Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp. in Water

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.     

Legionella contamination of dental-unit waters.

Water samples collected from 28 dental facilities in six U.S. states were examined for the presence of Legionella pneumophila and other Legionella spp. by the PCR-gene probe, fluorescent-antibody microscopic, and viable-plate-count detection methods. The PCR and fluorescent-antibody detection methods, which detect both viable and viable nonculturable Legionella spp., gave higher counts and rates of detection than the plate count method. By the PCR-gene probe detection method, Legionella spp. were detected in 68% of the dental-unit water samples and L. pneumophila was detected in 8%. Concentrations of Legionella spp. in dental-unit water reached 1,000 organisms per ml or more in 36% of the samples, and 19% of the samples were in the category of 10,000/ml or above. L. pneumophila, when present in dental-unit water, never reached concentrations of 1,000/ml or more. Microscopic examination with fluorescent-antibody staining indicated that the contamination was in the dental-unit water lines rather than in the handpieces. Legionella spp. were present in 61% of potable water samples collected for comparative analysis from domestic and institutional faucets and drinking fountains; this percentage was not significantly different from the rate of detection of Legionella spp. in dental-unit water. However, in only 4% of the potable water samples did Legionella spp. reach concentrations of 1,000 organisms per ml, and none was in the 10,000 organisms-per-ml category, and so health-threatening levels of Legionella spp. in potable water were significantly lower than in dental-unit water. L. pneumophila was found in 2% of the potable water samples, but only at the lowest detectable level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid enumeration of viable Legionella pneumophila serogroup 1

A rapid assay for the detection of viable Legionella pneumophila serogroup 1 was evaluated. A total of 431 environmental water samples were examined using an immunofluorescent assay (IFA) combined with the cell respiration stain iodonitrotetrazolium violet (INT) and the results compared with conventional culture. The IFA/INT assay was at least as sensitive and much quicker than culture for the detection of viable Legionella pneumophila serogroup 1 in most types of sample.     

Development of conventional and real-time NASBA for the detection of Legionella species in respiratory specimens

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneumophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1-1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1-10000 CFU of L. pneumophila serotype 1 depending on the background. After dilution of the nucleic acid extract prior to amplification, 1-10 CFU of L. pneumophila serotype 1 could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All 11 PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneumophila real-time NASBA and Legionella spp. real-time NASBA, respectively.     

Evaluation of the Duopath Legionella lateral flow assay for identification of Legionella pneumophila and Legionella species culture isolates

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila. In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [P < 0.001]). Since many countries' regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.     

Detection and quantification of Legionella pneumophila in water samples using competitive PCR

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.     

Comparative evaluation of two commercially available antigen enzyme immunoassays (EIA) for the detection of Legionella pneumophila urinary antigen in frozen non-concentrated urine samples

We evaluated two commercial enzyme immunoassay kits, Binax EIA (for detection of soluble antigen of Legionella pneumophila serogroup 1) and Biotest EIA (for detection of antigens of Legionella pneumophila serogroups and other Legionella spp.) in order to introduce this test routinely for the diagnosis of Legionnaires' disease (LD) in our Laboratory. Frozen non-concentrated urine samples belonging to 45 patients with and without LD were tested. The sensitivity of Binax EIA and Biotest EIA was 47.4% and 42.1% respectively, the specificity was 95% by both tests. Biotest did not detect antigen from a patient with culture-proven infection of L. pneumophila serogroup 6. The detection of urinary antigen by both EIA tests is a useful tool for rapid diagnosis of LD, especially when samples are unavailable for culture; the sensitivity may be increased if the assay is performed on unfrozen and concentrated samples.     

Dynamics of Legionella spp. and bacterial populations during the proliferation of L. pneumophila in a cooling tower facility

The dynamics of Legionella spp. and of dominant bacteria were investigated in water from a cooling tower plant over a 9-month period which included several weeks when Legionella pneumophila proliferated. The structural diversity of both the bacteria and the Legionella spp. was monitored by a fingerprint technique, single-strand conformation polymorphism, and Legionella spp. and L. pneumophila were quantified by real-time quantitative PCR. The structure of the bacterial community did not change over time, but it was perturbed periodically by chemical treatment or biofilm detachment. In contrast, the structure of the Legionella sp. population changed in different periods, its dynamics at times showing stability but also a rapid major shift during the proliferation of L. pneumophila in July. The dynamics of the Legionella spp. and of dominant bacteria were not correlated. In particular, no change in the bacterial community structure was observed during the proliferation of L. pneumophila. Legionella spp. present in the cooling tower system were identified by cloning and sequencing of 16S rRNA genes. A high diversity of Legionella spp. was observed before proliferation, including L. lytica, L. fallonii, and other Legionella-like amoebal pathogen types, along with as-yet-undescribed species. During the proliferation of L. pneumophila, Legionella sp. diversity decreased significantly, L. fallonii and L. pneumophila being the main species recovered.     

Species-specific detection of Legionella using polymerase chain reaction and reverse dot-blotting

Abstract Legionella pneumophila and some other Legionella species are capable of causing Legionnaire's disease, a potentially fatal pneumonia. The identification of legionellae by standard laboratory techniques such as culture is difficult and time-consuming. In the present work, the DNA sequence of the 23S-5S spacer region was determined for 43 Legionella isolates, and the sequence information was used to develop a species-specific detection system using PCR and reverse dot-blotting which employs just one PCR amplicon to perform genus- and species-specific detection. L. pneumophila serogroups 1–16 as well as 21 non- pneumophila isolates could be identified and differentiated at the species level using this system.     

Total and viable Legionella pneumophila cells in hot and natural waters as measured by immunofluorescence-based assays and solid-phase cytometry

A new method was developed for the rapid and sensitive detection of viable Legionella pneumophila. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viable L. pneumophila cells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter(-1), and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viable L. pneumophila cells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 × 10(3) viable but nonculturable L. pneumophila cells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viable L. pneumophila in both man-made water systems and environmental water samples.     

A colony based confirmation assay for Legionella and Legionella pneumophila employing the EnviroAmp™Legionella system and seroagglutination

Modifications to the EnviroAmp^™ Legionella detection system are described which permit the rapid analysis of bacterial colonies taken from Legionella selective media. Capillary PCR permitted twice the number of samples to be analysed with a single kit. When PCR was positive for Leg. pneumophila , this result was confirmed by seroagglutination. The reverse dot blot hybridization assay was only used where PCR indicated a Legionella sp. other than Leg. pneumophila , permitting further savings on detection system components. This technique and standard confirmation procedures were applied to 133 isolates arising from 63 water samples plated to Legionella isolation media. Results agreed except for two isolates which gave a positive result for Legionella spp. by PCR and hybridization but were negative using standard procedures. Raising the annealing/extension temperature of the PCR by 2 °C eliminated the false positive result with these two isolates but did not adversely effect the sensitivity of the assay, as determined by re-testing of 68 environmental isolates and testing of 69 new environmental isolates and 12 Legionella reference species. The modified technique provides a convenient and cost effective alternative to standard confirmation procedures.     

Rapid detection and enumeration of Legionella pneumophila in hot water systems by solid-phase cytometry

A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.     

Detection and identification of Legionella pneumophila by PCR-restriction fragment length polymorphism analysis of the RNA polymerase gene (rpoB)

The partial RNA polymerase beta-subunit coding gene (rpoB) sequences of 38 Legionella species (59 reference strains) were used to select both Legionella genus-specific and Legionella pneumophila species-specific primers to amplify the 347-bp and 217-bp DNAs, respectively. Enzyme restriction sites for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis were also generated by a computer program. Thirty-eight Legionella species were well differentiated by the identification scheme for Legionella genus-specific PCR-RFLP using HaeIII, AluI, CfoI, PstI, and MaeII. The most common and important pathogenic species, L. pneumophila, was differentiated into two subspecies (L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri) by both Legionella genus-specific PCR-RFLP and L. pneumophila species-specific PCR-RFLP using BamHI. Eighty-two Korean culture isolates could also be easily identified by both PCR-RFLP methods as 68 strains of L. pneumophila subsp. pneumophila, 11 strains of L. pneumophila subsp. fraseri, and three novel strains that were separately confirmed by 16S rDNA and rpoB sequence analysis. These results suggest that the rpoB PCR-RFLP for Legionella is a simple and convenient method, not only for specific detection, but also for the rapid identification of Legionella species.     

Detection of Legionella spp. in cooling tower water by the polymerase chain reaction method.

The presence of Legionella spp. in cooling tower water was investigated by using the polymerase chain reaction. Total Legionella spp. detection was performed with 20-mer 5S rRNA complementary DNA sequence primers, and specific Legionella pneumophila detection was performed with 20-mer and then 21-mer macrophage infectivity potentiator gene sequence primers. Of 27 cooling tower water samples, 25 were positive for Legionella spp., and 14 of these contained L. pneumophila.     

Legionella spp. in Puerto Rico cooling towers

Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.     

Legionella spp. in Puerto Rico cooling towers.

Water samples from air conditioning cooling towers receiving different treatment protocols on five large municipal buildings in San Juan, P.R., were assayed for various Legionella spp. and serogroups by using direct immunofluorescence. Several water quality parameters were also measured for each sample. Guinea pigs were inoculated with water samples to confirm pathogenicity and recover viable organisms. Legionella pneumophila serogroups 1 to 6, L. bozemanii, L. micdadei, L. dumoffii, and L. gormanii were observed in at least one of the cooling towers. L. pneumophila was the most abundant species; its density reached 10(5) cells per ml, which is within the range that is considered potentially pathogenic to humans. A significantly higher density of L. pneumophila was observed in the cooling tower water that was not being treated with biocides. Percent respiration (INT) and total cell activity (acridine orange direct count) were inversely correlated with bacterial density. This study demonstrates that Legionella spp. are present in tropical air-conditioning cooling systems and that, without continuous biocide treatment, they may reach densities that present a health risk.     

Identification and differentiation of Legionella pneumophila and Legionella spp. with real-time PCR targeting the 16S rRNA gene and species identification by mip sequencing

Fluorescent resonance energy transfer probes targeting the 16S rRNA gene were constructed for a sensitive and specific real-time PCR for identification and differentiation of Legionella pneumophila from other Legionella spp. For identification of non-L. pneumophila spp. by direct amplicon sequencing, two conventional PCR assays targeting the mip gene were established.     

PCR-酶切技术快速鉴定军团菌

[目的]建立一种新型的军团菌鉴定方法,并探讨该法在鉴定环境水源和临床标本军团菌菌株中的应用价值.[方法]根据军团菌16S rRNA基因保守序列设计引物,以分离培养得到的可疑军团菌菌株作为模板,采用PCR法对模板扩增,并用限制性内切酶对PCR产物进行酶切分析,建立一种嗜肺军团菌及非嗜肺军团菌的鉴定方法.对16株嗜肺军团菌、22株非嗜肺军团菌及12株其他细菌标准菌株进行检测,验证该方法的可靠性,最后用该法检测广州地区分离的169株可疑军团菌菌株并进行基因测序.[结果]该PCR方法检测嗜肺军团菌及非嗜肺军团菌所有标准菌株均为阳性,非军团菌检测结果均为阴性;进一步的Hinf Ⅰ酶切分析可准确的区分嗜肺军团菌标准菌株;广州地区分离的169株可疑军团菌菌株经该法检测发现160株为军团菌,其中79株为嗜肺军团菌,与基因测序检测结果一致.[结论]PCR-酶切技术可快速、特异地检测军团菌及嗜肺军团菌,适用于环境水源和临床标本可疑军团菌菌株的检测.     

Legionella pneumophila in commercial bottled mineral water

Sixty-eight commercial bottled mineral waters (64 brands, 68 different 'best-before dates') were tested for the presence of bacteria and fungi. Six samples were Legionella antigen positive and six were Legionella pneumophila PCR positive. Two samples were both Legionella antigen and L. pneumophila PCR positive. Legionella cultures were negative. Although the PCR might have detected only dead Legionella cells, the PCR has been described to detect specifically viable but not culturable (VBNC) L. pneumophila cells as well. Whether VBNC bacteria may be present in bottled mineral waters and the risk for infection this may pose for severely immunocompromised patients should be investigated.     

Cross-reactivity and sensitivity of two Legionella urinary antigen kits, Biotest EIA and Binax NOW, to extracted antigens from various serogroups of L. pneumophila and other Legionella species

Legionella antigen detection kits for diagnosing legionellosis from urine have become widely used, but basic information about reactivity of the kits to non-serogroup (SG) 1 L. pneumophila and other Legionella species remains incomplete. We evaluated Biotest EIA and the most recently developed Binax NOW by using in-vitro extracted antigens of 22 L. pneumophila SG 1 to 15 strains and of 27 other Legionella species. Both kits showed excellent sensitivity to L pneumophila SG 1 antigens, but reacted to different sets of non-SG I L. pneumophila with different sensitivity. No cross-reactivity was observed to Legionella species other than L. pneumophila.