paraoxydans (14%), Bacillus thuringensis (4%), Bacillus cereus (2%), Acinetobacter johnsonii (6%).
Conclusion: Results obtained in our study revealed a zero percent (0%) prevalence of Legionella in the water systems investigated, though other bacteria with pathogenic potential were isolated. This finding suggests that water systems in Benin metropolis may not provide vehicles for the transmission of diseases associated with Legionella species. Therefore, there is need for further study on Legionella in well, river and stream and a continuous assessment of borehole water in Benin metropolis and rural area in order to ascertain its presence and risk, this will facilitate the development of active prevention strategy for Legionnaire's disease.