A staphylococcal coagglutination test for detecting and serogrouping Legionella pneumophila

For detection of Legionella pneumophila and determination of its serogroup, the modified staphylococcal coagglutination test was studied in detail. Cross-reactions in serum-agglutination tests were observed among serogroups, but immunoglobulin-coated staphylococcal cells could detect L. pneumophila with high sensitivity (a cell concentration with an absorbance of 0.008 at 660 nm could be detected) and serogroups could be determined without cross-reactions. Moreover the coexistence of other bacteria did not affect the results of the test. These results suggest that the staphylococcal coagglutination test is useful for detection and identification of Legionella, especially in environmental samples, and for serogrouping of isolates of L. pneumophila from clinical specimens.     

一株与同种13个型抗原广泛交叉的嗜肺军团菌

从太原市郊晋阳湖水分离到一株细菌,命名为Jin-1。其形态染色性、营养需要、生长特性、生化反应性、DNA和蛋白质分析结果均符合嗜肺军团菌鉴定要求。该株在IgM介导的两种凝集反应中与嗜肺军团菌14个血清群(型)标准株抗原普遍交叉,在IgG介导的IFA、ELISA、dot-ELISA中则呈现嗜肺军团菌血清群5特异性。鉴定Jin-1为抗原结构较标准株复杂的嗜肺军团菌5型。种、型鉴定结果已为CDC证实。在种内型间抗原交叉范围如此广泛的嗜肺军团菌尚未见于以往文献。因此认为Jin-1的交叉性抗原可能属于胸腺非依赖性抗原。     

Temperature-dependent expression of flagella in Legionella

Legionella pneumophila, the causative agent of Legionnaires' disease, was analysed by electron microscopy for production of surface structures. Crystalline surface (S-) layers and fimbriae were not detected, but monotrichous flagellation was seen. Polyclonal antibodies specific for the 47 kDa flagellin subunit of L. pneumophila Philadelphia I were used in Western blots to confirm the presence of flagella subunits in various L. pneumophila strains tested, but the antiserum also reacted with flagellin subunits of L. micdadei, L. hackelia [serogroup (SG) 1 and SG2] and L. longbeachae (SG2). Flagellation of Legionellae was shown to be temperature regulated. When the growth temperature of virulent and avirulent of strain L. pneumophila Philadelphia I was shifted from 30 degrees C to either 37 or 41 degrees C, a decrease in the percentage of flagellated bacteria within the population was observed.     

The use of an immunoenzyme method for the rapid diagnosis of legionellosis

An enzyme immunoassay (EIA) system for the detection of L. pneumophila antigen in clinical material (sputum, urine, bronchial washings) has been developed. The use of EIA permits the detection of L. pneumophila antigen in the urine of 75-80% of patients during the first week of the disease. The specificity and sensitivity of EIA makes it possible to recommend this method for the rapid diagnosis of L. pneumophila infection.     

嗜肺军团菌实时荧光定量PCR快速检测方法的建立

目的：建立针对嗜肺军团菌Mip基因的实时荧光定量TaqMan PCR检测方法,并进行自来水和空调冷却水模拟标本的检测评价。方法：根据嗜肺军团菌Mip基因的特异性序列设计引物和TaqMan探针,建立嗜肺军团菌的实时荧光定量TaqMan PCR快速检测方法,对方法进行灵敏度及特异性评价,并对自来水和空调冷却水模拟标本中的嗜肺军团菌进行检测。结果：建立的方法对嗜肺军团菌的检测具有高度特异性,与3种非嗜肺军团菌和6种其他呼吸道病原均没有交叉反应;基因组DNA的检测灵敏度为1.6pg／μL,模拟自来水和空调冷却水标本的检测灵敏度为10CFU／mL。结论：建立的TaqMan荧光定量PCR方法特异、灵敏、快速,适于嗜肺军团菌的日常监测和暴发疫情的应急诊断。     

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.     

Biofilms as major sources of Legionella spp. in hydrothermal areas and their dispersion into stream water

Abstract Water and biofilms from two hydrothermal areas in central Portugal, and one hydrothermal area in New Mexico, USA, were examined for Legionella spp. In general, Legionella spp were isolated in higher numbers from biofilms than from water, although one biofilm with a temperature of 50°C, did not yield isolates of these organisms. In one area L. pneumophila serogroup (sg) 3 constituted the major population in the thermal discharge by the stream and the biofilm below it; however, L. pneumophila sg 1 was predominant in the sediments of the stream bed with minor thermal springs below the main discharge and in the water downstream. No Legionellae were isolated from water upstream of the hydrothermal area indicating that the thermal area was the source of the organisms in the stream water. In the other two hydrothermal areas, L. pneumophila sg 1 constituted the major population isolated, whereas L. pneumophila sg 3 was absent or isolated in low numbers. Isolates of L. micdadei were also recovered from one hydrothermal area, while ‘ L. londoniensis ’ was isolated from another.     

Use of a coagglutination reaction based on a Soviet staphylococcal reagent for serogrouping Streptococcus group B and meningococci

The coagglutination (COA) test is used for the serogrouping of streptococci, group B, and meningococci. The trial of a Soviet commercial preparation (staphylococcal reagent) has shown good prospects for its use in the COA test and high specificity of this test method. The availability of highly specific immune sera permits making standard kits for the identification of different infective agents in the COA test.     

Incubation of water samples containing amoebae improves detection of legionellae by the culture method.

Some protozoans isolated from aquatic habitats, including domestic water supplies, can support the intracellular replication of autochthonous legionellae in vitro. We studied the effect of incubating water samples containing amoebae on the sensitivity of culture for legionellae. Samples collected during investigations of legionellosis epidemics and shown by conventional culture procedures to contain amoebae, but not legionellae, were incubated at 35 degrees C and replated. Legionellae were recovered from 59 of 144 such samples. Species isolated included L. pneumophila, L. anisa, L. bozemanii, L. gormanii, L. micdadei, L. rubrilucens, L. sainthelensi, L. steigerwaltii, and an unnamed species. Acanthamoeba polyphaga, Acanthamoeba hatchetti, a Rosculus sp., Hartmannella vermiformis, and Vahlkampfia spp. were among the autochthonous amoebae identified. Legionellae were recovered by this procedure from only 3 of 63 samples that were negative for amoebae by conventional culture procedures. These results show that water samples negative for legionellae, but positive for amoebae, by standard culture techniques should be incubated and replated to maximize the sensitivity of culture for legionellae.     

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.     

Rapid detection and identification of Legionella pneumophila by a membrane immunoassay

Legionella pneumophila was detected and identified by an immunoblot assay using a monoclonal antibody specific to serogroups 1 to 8. Samples containing L. pneumophila were plated on buffered charcoal yeast extract agar supplemented with glycine, vancomycin, and polymyxin B. After incubation at 35 degrees C for 3 days, colonies were transferred to nitrocellulose membranes by blotting. Simultaneous detection and identification of L. pneumophila were done by treating the membrane with the monoclonal antibody and a peroxidase conjugate to mouse immunoglobulins. A diffuse cross-reaction was observed with Pseudomonas fluorescens colonies, but this was a low-level reaction that could easily be differentiated from the strong specific reactions to L. pneumophila.     

Incubation of water samples containing amoebae improves detection of legionellae by the culture method.

Some protozoans isolated from aquatic habitats, including domestic water supplies, can support the intracellular replication of autochthonous legionellae in vitro. We studied the effect of incubating water samples containing amoebae on the sensitivity of culture for legionellae. Samples collected during investigations of legionellosis epidemics and shown by conventional culture procedures to contain amoebae, but not legionellae, were incubated at 35 degrees C and replated. Legionellae were recovered from 59 of 144 such samples. Species isolated included L. pneumophila, L. anisa, L. bozemanii, L. gormanii, L. micdadei, L. rubrilucens, L. sainthelensi, L. steigerwaltii, and an unnamed species. Acanthamoeba polyphaga, Acanthamoeba hatchetti, a Rosculus sp., Hartmannella vermiformis, and Vahlkampfia spp. were among the autochthonous amoebae identified. Legionellae were recovered by this procedure from only 3 of 63 samples that were negative for amoebae by conventional culture procedures. These results show that water samples negative for legionellae, but positive for amoebae, by standard culture techniques should be incubated and replated to maximize the sensitivity of culture for legionellae.     

Detection of Legionella pneumophila antibodies in the blood serum of pneumonia patients by an immunoenzyme method

The conditions of making the enzyme immunoassay (EIA) for the detection of antibodies to L. pneumophila have been optimized. The use of L. pneumophila purified serotypic antigen at a concentration of 0.25 micrograms/ml for the sensitization of polystyrene plates has been shown to increase the sensitivity and specificity of the assay. 220 patients with severe pneumonia have been examined. As revealed in this investigation, antibodies to L. pneumophila can be detected in 12.2% of cases. A high degree of correlation (94.4%) between the results of EIA and the indirect immunofluorescence test has been shown.     

Monoclonal antibodies to Legionella pneumophila cytolysin

The fusion of spleen cells, taken from BALB/c mice immunized with the purified preparation of L. pneumophila cytolysin, with cells Sp2/0 and NP has been carried out. As a result, hybridoma cells producing IgG1, IgG3 and IgM antibodies to this protein have been obtained. All monoclonal antibodies (McAb) thus obtained react with L. pneumophila strain lysates in the precipitation test, while IgG3 and IgM antibodies react with erythrocyte diagnostic agents prepared from the lysate of L. pneumophila cells in the hemagglutination test. In the Western blot assay, McAb react with the 37 KD protein (cytolysin) and a number of other proteins from L. pneumophila cultures and L. pneumophila cell lysate, but do not react with the species-specific protein with a molecular weight of 29 KD, contained in the outer membrane of L. pneumophila, as well as with other species: L. bozemanii, L. dumoffii, L. longbeachae, L. micdadei. The possibility of using these McAb conjugated with FITC and peroxidase for the rapid diagnosis of Legionella infection is shown.     

Rapid detection and identification of Legionella pneumophila by a membrane immunoassay.

Legionella pneumophila was detected and identified by an immunoblot assay using a monoclonal antibody specific to serogroups 1 to 8. Samples containing L. pneumophila were plated on buffered charcoal yeast extract agar supplemented with glycine, vancomycin, and polymyxin B. After incubation at 35 degrees C for 3 days, colonies were transferred to nitrocellulose membranes by blotting. Simultaneous detection and identification of L. pneumophila were done by treating the membrane with the monoclonal antibody and a peroxidase conjugate to mouse immunoglobulins. A diffuse cross-reaction was observed with Pseudomonas fluorescens colonies, but this was a low-level reaction that could easily be differentiated from the strong specific reactions to L. pneumophila.     

Rapid detection of the gene of Legionella pneumophila using the fluorescence polarization with the asymmetric PCR.

We attempted the rapid detection method of Legionella pneumophila by the asymmetric PCR and the fluorescence polarization. Eleven extracted DNAs from L. pneumophila serogroup 1 to approximately 6, L. bozemanii, L. dumoffii, L. gormanii, L. micdadei, and Pseudomonas aeruginosa were amplified by asymmetric PCR, and the polarization of those products were measured. Only the polarization of L. pneumophila serogroup 1 to approximately 6 rose within a few minutes after the beginning of measurement. The sensitivity to L. pneumophila using this method was 10(3) cells.     

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.     

Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples

Legionella pneumophila, a bacterium that replicates within aquatic amoebae and persists in the environment as a free-living microbe, is the causative agent of Legionnaires' disease. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease, and within the 15 serogroups (Sg), L. pneumophila Sg1 causes more than 84% of Legionnaires' disease worldwide. Thus, rapid and specific identification of L. pneumophila Sg1 is of the utmost importance for evaluation of the contamination of collective water systems and the risk posed. Previously we had shown that about 20 kb of the 33-kb locus carrying the genes coding for the proteins involved in lipopolysaccharide biosynthesis (LPS gene cluster) by L. pneumophila was highly specific for Sg1 strains and that three genes (lpp0831, wzm, and wzt) may serve as genetic markers. Here we report the sequencing and comparative analyses of this specific region of the LPS gene cluster in L. pneumophila Sg6, -10, -12, -13, and -14. Indeed, the wzm and wzt genes were present only in the Sg1 LPS gene cluster, which showed a very specific gene content with respect to the other five serogroups investigated. Based on this observation, we designed primers and developed a classical and a real-time PCR method for the detection and simultaneous identification of L. pneumophila Sg1 in clinical and environmental isolates. Evaluation of the selected primers with 454 Legionella and 38 non-Legionella strains demonstrated 100% specificity. Sensitivity, specificity, and predictive values were further evaluated with 209 DNA extracts from water samples of hospital water supply systems and with 96 respiratory specimens. The results showed that the newly developed quantitative Sg1-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk L. pneumophila Sg1 in water and clinical samples.     

The action of interferon preparations on strains of Legionella of various serogroups and species

The action of different preparations of interferon on Legionella strains has been studied in vivo and in vitro. The preparations of leukinferon at a concentration of 500 international units (I.U.) and reaferon at a concentration of 10,000 I.U. have been found to produce an inhibiting effect on Legionella strains in vitro, in a medium with carbon-yeast agar. Leukinferon at a concentration of 125 I.U. suppresses the growth of L. pneumophila also in a liquid medium. The preparation of leukinferon at a minimal concentration of 100 I.U. has been found to suppress the development of lethal infection in chick embryos infected with L. pneumophila strain Philadelphia 1.     

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.