Enhancement of <i>Legionella pneumophila</i> Culture Isolation from Microenvironments by Macrophage Infectivity Potentiator (<i>mip</i>) Gene‐Specific Nested Polymerase Chain Reaction

Nintasen, Rungrat; Utrarachkij, Fuangfa; Siripanichgon, Kanokrat; Bhumiratana, Amaret; Suzuki, Yasuhiko; Suthienkul, Orasa

doi:10.1111/j.1348-0421.2007.tb03967.x

Cited by 7 publications

(7 citation statements)

References 22 publications

(14 reference statements)

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“…We confirm the large discrepancies between PCR and culture for L. pneumophila quantification in treated water samples (2,3,7,14,(23)(24)(25)(34)(35)(36). We show that L. pneumophila DNA detected by PCR in water samples treated with chlorine for 24 h includes VBNC forms and dead bacteria.…”

Section: Discussionsupporting

confidence: 68%

“…These viable bacteria were able to multiply in A. polyphaga and to recover their culturability. This explains the large discrepancies between PCR and culture for L. pneumophila quantification in chlorine-treated water samples (2,3,7,14,(23)(24)(25)(34)(35)(36). Different environmental conditions and disinfection processes could have different effects on VBNC generation.…”

Section: Discussionmentioning

confidence: 99%

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A PCR-Based Method for Monitoring Legionella pneumophila in Water Samples Detects Viable but Noncultivable Legionellae That Can Recover Their Cultivability

Dusserre

Ginevra

Hallier-Soulier³

et al. 2008

Appl Environ Microbiol

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Legionella pneumophila is the causative agent of Legionnaires' disease. This bacterium is ubiquitous in aqueous environments and uses amoebae as an intracellular replicative niche. Real-time PCR has been developed for rapid detection of Legionella DNA in water samples. In addition to culturable bacteria, this method may also detect dead and viable but noncultivable (VBNC) legionellae. In order to understand the significance of positive PCR results in this setting, we prepared water samples containing known concentrations of L. pneumophila and analyzed them comparatively by means of conventional culture, real-time PCR, viability labeling, and immunodetection (solidphase cytometry). We also examined the influence of chlorination on the results of the four methods. The different techniques yielded similar results for nonchlorinated water samples but not for chlorinated samples. After treatment for 24 h with 0.5 and 1 ppm chlorine, all cultures were negative, PCR and immunodetection showed about 10 6 genome units and bacteria/ml, and total-viable-count (TVC) labeling detected 10 5 and 10 2 metabolically active bacteria/ml, respectively. Thus, PCR also detected bacteria that were VBNC. The recoverability of VBNC forms was confirmed by 5 days of coculture with Acanthamoeba polyphaga. Therefore, some TVC-positive bacteria were potentially infective. These data show that L. pneumophila PCR detects not only culturable bacteria but also VBNC forms and dead bacterial DNA at low chlorine concentrations.Legionella pneumophila, the bacterium responsible for Legionnaires' disease and Pontiac fever, is ubiquitous in natural and man-made aqueous environments and requires free-living amoebae for its intracellular replication (1,15,31). Under appropriate conditions, L. pneumophila can also survive for long periods as a free organism in low-nutrient environments (4, 30). Regular monitoring of potentially contaminated water sources is essential to prevent legionellosis outbreaks (21,27). Culture with selective media is the standard method for the detection, isolation, and identification of L. pneumophila in clinical and environmental samples (18,19), but it can take more than 7 days. Cost-effective and reliable real-time quantitative PCR methods have been developed for rapid detection/quantification of Legionella DNA in water samples and are often used as a routine monitoring tool (14, 36). The results are expressed as the number of genome units (GU) per liter, but the precise equivalence with the number of CFU has not been established. Culture and PCR agree well on samples from hot water systems but not from cooling towers. Culture is always less sensitive than PCR (2,23,36).Discrepancies between PCR and culture results can be explained by several factors. Legionella growth can be inhibited or masked by overgrowth of contaminating microorganisms (18). Furthermore, L. pneumophila can enter a viable but noncultivable (VBNC) state, from which it can recover after passage in amoebae (12,30). These VBNC legionellae may be detected by PC...

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Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%

A PCR-Based Method for Monitoring Legionella pneumophila in Water Samples Detects Viable but Noncultivable Legionellae That Can Recover Their Cultivability

Dusserre

Ginevra

Hallier-Soulier³

et al. 2008

Appl Environ Microbiol

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“…Furthermore, MEGAN with NCBI-NT, Kraken, and CLARK with RefSeq database detected a larger number of L. pneumophila sequences in PCR-negative samples such as HKU_B and HKU_C than in PCR-positive samples including HKU_G, HKU_H, and HKU_I ( Table 2 ). Since the sensitivity of nested PCR assay with the employed primer sets is known to be 10 fg or 10 CFU per ml ( Nintasen et al, 2007 ), the inconsistency is probably attributed to false positive detections due to the low specificity of these database search methods in detecting L. pneumophila .…”

Section: Discussionmentioning

confidence: 99%

“…The conventional method could be used to enumerate the number of L. pneumophila in a water sample. Based on the sensitivity the L. pneumophila -specific nested PCR ( Nintasen et al, 2007 ), the number of L. pneumophila were estimated as at least 10 CFU/ml. Another limitation of this study was the number of reads generated by Miseq.…”

Section: Discussionmentioning

confidence: 99%

Comparison of Database Search Methods for the Detection of Legionella pneumophila in Water Samples Using Metagenomic Analysis

et al. 2018

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Metagenomic analysis has become a powerful tool to analyze bacterial communities in environmental samples. However, the detection of a specific bacterial species using metagenomic analysis remains difficult due to false positive detections of sequences shared between different bacterial species. In this study, 16S rRNA amplicon and shotgun metagenomic analyses were conducted on samples collected along a stream and ponds in the campus of Hokkaido University. We compared different database search methods for bacterial detection by focusing on Legionella pneumophila. In this study, we used L. pneumophila-specific nested PCR as a gold standard to evaluate the results of the metagenomic analysis. Comparison with the results from L. pneumophila-specific nested PCR indicated that a blastn search of shotgun reads against the NCBI-NT database led to false positive results and had problems with specificity. We also found that a blastn search of shotgun reads against a database of the catalase-peroxidase (katB) gene detected L. pneumophila with the highest area under the receiver operating characteristic curve among the tested search methods; indicating that a blastn search against the katB gene database had better diagnostic ability than searches against other databases. Our results suggest that sequence searches targeting long genes specifically associated with the bacterial species of interest is a prerequisite to detecting the bacterial species in environmental samples using metagenomic analyses.

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“…12,13 A PCR-based detection, an attractive and sensitive technique, has been suggested as a way to overcome problems of the culture method. 4,12,14 For the reason, we developed and evaluated the duplex PCR (dPCR) assay for simultaneous detection of Legionella sp. and L. pneumophila in cooling tower water samples, and compared the sensitivity of the dPCR assay with the sensitivity of the culture method as a gold standard.…”

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confidence: 99%

Simultaneous detection of Legionella species and Legionella pneumophila by duplex PCR (dPCR) assay in cooling tower water samples from Jakarta, Indonesia

Yasmon¹,

Yusmaniar²,

Anis³

et al. 2010

Med J Indones

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Tujuan: Metode kultur memiliki sensitivitas yang rendah dan memerlukan waktu yang lama untuk mendeteksi bakteri Legionella. Oleh karena itu, dalam penelitian ini dikembangkan uji PCR duplex (dPCR) untuk deteksi Legionella sp. dan L. peneumophila secara simultan pada sampel air tower. Metode kultur digunakan sebagai baku emas. Metode: Dilakukan optimasi metode dPCR untuk mendapatkan teknik uji yang memiliki sensitivitas dan spesifi sitas tinggi. Metode kemudian diuji pada 9 sampel air tower yang diperoleh dari 9 gedung di Jakarta. Untuk metode kultur, bakteri ditumbuhkan pada media selektif 'growth factor supplemented-buffered charcoal yeast extract' (BCYE). Hasil: Dari 9 sampel yang diuji dengan dPCR, 6 menunjukkan positif Legionella sp., 1 positif L. pneumophila, dan 2 menunjukkan hasil uji negatif. Untuk sampel yang sama, metode kultur menunjukkan hasil uji negatif. Kesimpulan: Uji dPCR adalah uji yang sangat sensitif dibandingkan dengan metode kultur, dan uji dPCR ini dapat digunakan untuk pemeriksaan rutin Legionella sp. dan L. pneumophila pada sampel air dari 'tower'.

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Enhancement of Legionella pneumophila Culture Isolation from Microenvironments by Macrophage Infectivity Potentiator (mip) Gene‐Specific Nested Polymerase Chain Reaction

Cited by 7 publications

References 22 publications

A PCR-Based Method for Monitoring Legionella pneumophila in Water Samples Detects Viable but Noncultivable Legionellae That Can Recover Their Cultivability

A PCR-Based Method for Monitoring Legionella pneumophila in Water Samples Detects Viable but Noncultivable Legionellae That Can Recover Their Cultivability

Comparison of Database Search Methods for the Detection of Legionella pneumophila in Water Samples Using Metagenomic Analysis

Simultaneous detection of Legionella species and Legionella pneumophila by duplex PCR (dPCR) assay in cooling tower water samples from Jakarta, Indonesia

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