LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.     

Entry of poliovirus into cells does not require a low-pH step.

The requirement of a low-pH step during poliovirus entry was investigated by using the macrolide antibiotic bafilomycin A1, which is a powerful and selective inhibitor of the vacuolar proton-ATPases. Thus, viruses such as Semliki Forest virus and vesicular stomatitis virus that enter cells through endosomes and need their acidification, are potently inhibited by bafilomycin A1, whereas poliovirus infection is not affected by the antibiotic. The presence of lysosomotropic agents such as chloroquine, amantadine, dansylcadaverine, and monensin during poliovirus entry did not inhibit infection, further supporting the idea that poliovirus does not depend on a low-pH step to enter the cytoplasm. The effect of bafilomycin A1 on other members of the Picornaviridae family was also assayed. Encephalomyocarditis virus entry into HeLa cells was not affected by the macrolide antibiotic, whereas rhinovirus was sensitive. Coentry of toxins, such as alpha-sarcin, with viral particles was potently inhibited by bafilomycin A1, indicating that an active vacuolar proton-ATPase is necessary for the early membrane permeabilization (coentry of alpha-sarcin) induced by poliovirus to take place.     

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.     

Detection of Legionella spp. and Legionella pneumophila in water samples of Spain by specific real-time PCR

Legionella pneumophila is the primary cause of the legionellosis diseases (90 %) (Yu et al. in J Infect Dis 186:127–128, 2002; Doleans et al. in J Clin Microbiol 42:458–460, 2004; Den Boer et al. in Clin Microbiol Infect 14:459–466, 2008). In this study, methodologies based on molecular biology were developed in order to provide a quick diagnosis of the bacterial presence in water samples of Spain. Multiplex real-time polymerase chain reaction assays were realized to target the 16S rRNA and macrophage infectivity potentiator (mip) genes of, respectively, Legionella spp. and L. pneumophila including in the design of an internal control. The results obtained by the culture and the gene amplification methods agreed in 94.44 % for the 16S rRNA gene, and a concordance of 66.67 % of the cases was obtained for the mip gene.     

The DotA protein from Legionella pneumophila is secreted by a novel process that requires the Dot/Icm transporter.

Legionella pneumophila requires the dot/icm genes to create an organelle inside eukaryotic host cells that will support bacterial replication. The dot/icm genes are predicted to encode a type IV-related secretion apparatus. However, no proteins have been identified that require the dot/icm genes for secretion. In this study we show that the DotA protein, which was previously found to be a polytopic membrane protein, is secreted by the Dot/Icm transporter into culture supernatants. Secreted DotA protein was purified and N-terminal sequencing of the purified protein revealed that a 19 amino acid leader peptide is removed from DotA prior to secretion. Extracellular DotA protein did not fractionate in membrane vesicles. Structures containing secreted DotA protein were visualized by electron microscopy and were shaped like hollow rings. These data indicate that the large poly topic membrane protein DotA is secreted from L.pneumophila by a unique process. This represents the first target secreted by the dot/icm-encoded apparatus and demonstrates that this transporter is competent for protein secretion.     

Initiation of adenovirus DNA replication does not occur via a hairpin mechanism.

Models have been proposed suggesting that initiation of adenovirus DNA replication might occur via a hairpin mechanism. A consequence of the models is a covalent linkage of progeny and parental DNA in newly completed molecules. Analysis of mature molecules from KB cells infected with adenovirus type 5 indicates that, if a hairpin mechanism is involved, the length of the hairpin must be shorter than 50 basepairs long. Recent nucleotide sequence analysis of the termini of adenovirus type 5 DNA (P.H.Steenbergh et al. (1977) Nucl. Acids Res. 4, 4371-4389) has shown that a hairpin of this size does not exist and that therefore a hairpin mechanism is unlikely.     

Induction of apoptosis by Sindbis virus occurs at cell entry and does not require virus replication

Sindbis virus (SV) is an alphavirus that causes encephalitis in mice and can lead to the apoptotic death of infected cells. To determine the step in virus replication during which apoptosis is triggered, we used UV-inactivated SV, chemicals that block virus fusion or protein synthesis, and cells that do and do not express heparan sulfate, the initial binding molecule for SV infection of many cells. In initial experiments, UV-inactivated neuroadapted SV (NSV) induced apoptosis in Chinese hamster ovary (CHO) cells lacking heparan sulfate in the presence of cycloheximide. When fusion of prebound UV-inactivated NSV was rapidly induced at the plasma membrane by exposure to acidic pH, apoptosis was induced in CHO cells with or without heparan sulfate in the presence or absence of cycloheximide in a virus dose-dependent manner. In N18 neuroblastoma cells, the relative virulence of the virus strain was an important determinant of apoptosis induced by UV-inactivated SV. Treatment of N18 cells with monensin to prevent endosomal acidification an hour before, but not 2 h after, exposure to live NSV blocked the induction of cell death, as did treatment with NH(4)Cl or bafilomycin A1. These studies indicate that SV can induce apoptosis at the time of fusion with the cell membrane and that virus replication is not required.     

The effect of water activity on Legionella spp. growth and survival

Growth and survival of Legionella spp. at various water activity (aw) levels were determined. Compared with Escherichia coli, the growth of Legionella spp. was limited to a high aw environment (greater than or equal to 0.98).     

The effect of water activity on Legionella spp. growth and survival

Growth and survival of Legionella spp. at various water activity (a_w) levels were determined. Compared with Escherichia coli , the growth of Legionella spp. was limited to a high a_w environment (≥0.98).     

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.     

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.     

RCN1-regulated phosphatase activity and EIN2 modulate hypocotyl gravitropism by a mechanism that does not require ethylene signaling

The roots curl in naphthylphthalamic acid1 (rcn1) mutant of Arabidopsis (Arabidopsis thaliana) has altered auxin transport, gravitropism, and ethylene response, providing an opportunity to analyze the interplay between ethylene and auxin in control of seedling growth. Roots of rcn1 seedlings were previously shown to have altered auxin transport, growth, and gravitropism, while rcn1 hypocotyl elongation exhibited enhanced ethylene response. We have characterized auxin transport and gravitropism phenotypes of rcn1 hypocotyls and have explored the roles of auxin and ethylene in controlling these phenotypes. As in roots, auxin transport is increased in etiolated rcn1 hypocotyls. Hypocotyl gravity response is accelerated, although overall elongation is reduced, in etiolated rcn1 hypocotyls. Etiolated, but not light grown, rcn1 seedlings also overproduce ethylene, and mutations conferring ethylene insensitivity restore normal hypocotyl elongation to rcn1. Auxin transport is unaffected by treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid in etiolated hypocotyls of wild-type and rcn1 seedlings. Surprisingly, the ethylene insensitive2-1 (ein2-1) and ein2-5 mutations dramatically reduce gravitropic bending in hypocotyls. However, the ethylene resistant1-3 (etr1-3) mutation does not significantly affect hypocotyl gravity response. Furthermore, neither the etr1 nor the ein2 mutation abrogates the accelerated gravitropism observed in rcn1 hypocotyls, indicating that both wild-type gravity response and enhanced gravity response in rcn1 do not require an intact ethylene-signaling pathway. We therefore conclude that the RCN1 protein affects overall hypocotyl elongation via negative regulation of ethylene synthesis in etiolated seedlings, and that RCN1 and EIN2 modulate hypocotyl gravitropism and ethylene responses through independent pathways.     

Legionella pneumophila Strain 130b Possesses a Unique Combination of Type IV Secretion Systems and Novel Dot/Icm Secretion System Effector Proteins

Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.Many bacterial pathogens use specialized protein secretion systems to deliver into host cells virulence effector proteins that interfere with the antimicrobial responses of the host and facilitate the survival of the pathogen (5, 10, 22, 76). The components of these secretion systems are highly conserved. Comparative bioinformatic analysis of pathogen genomes revealed an ever-increasing number of proteins that are likely to be translocated virulence effectors. Only a few effectors have been characterized, and their biochemical functions are unknown, yet the identification of translocated effector proteins and their mechanism of action is fundamental to understanding the pathogenesis of many bacterial infections.Legionella pneumophila is the etiological agent of Legionnaires’ disease, which is an acute form of pneumonia (34, 66). L. pneumophila serogroup 1 accounts for more than 90% of all cases worldwide. Although L. pneumophila is an environmental organism, its ability to survive and replicate in amoebae, such as Acanthamoeba castellanii, has equipped the organism with the capacity to replicate in human cells (45, 58, 68, 80). Following the inhalation of aerosols containing L. pneumophila into the human lung, the bacteria promote their uptake by alveolar macrophages and epithelial cells (21, 44, 71), where they replicate within an intracellular vacuole that avoids fusion with the endocytic pathway (46, 47). L. pneumophila evades endosome fusion by establishing a replicative vacuole that shares many characteristics with the endoplasmic reticulum (ER) (48, 53, 89). The formation of the unique Legionella-containing vacuole (LCV) requires the Dot (defective in organelle trafficking)/Icm (intracellular multiplication) type IV secretion system (T4SS) (85, 91).Type IV secretion systems are versatile multiprotein complexes that can transport DNA and proteins to recipient bacteria or host cells (19, 36). Based on structural and organizational similarity, three main T4SS classes have been distinguished: T4SSA, T4SSB, and genomic island-associated T4SS (GI-T4SS) (3, 51). The genetic organization and components of T4SSA have high similarity to the classical VirB4/VirD4 transfer DNA (T-DNA) transfer system of Agrobacterium tumefaciens (3). In the sequenced L. pneumophila strains, three distinct T4SSAs with different prevalences among strains have been described: Lvh, Trb-1, and Trb-2 (37, 83, 86). The Lvh (Legionella vir homologues) T4SSA is not required for intracellular bacterial replication in macrophages and amoebae but seems to contribute to infection at lower temperatures and inclusion in Acanthamoeba castellanii cysts (6, 78, 86).The Dot/Icm T4SSB secretes and translocates multiple bacterial effector proteins into the vacuolar membrane and cytosol of the host cell (31, 70). The functions of the great majority of these proteins are unknown. Several effectors have similarity to eukaryotic proteins or carry eukaryotic motifs (7, 16, 25). They are predicted to allow L. pneumophila to manipulate host cell processes by functional mimicry (31, 70). Many of the effectors have paralogues or belong to related protein families that are likely to have overlapping functions.Comparative analysis of the recent L. pneumophila genome sequences has revealed their diversity and plasticity (16, 18, 88). This plasticity enables the bacterium to acquire new genetic factors, including new effector proteins that enhance bacterial replication and survival in eukaryotic cells. This has resulted in a diverse species in which 7 to 11% of the genes in each L. pneumophila isolate are strain specific (38). Some of the diversity occurs among genes encoding Dot/Icm effectors, including those within the same family. For example some ankyrin repeat and F-box effector genes are highly conserved, while others vary considerably between L. pneumophila isolates (16, 41, 62, 73, 75). Even though it is not experimentally proven, the subsequent selection of Dot/Icm effectors among different L. pneumophila isolates might reflect their usefulness in host-pathogen interactions, whereby different effector repertoires are maintained during adaptation to different environmental niches or hosts. This may then translate into differences in virulence during opportunistic infection.In this study, we sequenced the genome of L. pneumophila serogroup 1 strain 130b (ATCC BAA-74, also known as Wadsworth or AA100) (29, 30) and analyzed the sequence for T4SSs and novel Dot/Icm effectors. This analysis established that the strain encodes a unique combination of T4SSs and a set of Dot/Icm effectors that had not been described previously but that are present in a range of clinical and environmental L. pneumophila isolates. The new effectors represent the latest members of an ever-growing list of T4SS substrates and presumably reflect the great capacity of L. pneumophila for adaptation to a variety of hosts.     

Introduction of Monochloramine into a Municipal Water System: Impact on Colonization of Buildings by Legionella spp.

Legionnaires' disease (LD) outbreaks are often traced to colonized potable water systems. We collected water samples from potable water systems of 96 buildings in Pinellas County, Florida, between January and April 2002, during a time when chlorine was the primary residual disinfectant, and from the same buildings between June and September 2002, immediately after monochloramine was introduced into the municipal water system. Samples were cultured for legionellae and amoebae using standard methods. We determined predictors of Legionella colonization of individual buildings and of individual sampling sites. During the chlorine phase, 19 (19.8%) buildings were colonized with legionellae in at least one sampling site. During the monochloramine phase, six (6.2%) buildings were colonized. In the chlorine phase, predictors of Legionella colonization included water source (source B compared to all others, adjusted odds ratio [aOR], 6.7; 95% confidence interval [CI], 2.0 to 23) and the presence of a system with continuously circulating hot water (aOR, 9.8; 95% CI, 1.9 to 51). In the monochloramine phase, there were no predictors of individual building colonization, although we observed a trend toward greater effectiveness of monochloramine in hotels and single-family homes than in county government buildings. The presence of amoebae predicted Legionella colonization at individual sampling sites in both phases (OR ranged from 15 to 46, depending on the phase and sampling site). The routine introduction of monochloramine into a municipal drinking water system appears to have reduced colonization by Legionella spp. in buildings served by the system. Monochloramine may hold promise as community-wide intervention for the prevention of LD.     

PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB

A total of 25 gyrB gene sequences from 20 Legionella pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were obtained and analyzed, and a multiplex PCR for the simultaneous detection of Legionella bozemanae, Legionella longbeachae, Legionella micdadei and Legioenella pneumophila, and two single PCRs for the differentiation of L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri were established. The multiplex PCR method was shown to be highly specific and reproducible when tested against 41 target strains and 17 strains of other bacteria species. The sensitivity of the multiplex PCR was also analyzed and was shown to detect levels as low as 1 ng of genomic DNA or 10 colony-forming units (CFUs) per milliliter in mock water samples. Sixty-three air conditioner condensed water samples from Shanghai City were examined, and the result was validated using 16S rRNA sequencing. The data reported here demonstrate that the multiplex PCR method described is efficient and convenient for the detection of Legionella species in water samples. Twenty L. pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were used for the validation of the two L. pneumophila subspecies-specific PCR methods, and the results indicated that the two PCR methods were both highly specific and convenient for the identification of L. pneumophila at the subspecies level.     

Introduction of monochloramine into a municipal water system: impact on colonization of buildings by Legionella spp

Legionnaires' disease (LD) outbreaks are often traced to colonized potable water systems. We collected water samples from potable water systems of 96 buildings in Pinellas County, Florida, between January and April 2002, during a time when chlorine was the primary residual disinfectant, and from the same buildings between June and September 2002, immediately after monochloramine was introduced into the municipal water system. Samples were cultured for legionellae and amoebae using standard methods. We determined predictors of Legionella colonization of individual buildings and of individual sampling sites. During the chlorine phase, 19 (19.8%) buildings were colonized with legionellae in at least one sampling site. During the monochloramine phase, six (6.2%) buildings were colonized. In the chlorine phase, predictors of Legionella colonization included water source (source B compared to all others, adjusted odds ratio [aOR], 6.7; 95% confidence interval [CI], 2.0 to 23) and the presence of a system with continuously circulating hot water (aOR, 9.8; 95% CI, 1.9 to 51). In the monochloramine phase, there were no predictors of individual building colonization, although we observed a trend toward greater effectiveness of monochloramine in hotels and single-family homes than in county government buildings. The presence of amoebae predicted Legionella colonization at individual sampling sites in both phases (OR ranged from 15 to 46, depending on the phase and sampling site). The routine introduction of monochloramine into a municipal drinking water system appears to have reduced colonization by Legionella spp. in buildings served by the system. Monochloramine may hold promise as community-wide intervention for the prevention of LD.     

Cutting edge: Inhibition of hepatitis B virus replication by activated NK T cells does not require inflammatory cell recruitment to the liver.

We have previously reported that intrahepatic NK T cells activated by alpha-galactosylceramide inhibit hepatitis B virus replication noncytopathically in the liver of transgenic mice. This effect is mediated by antiviral cytokines directly produced by activated NK T cells and/or by other cytokine-producing inflammatory cells that are recruited into the liver. In this study, we demonstrated that IFN-gamma produced by activated NK T cells induced parenchymal and nonparenchymal cells of the liver to produce high levels of CXC chemokine ligands 9 and 10, which mediated the intrahepatic recruitment of lymphomononuclear inflammatory cells. Recruitment of these cells was not necessary for the antiviral activity, indicating that direct activation of the intrahepatic resident NK T cell is sufficient to control viral replication in this model.