Identification methods for Legionella from environmental samples

Bartie, Catheleen; Venter, Stephanus N.; Nel, Louis Hendrik

doi:10.1016/s0043-1354(02)00220-8

Cited by 46 publications

(39 citation statements)

References 11 publications

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“…Looking for better recovery results, Sanden et al [57] observed that preincubation of water samples with free-living amoebae for several days (seven days) increased the recovery of Legionella spp., which was related with proliferation of amoebae in the samples. The same observations were done by Bartie et al [30], who showed that re-incubation of water samples concentrates with autochthonous amoebae improved the culturability of legionellae in a selective medium. Although these approaches are quite selective, due to the improvement of limit of detection or interference with other bacteria, these methods can be time expensive.…”

Section: Discussionsupporting

confidence: 72%

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Detection of <i>Legionella</i> spp. in Natural and Man-made Water Systems Using Standard Guidelines

Borges¹,

Simes²,

Martínez‐Murcia³

et al. 2012

MICROBIOLOGY

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Infections caused by Legionella spp. are considered at the present time, an emerging public health problem and are linked to high rates of mortality and morbidity, if not properly treated. In this study were analyzed 54 samples of water from 8 counties at Northern Portugal, with the aim of obtaining a collection of strains of the genus Legionella and to characterize them genetically and phenotypically. Another objective of this study was to evaluate the effectiveness of the technique of cultivation, a standard method according to International Organization for Standardization ISO 11731:1998, for detection and enumeration of species of Legionella. For laboratory processing, after the filtration of samples (1 L), the filtrate was resuspended in sterile distilled water (5 ml). Heat treatment for selective inhibition of non-Legionella bacteria was performed. Subsequently, 100 μl of the suspension was spread in GVPC selective agar medium, and incubated (7 to 10 days) at 37 ℃. Colonies that were morphologically characteristic of the genus were sub-cultured onto BCYE agar and blood agar for verification. According to the procedure recommended by the standard method, only the colonies which grew in BCYE agar and not on blood agar were considered as suspected Legionella strains. The identification of these initially selected colonies was performed by sequencing the 16S rRNA gene, which revealed that none of the isolates were identified as belonging to the genus Legionella. However, through the ISO 11731:1998 they were interpreted as positive, corresponding therefore to "false-positive" results. The methods used in this study allowed the isolation of a number of isolates (40), which form an independent group of all genus of the family Chitinophagaceae outlined so far, and that by their phylogenetic distance might be a genus not yet described and therefore a new species. The results obtained, highlighted the importance of using culture and genetic methods in parallel for the proper identification of microorganisms.

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Section: Discussionsupporting

confidence: 72%

“…After filtration, bacteria collected on the membranes were resuspended in 5 ml of water and shaken vigorously. Heat treatment for selective inhibition of non-Legionella bacteria were performed as described previously [21,24,29,30]. Briefly, 1 ml of the concentrate was treated at 50 ± 1℃ for 30 ± 1 min.…”

Section: Isolation Of Legionellae From Water Samplesmentioning

confidence: 99%

Detection of <i>Legionella</i> spp. in Natural and Man-made Water Systems Using Standard Guidelines

Borges¹,

Simes²,

Martínez‐Murcia³

et al. 2012

MICROBIOLOGY

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“…The cell suspension was placed in a 50ºC water bath during 30 min. The heat treatment of concentrated water samples was used as a selective method to reduce the numbers of non-Legionella bacteria as previously described (5). These represented the prepared samples.…”

Section: Sample Collection and Concentrationmentioning

confidence: 99%

“…Genetic markers have been proposed to identify Legionella strains to the species level, including 16S ribosomal RNA, macrophage infectivity potentiator (mip), defect organelle trafficking (dotA), RNA polymerase β-subunit (rpoB) genes or 23S-5S ribosomal intergenic spacer region (23S-5S ISR) (20,21,27,28,43). Other techniques have been proposed as alternatives to PCR and growth procedures to identify Legionella species, including direct fluorescent antibody (DFA) staining and latex agglutination (5). The present study aimed to compare the results obtained using the standard culture procedure, the direct fluorescent antibody (DFA) assay and the amoebic coculture procedure, in order to establish which one is the best method for the isolation and identification of Legionella spp from environmental samples.…”

Section: Introductionmentioning

confidence: 99%

Detection of Legionella pneumophila in water and biofilm samples by culture and molecular methods from man-made systems in São Paulo - Brazil

Carvalho

Foronda

Pellizari³

2007

Braz. J. Microbiol.

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Legionella pneumophila is a pathogenic bacteria associated to aquatic habitat of natural and artificial environments. Clinical cases of legionellosis have been reported in Brazil but there is a lack of information about the incidence and concentration of this bacterium in environmental sources. Thus, the present study was designed to evaluate the occurrence of legionellae in São Paulo city, Brazil, using different methods of detection and identification. Sixty-seven water and biofilm samples from natural reservoirs and man-made systems were collected and analyzed for the presence of Legionella spp by culturing onto a selective medium, coculture in axenic free-living amoebae and direct fluorescent antibody (DFA) assay. Results showed that freshwater of reservoirs did not contain legionellae, Legionella pneumophila was isolated from man-made systems, with predominance of Legionella pneumophila serogroup 1 strains. Although there was no statistical difference among the proposed detection methods, the plate culture method yielded a higher number of L. pneumophila positive samples, followed by amoebic coculture procedure and direct fluorescent antibody assay. Results of PCR and sequencing reactions revealed that application of macrophage infectivity potentiator gene as a molecular marker was an important tool for the identification of environmental isolates of L. pneumophila. The agreement among the three detection methods-when all methods yielded similar results-and the prevalence of a single Legionella species in the sampled man-made systems could suggest that the occurrence of this bacterium had been influenced by the higher concentration of metallic ions dissociated in water of those systems than in natural reservoirs. Thus, the results of this study revealed that the water of man-made systems in Sao Paulo may serve as a reservoir for L. pneumophila and other microorganism, including free-living protozoans.

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“…Apart from this, many researchers all over the world including South Africa have reported the detection and isolation of Legionellae in their aquatic environments (Bartie et al, 2003;Erdogan and Arslan, 2007;Kuroki et al, 2009). It is inconceivable to think that L. pneumophila is not present in the authors' aquatic environment.…”

Section: Introductionmentioning

confidence: 99%

Isolation of Legionella pneumophila from surface and ground waters in Osogbo, Nigeria

Alli¹,

Olusoga²,

Adedokun³

et al. 2011

Afr. J. Microbiol. Res.

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Legionella is a ubiquitous water environmental organism. Isolation/detection of Legionellae has been reported worldwide. However, there is no reported isolation of Legionellae in countries in Africa with the exception of South Africa. The aim of this study was to survey the surface, ground waters and air conditioner water systems (ACWs) in Osogbo, Nigeria for the presence of Legionella pneumophila by cultural isolation method. A total of 313 water samples from the streams (surface water), wells (groundwater) and ACWS were cultured on buffered charcoal yeast extract (BCYE) medium. The isolates were identified by Gram staining techniques, biochemical tests and demonstration of pore forming activity. Polymerase chain reaction for amplification of 0.52 kb fragment of the part of dot/icm region of L. pneumophila was used for the final identification. The results obtained showed that the prevalence of L. pneumophila from streams (8%) was statistically significant when compared to wells (1%) (Chi square, P < 0.05). L. pneumophila was not isolated from any of the water samples taken from air-conditioner water systems. The study concludes that L. pneumophila is present in natural aquatic environment in Nigeria.

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Identification methods for Legionella from environmental samples

Cited by 46 publications

References 11 publications

Detection of <i>Legionella</i> spp. in Natural and Man-made Water Systems Using Standard Guidelines

Detection of <i>Legionella</i> spp. in Natural and Man-made Water Systems Using Standard Guidelines

Detection of Legionella pneumophila in water and biofilm samples by culture and molecular methods from man-made systems in São Paulo - Brazil

Isolation of Legionella pneumophila from surface and ground waters in Osogbo, Nigeria

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