Legionella oakridgensis: unusual new species isolated from cooling tower water.

We describe a new species of Legionella represented by 10 strains isolated from industrial cooling towers. Legionella oakridgensis differed genetically from the other seven species of Legionella in DNA hybridization studies and differed serologically in direct fluorescent-antibody tests. The new species, unlike all other species except L. jordanis, did not require added L-cysteine for growth in serial transfer on charcoal-yeast extract agar. L. oakridgensis, as well as three other species tested, required L-cysteine for primary isolation from animal tissues. L. oakridgensis was the only species of Legionella that failed to produce alkaline phosphatase at pH 8.5. In all other respects, it resembled other species of Legionella, including having a high content of branched-chain cellular fatty acids and being pathogenic for guinea pigs. These bacteria have not yet been associated with human disease, but they are potential causes of legionellosis.     

Distribution of Legionella longbeachae serogroup 1 and other legionellae in potting soils in Australia.

Legionella longbeachae serogroup 1 and other Legionella spp. were isolated from 73% of 45 potting soils made in Australia by 13 manufacturers but were not detected in 19 potting soils made in Greece, Switzerland, and the United Kingdom examined between March 1989 and May 1990. Several Legionella species were isolated from a small number of samples of uncomposted pine sawdusts, but it is not known whether sawdust was the source of some of the legionellae found in potting soils. Legionella spp. persisted for periods ranging from 3 to 10 months in a potting soil held at temperatures between -20 and 35 degrees C. Isolates of L. longbeachae serogroup 1 from soil did not grow at 43 degrees C, a temperature which was also lethal for this species in soil. Most Legionella spp. isolated from potting and natural soils belonged to one distinct group according to analysis of ubiquinones and were serologically related to several known species in this group. A small number of potting soils contained L. pneumophila and L. micdadei.     

Distribution of Legionella longbeachae serogroup 1 and other legionellae in potting soils in Australia

Legionella longbeachae serogroup 1 and other Legionella spp. were isolated from 73% of 45 potting soils made in Australia by 13 manufacturers but were not detected in 19 potting soils made in Greece, Switzerland, and the United Kingdom examined between March 1989 and May 1990. Several Legionella species were isolated from a small number of samples of uncomposted pine sawdusts, but it is not known whether sawdust was the source of some of the legionellae found in potting soils. Legionella spp. persisted for periods ranging from 3 to 10 months in a potting soil held at temperatures between -20 and 35 degrees C. Isolates of L. longbeachae serogroup 1 from soil did not grow at 43 degrees C, a temperature which was also lethal for this species in soil. Most Legionella spp. isolated from potting and natural soils belonged to one distinct group according to analysis of ubiquinones and were serologically related to several known species in this group. A small number of potting soils contained L. pneumophila and L. micdadei.     

Antibodies isolated by using cloned surface antigens recognize antigenically related components of Legionella pneumophila and other Legionella species

We studied the antigenic cross-reactivity of surface proteins among various strains of Legionella pneumophila and other Legionella species by using a novel method of antibody purification. Anti-bacterial antibodies in hyperimmune sera were adsorbed to and eluted from the surface of recombinant E. coli cells that express individual L. pneumophila antigens on their surface. These affinity-purified antibodies were then used to probe protein immunoblots prepared from the test strains to detect cross-reactive domains. We found that antigenic proteins are generally conserved in all L. pneumophila serogroups. Although some of these antigenic domains are shared with members of other Legionella species, they are associated with proteins of different molecular mass. Our approach to the study of antigenic cross-reactivity has potential advantages over similar studies that use either monoclonal antibodies or monospecific antibodies prepared by immunization with purified antigens.     

A pair of highly conserved two-component systems participates in the regulation of the hypervariable FIR proteins in different Legionella species

Legionella pneumophila and other pathogenic Legionella species multiply inside protozoa and human macrophages by using the Icm/Dot type IV secretion system. The IcmQ protein, which possesses pore-forming activity, and IcmR, which functions as its chaperone, are two essential components of this system. It was previously shown that in 29 Legionella species, a large hypervariable-gene family (fir genes) is located upstream from a conserved icmQ gene, but although nonhomologous, the FIR proteins were found to function similarly together with their corresponding IcmQ proteins. Alignment of the regulatory regions of 29 fir genes revealed that they can be divided into three regulatory groups; the first group contains a binding site for the CpxR response regulator, which was previously shown to regulate the L. pneumophila fir gene (icmR); the second group, which includes most of the fir genes, contains the CpxR binding site and an additional regulatory element that was identified here as a PmrA binding site; and the third group contains only the PmrA binding site. Analysis of the regulatory region of two fir genes, which included substitutions in the CpxR and PmrA consensus sequences, a controlled expression system, as well as examination of direct binding with mobility shift assays, revealed that both CpxR and PmrA positively regulate the expression of the fir genes that contain both regulatory elements. The change in the regulation of the fir genes that occurred during the course of evolution might be required for the adaptation of the different Legionella species to their specific environmental hosts.     

Second serogroup of Legionella quinlivanii isolated from two unrelated sources in the United Kingdom.

A series of strains, presumptively identified as legionellas on the basis of their nutritional requirements and biochemical reactivity, were isolated from two unrelated environmental sources in the UK. Representatives of each of these series had a restriction endonuclease digest pattern indistinguishable from that of the Legionella quinlivanii type strain (1442-AUS-E) and the identity of these strains was confirmed by DNA homology studies. Serological examination of the two strains showed that they were distinct from the type strain 1442-AUS-E but indistinguishable from each other. A second serogroup, L. quinlivanii serogroup 2 (type strain LC870; NCTC 12434), is proposed to accommodate these strains.     

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.     

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.     

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.     

Legionella: from environmental habitats to disease pathology, detection and control

Studies on Legionella show a continuum from environment to human disease. Legionellosis is caused by Legionella species acquired from environmental sources, principally water sources such as cooling towers, where Legionella grows intracellularly in protozoa within biofilms. Aquatic biofilms, which are widespread not only in nature, but also in medical and dental devices, are ecological niches in which Legionella survives and proliferates and the ultimate sources to which outbreaks of legionellosis can be traced. Invasion and intracellular replication of L. pneumophila within protozoa in the environment play a major role in the transmission of Legionnaires' disease. Protozoa provide the habitats for the environmental survival and reproduction of Legionella species. L. pneumophila proliferates intracellularly in various species of protozoa within vacuoles studded with ribosomes, as it also does within macrophages. Growth within protozoa enhances the environmental survival capability and the pathogenicity (virulence) of Legionella . The growth requirements of Legionella , the ability of Legionella to enter a viable non-culturable state, the association of Legionella with protozoa and the occurrence of Legionella within biofilms complicates the detection of Legionella and epidemiological investigations of legionellosis. Polymerase chain reaction (PCR) methods have been developed for the molecular detection of Legionella and used in environmental and epidemiological studies. Various physical and chemical disinfection methods have been developed to eliminate Legionella from environmental sources, but gaining control of Legionella in environmental waters, where they are protected from disinfection by growing within protozoa and biofilms, remains a challenge, and one that must be overcome in order to eliminate sporadic outbreaks of legionellosis.     

用绿色荧光蛋白研究军团菌与寄主的相互关系

原生动物作为军团菌的天然寄主,在军团菌的生存、增殖、毒力和抗逆性等方面起着重要的作用。通过多次转化筛选,获得了一种高量表达绿色荧光蛋白基因gfpmut2的自发突变质粒。该质粒在嗜肺军团菌细胞内稳定复制和表达;转化了该质粒的嗜肺军团菌在自然光下即可发出明亮的绿色荧光。以转化菌饲喂嗜热四膜虫BF1株后,在荧光显微镜下能清楚观察到细菌在细胞内的形态变化、增殖和裂解宿主细胞的过程。为研究嗜肺军团菌与原生动物寄主的相互关系提供了一种简单而直观的方法。     

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.     

The potential of in situ hybridization and an immunogold assay to identify Legionella associations with other microorganisms.

Based on in vitro studies, bacteria in the genus Legionella are believed to multiply within protozoa such as amoebae in aquatic environments. Current methods used for detection of Legionella species, however, are not designed to show this relationship. Thus the natural intimate association of Legionella with other microorganisms remains to be clearly documented and the extent to which protozoa might be infected with Legionella species remains undefined. In this report we describe methods based on the use of Legionella specific reagents that would prove useful in describing its associations with other microorganisms. An immunogold and in situ hybridization technique have the potential to demonstrate the natural occurrence of Legionella species in free-living amoebae. In preliminary observations, however, bacteria reactive with Legionella specific reagents were often not intimately associated with amoebae. Bacteria occurred as free single cells, as cell aggregates, in proximity to other cells and debris, and only occasionally in close proximity to amoebae. Although some Legionella species replicate within amoebae, these preliminary observations suggest the bacteria may be encountered most frequently as extracellular microorganisms, either free-floating or in association with other structures or microorganisms. The future use of these techniques will aid in the elucidation of any naturally occurring relationships between Legionella species and other microorganisms.     

Problems associated with identification of Legionella species from the environment and isolation of six possible new species

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at -70 degrees C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionella jordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.     

High-performance liquid chromatographic analysis of ubiquinones from new Legionella species

Abstract The ubiquinone composition of Legionella feeleii, L. oakridgensis, L. rubrilucens, L. sainthelensi, L. wadsworthii and some environmental legionella isolates was investigated by high performance liquid chromatography. The results of the present study indicate ubiquinones are valuable criteria for the rapid identification of Legionella species and the recognition of new taxa.     

Vaccination with the major secretory protein of Legionella induces humoral and cell-mediated immune responses and protective immunity across different serogroups of Legionella pneumophila and different species of Legionella.

In a previous study, we demonstrated that immunization of guinea pigs with the major secretory protein (MSP) of Legionella pneumophila, serogroup 1 induced humoral and cell-mediated immune responses to MSP and protective immunity against lethal aerosol challenge with this serogroup of L. pneumophila. Although serogroup 1 L. pneumophila cause most cases of Legionnaires' disease, other serogroups of L. pneumophila and species of Legionella cause many cases. In this study, we have examined if immunization with MSP induces humoral and cell-mediated immune responses and protective immunity across different serogroups of L. pneumophila and species of Legionella. By immunoblot analysis, MSP from L. pneumophila serogroup 1 (Lp1 MSP), L. pneumophila serogroup 6 (Lp6 MSP), and Legionella bozemanii (Lb MSP) shared common epitopes recognized by guinea pig anti-Lp1 MSP antiserum. These MSP molecules, however, were not identical as they had different apparent m.w. Immunization of guinea pigs with MSP induced strong cell-mediated immune responses across the different serogroups and species, as indicated by splenic lymphocyte proliferation and cutaneous delayed-type hypersensitivity in response to both homologous and heterologous MSP. Immunization with MSP induced strong protective immunity across two serogroups of L. pneumophila; overall, 9 survived aerosol challenge with L. pneumophila serogroup 1 compared to 0 of 12 (0%) sham-immunized control animals (p = 3 x 10(-4), Cochran-Mantel-Haenzel chi 2 statistic for pooled data). Immunization with MSP also induced protective immunity across species of Legionella but protection was species-specific. Whereas immunization with Lb MSP induced protective immunity against L. pneumophila, neither immunization with Lp1 MSP nor immunization with Lb MSP induced protective immunity against L. bozemanii, which produces MSP. Not surprisingly, immunization with MSP did not induce protective immunity against MSP-negative Legionella micdadei. In the case of both L. bozemanii and L. micdadei, immunization with a sublethal dose did confer protective immunity to aerosol challenge indicating that these species do contain immunoprotective components. This study demonstrates that immunization with MSP induces humoral and cell-mediated immune responses across different serogroups of L. pneumophila and species of Legionella, but that the capacity of MSP immunization to induce protective immunity is species-specific. Nevertheless, an MSP vaccine has the potential to induce protective immunity against the great majority of cases of Legionnaires' disease.     

Biofilm formation by Legionella spp. in experiment

Ability of biofilm formation was studied in 28 strains belonging to 12 species of Legionella. Optimal conditions for formation of biofilms were ascertained using reference strain Legionella pneumophila Philadelphia 1. Comparative assessment of the ability of Legionella spp. to form biofilms was performed by cultivation in proteosopepton broth (for 96 hours) and in water (for up to 2 weeks). Highest rates of biofilm formation were observed for strains of L. pneumophila and L. longbeachae. Between L. pneumophila strains the most prominent ability to form biofilms was observed in newly isolated strains BLR-05 and TOTAL 1. Opportunity to use different ability of Legionella species to biofilm formation as a epidemiologically significant marker and for modeling of biofilms of Legionella in association with other microorganisms was discussed.     

Problems associated with identification of Legionella species from the environment and isolation of six possible new species.

Following investigation of an outbreak of legionellosis in South Australia, numerous Legionella-like organisms were isolated from water samples. Because of the limited number of commercially available direct fluorescent-antibody reagents and the cross-reactions found with some reagents, non-pneumophila legionellae proved to be difficult to identify and these isolates were stored at -70 degrees C for later study. Latex agglutination reagents for Legionella pneumophila and Legionella anisa developed by the Institute of Medical and Veterinary Science, Adelaide, Australia, were found to be useful as rapid screening aids. Autofluorescence was useful for placing isolates into broad groups. Cellular fatty acid analysis, ubiquinone analysis, and DNA hybridization techniques were necessary to provide definitive identification. The species which were isolated most frequently were L. pneumophila, followed by L. anisa, Legionella jamestowniensis, Legionella quinlivanii, Legionella rubrilucens, Legionella spiritensis, and a single isolate each of Legionella erythra, Legionella jordanis, Legionella birminghamensis, and Legionella cincinnatiensis. In addition, 10 isolates were found by DNA hybridization studies to be unrelated to any of the 26 currently known species, representing what we believe to be 6 possible new species.     

Second serogroup of Legionella quinlivanii isolated from two unrelated sources in the United Kingdom

R.J. BIRTLES, N. DOSHI, N.A. SAUNDERS AND T.G. HARRISON. 1991. A series of strains, presumptively identified as legionellas on the basis of their nutritional requirements and biochemical reactivity, were isolated from two unrelated environmental sources in the UK. Representatives of each of these series had a restriction endonuclease digest pattern indistinguishable from that of the Legionella quinlivanii type strain (1442-AUS-E) and the identity of these strains was confirmed by DNA homology studies. Serological examination of the two strains showed that they were distinct from the type strain 1442-AUS-E but indistinguishable from each other. A second serogroup, L. quinlivanii serogroup 2 (type strain LC870; NCTC 12434), is proposed to accommodate these strains.     

Occurrence and genetic diversity of uncultured Legionella spp. in drinking water treated at temperatures below 15 degrees C

Representatives of the genus Legionella were detected by use of a real-time PCR method in all water samples collected directly after treatment from 16 surface water (SW) supplies prior to postdisinfection and from 81 groundwater (GW) supplies. Legionella concentrations ranged from 1.1 x 10(3) to 7.8 x 10(5) cells liter(-1) and were significantly higher in SW treated with multiple barriers at 4 degrees C than in GW treated at 9 to 12 degrees C with aeration and filtration but without chemical disinfection. No Legionellae (<50 CFU liter(-1)) were detected in treated water by the culture method. Legionella was also observed in untreated SW and in untreated aerobic and anaerobic GW. Filtration processes in SW and GW treatment had little effect or increased the Legionella concentration, but ozonation in SW treatment caused about 1-log-unit reduction. A phylogenetic analysis of 16S rRNA gene sequences of 202 clones, obtained from a selection of samples, showed a high similarity (>91%) with Legionella sequences in the GenBank database. A total of 40 (33%) of the 16S rRNA gene sequences obtained from treated water were identified as described Legionella species and types, including L. bozemanii, L. worsleiensis, Legionella-like amoebal pathogen types, L. quateirensis, L. waltersii, and L. pneumophila. 16S rRNA gene sequences with a similarity of below 97% from described species were positioned all over the phylogenetic tree of Legionella. Hence, a large diversity of yet-uncultured Legionellae are common members of the microbial communities in SW and GW treated at water temperatures of below 15 degrees C.