The ability of aquatic bacteria Pseudomonas fluorescens SSD (Ps-D) and Pseudomonas putida SSC (Ps-C) to support the persistence of Legionella pneumophila (Lp-1) in an artificial water microcosm was investigated for 42 day, at two different incubation temperatures. At 4 °C, individually suspended Lp-1 was no longer detectable just after 24 hours, while in co-cultures with Pseudomonas, Lp1 showed a better survival capability. At 30 °C, Lp-1 alone displayed high survival rates over the entire period of observation. When Lp-1 was inoculated with Ps-C and Ps-D, its count showed a marked decrease, followed by a gradual and costant decline

BONDI, Moreno

BORELLA, Paola

GUERRIERI, Elisa

MESSI, Patrizia

English

Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia

NEW MICROBIOLOGICA, 30, 247-251, 2007Influence of aquatic microorganismson Legionella pneumophila survivalElisa Guerrieri1, Moreno Bondi1, Paola Borella2, Patrizia Messi11Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy2Department of Public Health Sciences, University of Modena and Reggio Emilia, Modena, ItalyThe ability of aquatic bacteria Pseudomonas fluorescens SSD (Ps-D) and Pseudomonas putida SSC (Ps-C) to sup-port the persistence of Legionella pneumophila (Lp-1) in an artificial water microcosm was investigated for 42 day,at two different incubation temperatures. At 4 °C, individually suspended Lp-1 was no longer detectable just after24 hours, while in co-cultures with Pseudomonas, Lp1 showed a better survival capability. At 30 °C, Lp-1 alone dis-played high survival rates over the entire period of observation. When Lp-1 was inoculated with Ps-C and Ps-D, itscount showed a marked decrease, followed by a gradual and costant decline.KEY WORDS: Legionella pneumophila, Pseudomonas, Survival, Aquatic habitatsSUMMARYReceived February 12, 2007 Accepted March 19, 2007A number of studies are available on the abili-ty of Legionella pneumophila to survive inaquatic systems and the role of amoebal hostsin its ecology is widely documented (Borella etal., 2005). Nevertheless, few studies are report-ed to date on the relationship between Legionellaand other water-borne microorganisms, fre-quently isolated from the same samples(Sommerse et al., 1996). The microbial flora ofaquatic environments is generally made up of sev-eral species of bacteria, fungi and protozoa.Within this complex community, a large num-ber of interactions occur, in particular among dif-ferent bacteria (Messi et al., 2003; Messi et al.,2005). Thus, we cannot exclude that legionellasurvival may also be positively or negatively influ-enced by other aquatic germs. A mutualisticmechanism was observed in complex media lack-ing cysteine or iron salts, unable to support thegrowth of L. pneumophila, where the bacteriumformed satellite colonies around strains ofsome common aquatic species, includingFlavobacterium, Pseudomonas, Alcaligenes andAcinetobacter (Wadowsky et al., 1985). A role ofaquatic bacteria in supporting the growth orinfluencing the survival of L. pneumophila in sim-ple media or water has been supposed. In a studyon cooling towers, the highest counts of legionel-lae were observed in samples with high con-centrations of heterotrophic bacteria, even if thecorrelation between the occurrence of thepathogen and heterotrophic bacteria was not sig-nificant (Yamamoto et al., 1992). In this case theauthors suggested an indirect or synergistic rela-tionship, rather than a direct relationship withspecific bacteria. Wadowsky et al. (1991)observed that killed cells of Pseudomonas pauci-mobilis represented a source of nutrients for hostprotozoa, thus playing a positive role in legionel-la survival. Surman et al. (1994) demonstratedthat bacteria isolated from biofilms grown in acontinous culture model system enhanced theviability of L. pneumophila when inoculated ontoCorresponding authorPatrizia MessiDipartimento di Scienze BiomedicheUniversità degli Studi di Modena e Reggio EmiliaVia Campi 287, 41100 Modena, ItalyE-mail: messi.patrizia@unimore.itR2A medium, a phenomenon not observed whenheat-killed and cell-free extracts were used. Leeand West (1991) confirmed that other bacteriacould represent an essential nutrient for the pro-tozoa, although the ability of these microor-ganisms to support the growth of legionellasunder natural conditions is not proven.In this study, the survival capability of a strainof L. pneumophila was investigated in an artifi-cial water microcosm, in the presence of otherwater-borne bacteria to better define their abil-ity to support/inhibit the environmental per-sistence of the pathogen. Two strains belongingto the genus Pseudomonas, the most frequentlypresent in water habitat, were chosen as repre-sentative of the natural microflora of aquaticenvironments.L. pneumophila serogroup 1 strain (Lp-1), iso-lated from hot tap water and already employedin previous studies (Guerrieri et al., 2005), wassubcultured on buffered charcoal-yeast extractagar supplemented with a-ketoglutarate (aBCYE,OXOID Milan, Italy) (Edelstein, 1981) andincubated for 4 d at 36 °C in a moist chamberwith 2.5% CO2 added.Pseudomonas fluorescens SSD (Ps-D) andPseudomonas putida SSC (Ps-C), isolated in ourlaboratory from mineral water samples (Messiet al., 2002), were used as aquatic bacteria. Theisolates were recovered by using standard pro-cedures for screening of coliforms in water andwastewater (APHA, 1995), and were tested foroxidase production (NN-dimethyl-p-phenylene-diamine dihydrochloride, Sigma Chemical Co.,St. Louis, MO, USA). Identification was madeaccording to standardized tests API 20 NE strips(bioMèrioux, Marcy l’Etoile, France). Cultures ofPseudomonas were then grown in Tryptic SoyBroth (TSB, Difco Laboratories, Detroit, MI) sup-plemented with 0.6% yeast extract (TSB-YE)(Difco) at 30 °C for 18 h.All the strains were stored in phosphate-bufferedsaline (PBS; 8 g NaCl, 0.2 g KCl, 2.9 gNa2HPO4·12H2O, 0.2 g KH2PO4 with 1 l of dis-tilled water) supplemented with 30% (vol./vol.)glycerine at -80 °C.Cell suspensions of Lp-1 were obtained by col-lecting colonies, grown on aBCYE agar plates,with a sterile swab and suspending the same in5 ml of sterile deionized water. For Pseudomonasovernight cultures grown in TSB-YE at 30 °C for18 h were used. All cultures were centrifuged at2,000 x g for 20 min. After discarding the super-natant fluid, the pellets were re-suspended in 5 mlsterile deionized water. Centrifugation, super-natant discard and re-suspension were repeat-ed three times. The density of the final suspen-sions was measured by the plate count method.The survival experiments were carried out by thepreviously described “macromethod” (Guerrieriet al., 2005) on samples of tap water filteredthrough membrane filters (0.45-mm-pore-sizeSartorius AG, Goettingen, Germany) to removeautochthonous microbiota. Eight hundred ml-volumes of the water were added to sterile Pyrexglass flasks (1 l), cleaned with acid and autoclavedbefore use.The flasks were then inoculated with 0.8 ml ofthe test strain suspensions in different combi-nations (Lp-1 alone, Lp-1 with Ps-D, Lp-1 withPs-C, Ps-D alone and Ps-C alone) to yield a finalbacterial count of 4 x 106 cfu ml-1 for Lp-1, 3 x105 cfu ml-1 for Ps-D and 5 x 105 cfu ml-1 for Ps-C. The flasks were incubated for 42 days at 4 °Cand 30 °C.At regular intervals, daily during the first weekand every three days afterwards, the bacterialcount was determined by spreading 0.1 ml of seri-al tenfold dilution samples on appropriate agarplates. The medium α-BCYE was used for singlyinoculated legionella counts, while the moreselective MWY and GVPC (OXOID Milan, Italy)were used to enumerate legionella cells in thepresence of other bacteria; Tryptic Soy Agar (TSA,Difco Laboratories, Detroit, MI) was used for theother bacterial counts.Plates were incubated at 36 °C in a moist cham-ber with 2.5% CO2 added for Lp-1 and at 30 °Cfor pseudomonas. During incubation, plates wereexamined daily for up to 7 d, and finally thegrown colonies counted. The experiment wasconducted in triplicate and the bacterial countwas performed on three plates. The arithmeti-cal mean of three sample determinations,expressed as log bacterial count, was plottedagainst incubation time (days).The results obtained at 4 °C and 30 °C are shownin Figures 1 and 2. Reported data refer to theexperiments performed with the Ps-D strain, asthe results obtained employing Ps-C were simi-lar. At 4 °C (Figure 1), individually suspended Lp-1 was no longer detectable after just 24 hours.248 E. Guerrieri, M. Bondi, P. Borella, P. MessiLp1, when inoculated with Ps-D, showed an ini-tial decrease (about 0.5 log) and then remainednearly constant until the 4th day of incubation.Successively it showed a progressive decrease,with a final viable count of about 102 cfu ml-1 after42 days of incubation. This trend supports the evi-dence of a favouring role of Pseudomonas on thesurvival capability of Legionella under stressingconditions (very low temperature). We suggestthat this positive effect may be an indirect actiondue to increased organic compounds in the medi-um. These substances derive from the metabol-ic activity of Pseudomonas, which is able to active-ly multiply also under low temperature conditions.Indeed, for both the single inoculated and the co-cultured experiments, after an initial decline (2-3 log), the viable count of Ps-D graduallyincreased and remained nearly constant (about105-6 cfu ml-1) until the 42th day. The good sur-vival capability of Pseudomonas strains in arti-ficial water microcosms has been previouslydemonstrated (Messi et al., 2002) and the pres-ent investigation confirms that this happens alsounder unfavourable physical conditions.In the experiments carried out at 30 °C (Figure2), after the initial physiological decrease, sin-Aquatic microorganisms and L. pneumophila 249FIGURE 1 - Viable cell count ofLegionella pneumophila serogroup1 (Lp-1) and Pseudomonas fluo-rescens SSD (Ps-D) at 4°C.  Lp-1 alone; r Lp-1 in co-culture withPs-D;  Ps-D alone; £ Ps-D in co-culture with Lp-1.FIGURE 2 - Viable cell count ofLegionella pneumophila serogroup1 (Lp-1) and Pseudomonas fluo-rescens SSD (Ps-D) at 30°C.  Lp-1 alone; r Lp-1 in co-culture withPs-D; < Ps-D alone;  Ps-D in co-culture with Lp-1.gle inoculated Lp-1 showed high survival ratesover the entire period of observation (104 cfu ml-1 after 42 days). In this case Ps-D displayed a neg-ative influence on the survival capability of Lp1,which showed a marked initial decrease, if com-pared to the control, followed by a gradual andcostant decline. However, Legionella was stillrecovered with a count of c.a. 102 cfu ml-1 at theend of the experiment. In this case a competi-tion for nutrients, resulting more favourable forPseudomonas, may be suggested. With regard toPs-D, the viable count of both the single inocu-lated and the co-cultured strain, after a physio-logical initial decrease, remained nearly constantuntil the 21st day. Thereafter Ps-D alonedecreased more than Ps-D in co-colture.The results of the present study suggest that thebacteria belonging to autocthonous microfloraof the aquatic habitat may directly influence L.pneumophila and therefore play a role in its ecol-ogy, depending on the environmental conditions.Actually, the survival capability of the pathogenwas enhanced by the presence of Pseudomonasstrains at a temperature of 4 °C, while it result-ed negatively influenced at a growing tempera-ture of 30 °C. This could be in accordance withLeoni et al. (2001) who reported a statisticallysignificant inverse correlation between legionel-lae and Gram negative bacteria in samples fromthe showers of several swimming pool estab-lishments. The water temperature was higherthan that employed in this study, but in any casethis phenomenon was observed only when thetemperature was under 43 °C and other com-petitor microorganisms were not present.Obviously, our experiments cannot completelyreproduce the real conditions of natural habitats,characterized by a more complex community inwhich both the presence of protozoa and thebiofilm formation represent essential features inLegionella ecology (Borella et al., 2005). As manyaquatic bacteria, including Pseudomonas, are ableto survive and multiply within protozoa or mayact as intracellular parasites (Inglis et al., 2000;Winiecka-Krusnell and Linder, 2001), a compe-tition for the protozoan host may exist under nat-ural conditions. This aspect deserves special con-sideration also because the capability of intra-cellular life is considered an important virulencefactor, besides other traits commonly shown byaquatic bacteria (Bondi et al., 2000).Furthermore, the interactions which occurwithin biofilms between L. pneumophila andaquatic bacteria have recently been studied. Inbiofilms formed under artificial conditions, thepresence of bacteria like Empedobacter breve orMicobacterium spp. favoured the adherence andpersistance of L. pneumophila. On the contrary,Pseudomonas spp., Corynebacterium glutam-icum and Klebsiella pneumoniae reduced theadherence of Legionella and accelerated itsdetachment from the biofilm (Mampel et al.,2006). Based on these observations, the authorssuggest the important role of the other bacteriain modulating the behaviour of L. pneumophi-la. As Pseudomonas represents the major genusinvolved in biofilm formation in aquatic envi-ronments (Donlan, 2002), studies are in progressto investigate the interactions betweenpseudomonas or pseudomonas-like bacteriaand L. pneumophila occurring in biofilms.ACKNOWLEDGMENTSWe are sincerely grateful to the Department ofMicrobiology-University of Parma, for providingAcanthamoeba polyphaga UP strain.This work was supported by the funds of the ItalianUniversity Ministry (MIUR), years 2000 and 2002.REFERENCESAPHA -AMERICAN PUBLIC HEALTH ASSOCIATION (1995).Standard methods for the examination of water andwastewater, 19th ed. American Public HealthAssociation, Washington, DC. 953-964.BONDI, M., MESSI, P., GUERRIERI, E., AND BITONTE, F.(2000). Virulence profiles and other biological char-acters in water isolated Aeromonas hydrophila. NewMicrobiol 23, 347-356.BORELLA, P., GUERRIERI, E., MARCHESI, I., BONDI, M.,AND MESSI, P. (2005). 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Res Microbiol 152, 613–619.YAMAMOTO, H., SUGIURA, M., KUSUNOKI, S., EZAKI, T.,IKEDO, M., AND YUBUUCHI, E. (1992). Factors stim-ulating propagation of Legionellae in coolingtower water. Appl Environ Microbiol 58, 1394-1397.Aquatic microorganisms and L. pneumophila 251

Influence of aquatic microorganisms on Legionella pneumophila survival

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Influence of aquatic microorganisms on Legionella pneumophila survival

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