High Prevalence,Genetic Diversity and Intracellular Growth Ability of Legionella in Hot Spring Environments

Background

Legionella is the causative agent of Legionnaires'' disease, and hot springs are a major source of outbreaks of this disease. It is important from a public health perspective to survey hot spring environments for the presence of Legionella.

Methods

Prospective surveillance of the extent of Legionella pollution was conducted at three hot spring recreational areas in Beijing, China in 2011. Pulsed-field gel electrophoresis (PFGE) and sequence-based typing (SBT) were used to describe the genetic polymorphism of isolates. The intracellular growth ability of the isolates was determined by interacting with J774 cells and plating the dilutions onto BCYE agar plates.

Results

Overall, 51.9% of spring water samples showed Legionella-positive, and their concentrations ranged from 1 CFU/liter to 2,218 CFU/liter. The positive rates of Legionella were significantly associated with a free chlorine concentration of ≥0.2 mg/L, urea concentration of ≥0.05 mg/L, total microbial counts of ≥400 CFU/ml and total coliform of ≥3 MPN/L (p<0.01). The Legionella concentrations were significantly associated with sample temperature, pH, total microbial counts and total coliform (p<0.01). Legionella pneumophila was the most frequently isolated species (98.9%), and the isolated serogroups included serogroups 3 (25.3%), 6 (23.4%), 5 (19.2%), 1 (18.5%), 2 (10.2%), 8 (0.4%), 10 (0.8%), 9 (1.9%) and 12 (0.4%). Two hundred and twenty-eight isolates were analyzed by PFGE and 62 different patterns were obtained. Fifty-seven L. pneumophila isolates were selected for SBT analysis and divided into 35 different sequence types with 5 main clonal groups. All the 57 isolates had high intracellular growth ability.

Conclusions

Our results demonstrated high prevalence and genetic polymorphism of Legionella in springs in Beijing, China, and the SBT and intracellular growth assay results suggested that the Legionella isolates of hot spring environments were pathogenic. Improved control and prevention strategies are urgently needed.     

Intracellular multiplication ofLegionella pneumophila in amoebae isolated from hospital hot water tanks

We studied the ability ofLegionella to multiply in potable water samples obtained from investigations of nosocomial legionellosis. AutochthonousLegionella multiplied in three of 14 hospital water samples after incubation at 35°C and 42°C. All three samples were from hot water tanks. Multiplication did not occur when a selected sample was filtered through a 0.45-m membrane and reinoculated with indigenousLegionella. We isolated bothLegionella pneumophila and one or more species of free-living amoebae, primarity members of theHartmannellidae, from each of these hot water tank samples. Amoebae from a total of six hot water tank samples were used for cocultivation studies withL. pneumophila. All amoebae supported multiplication ofLegionella in coculture at 35°C. Four of six isolates of amoebae supported multiplication oflegionella at 42°C, while none supported multiplication at 45°C. Gimenez staining and electron microscopy showed thatLegionella multiplied intracellularly in amoebae. Control of these amoebae in potable water may prevent colonization and multiplication ofLegionella in domestic hot water systems.     

Distribution of Legionella Species from Environmental Water Sources of Public Facilities and Genetic Diversity of L. pneumophila Serogroup 1 in South Korea

A total of 560 Legionella species were isolated from environmental water sources from public facilities from June to September 2008 throughout South Korea. The distribution of Legionella isolates was investigated according to geographical region, facility type, and sample type. The genetic diversity of 104 isolates of Legionella pneumophila serogroup 1 (sg 1) was analyzed by sequence-based typing (SBT). L. pneumophila was distributed broadly throughout Korea, accounting for 85.0% of the isolates, and L. pneumophila sg 1 predominated in all of the public facilities except for the springs. Legionella anisa and Legionella bozemanii predominated among non-L. pneumophila species (48.1% and 21.0%, respectively). The second most dominant strain differed depending on the facility type: L. anisa was the second most dominant strain in the buildings (10.8%), L. pneumophila sg 5 in public baths (21.6%), L. pneumophila sg 6 in factories (12.0%), and L. pneumophila sg 7 in hospitals (13.1%). In the SBT analysis, 104 L. pneumophila sg 1 isolates were differentiated into 26 sequence types (STs) and categorized into 3 clonal groups (CGs) and 10 singleton STs via the eBURST V3 program. ST1, a potential founder of major CG1, was commonly distributed (48.1%). The dominant ST in hot water was ST-K1 (7, 12, 17, 3, 35, 11, 11), which was designated in this study (36.1%). The second most dominant strain differed depending on the type of facility from which the samples were obtained. The unique allelic profile of ST-K1, obtained from hot water, was not found in the European Working Group for Legionella Infections (EWGLI) SBT database.Legionella species, ubiquitous Gram-negative bacteria, are found in a variety of artificial water systems, natural freshwaters, and soils. Currently, the Legionella genus includes 52 species and more than 70 different serogroups, and more than 20 species have been proven to be causative agents of Legionnaires'' disease (LD). The species Legionella pneumophila accounts for approximately 90% of confirmed cases of legionellosis, and L. pneumophila serogroup 1 (sg 1) has been recognized as the most important agent in this regard, as that specific strain was initially implicated as the pathogen causative of LD in 1977 (15; http://www.bacterio.cict.fr/l/legionellaceae.html). The other non-L. pneumophila sg 1 strains, sg 2 to 15, accounted for 7.4% of cases, and Legionella longbeachae (3.9%) and Legionella bozemanii (2.4%) have also been associated with the pathogen of LD. In particular, L. longbeachae has been recognized as accounting for 30.4% of community-acquired Legionella isolates in Australia and New Zealand (53).The most common transmission mechanism of legionellosis is the inhalation of aerosols from the water systems of artificial facilities, including large buildings, hotels, hospitals, public baths, spas, or decorative fountains contaminated by Legionella species (1). Therefore, hot water and water from cooling towers have been perceived as sources of infection in cases of community-acquired, nosocomially acquired, or travel-associated LD (15, 26, 31, 37, 38, 39, 41, 43). Thus, it is important from a public health perspective to continually survey environmental water systems for the presence of Legionella species (2, 34, 35). In particular, hot-water systems used as public baths, such as springs, spas, or tubs, have become a popular means of recreation in a lot of countries, including South Korea. The contamination of hot-water systems has gradually become recognized as an important risk factor all over the world (4, 12, 18, 23, 42, 50), as sources of legionellosis have been detected increasingly since 1982 (52) and many cases of nosocomially acquired (32, 51) and community-acquired (6, 7, 48) LD have been detected in Legionella-contaminated hot-water systems or hot springs.In South Korea, several cases of nosocomial infection and community-acquired pneumonia have occasionally been reported (9, 45) since the first recognized outbreak in South Korea in 1984, which was associated with Legionella gormanii (27). Since 2006, the Korean National Infectious Disease Surveillance (NIDS) program (http://dis.cdc.go.kr/) has reported an average of 20 cases of LD per year (29). In South Korea, surveys of Legionella acquired from environmental water in public facilities such as hot springs and public baths has been gradually enhanced since 2007. An annual training program for the detection of Legionella species from environmental water systems and clinical specimens is currently conducted for the personnel of 16 Provincial Institute of Health and Environment locations (PIHEs) throughout South Korea. Recently, the rate of detection of environmental Legionella bacteria has been gradually increasing (8.1% in 2006, 9.4% in 2007, and 10.3% in 2008).The principal objectives of this study were to assess the current distribution of Legionella species from environmental water sources from public facilities such as buildings, hotels, public baths, springs, hospitals, or factories throughout South Korea. Additionally, the molecular typing of L. pneumophila sg 1 isolates was conducted using sequence-based typing (SBT) to assess the genetic diversity among the isolates.     

Polyadenylated mRNA for the light-harvesting chlorophyll a/b protein

In thylakoid membranes isolated from green plants of parsley, pea, and barley, the light-harvesting chlorophyll a/b protein complex (LHCP, mol. weight: 25,000), is a major constituent. Poly(A)RNA isolated from these species was translated in a wheat germ, cell-free system. The in vitro translation products were treated with antibodies raised against the LHCP. This treatment resulted in the precipitation of a precursor protein (mol. weight: 29,000). Poly(A)RNA was also prepared from a cell culture ofPetroselinum that does not develop chloroplasts upon illumination. This poly(A)RNA is capable of stimulating amino acid incorporation in the in vitro translation system, however, it does not direct the synthesis of LHCP.     

Burkholderia sp. Induces Functional Nodules on the South African Invasive Legume Dipogon lignosus (Phaseoleae) in New Zealand Soils

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678^T which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.     

Close Genetic Relationship between Legionella pneumophila Serogroup 1 Isolates from Sputum Specimens and Puddles on Roads,as Determined by Sequence-Based Typing

We investigated the prevalence of Legionella species isolated from puddles on asphalt roads. In addition, we carried out sequence-based typing (SBT) analysis on the genetic relationship between L. pneumophila serogroup 1 (SG 1) isolates from puddles and from stock strains previously obtained from sputum specimens and public baths. Sixty-nine water samples were collected from puddles on roads at 6 fixed locations. Legionella species were detected in 33 samples (47.8%) regardless of season. Among the 325 isolates from puddles, strains of L. pneumophila SG 1, a major causative agent of Legionnaires'' disease, were the most frequently isolated (n = 62, 19.1%). Sixty-two isolates of L. pneumophila SG 1 from puddles were classified into 36 sequence types (STs) by SBT. ST120 and ST48 were identified as major STs. Environmental ST120 strains from puddles were found for the first time in this study. Among the 14 STs of the clinical isolates (n = 19), 4 STs (n = 6, 31.6%), including ST120, were also detected in isolates from puddles on roads, and the sources of infection in these cases remained unclear. The lag-1 gene, a tentative marker for clinical isolates, was prevalent in puddle isolates (61.3%). Our findings suggest that puddles on asphalt roads serve as potential reservoirs for L. pneumophila in the environment.     

Cytological evidence for somatic diploidization in dikaryotic cells ofArmillariella mellea

Dikaryotic hyphae isolated from basidiocarps ofArmillariella mellea are unstable in aseptic culture and change into monokaryotic hypae. During monokaryotization the nuclei of a dikaryon fuse and fusion nucleus immediately divides resulting in two uninucleate cells, from which the monokaryotic mycelium originates. Similar fusion of two nuclei takes place in matings of compatible singlespore isolates. It is concluded that the resulting monokaryotic mycelium is diploid.     

Serologic grouping and sexual compatibility of airbornecryptococcus neoformans

Through the use of Anderson air samplers, 214 isolates ofCryptococcus neoformans were cultured from the air in a vacant tower in a large complex of buildings in Oklahoma City. The tower contained hundreds of pigeons, a massive amount of droppings, nests with eggs and young, dying and dead pigeons. All isolates were serotype A-D and self-sterile for the production of basidiospores. Among these, 193 were of the ‘alpha’ mating type, producing basidiospores when paired with ‘a’ mating type. No isolates of ‘α’ mating type were found. The remaining 21 isolates were untypable for their mating type. These findings imply that the infectious particles ofC. neoformans in nature are relatively small, nonencapsulated yeast cells andnot basidiospores.     

Antimicrobial activity of some Indian plants

Antimicrobial activity of 105 Indian plant species was tested. Among them, 30 showed antibacterial activity; 20 of these exhibited antifungal action as well. Seeds ofCarum copticum, stem ofPinus longifolia, roots ofPlumbago zeylanica andSaussurea lappa, and rhizome ofAlpinia officinarum have considerable antifungal activity, especially against pathogenic fungi. Antibiotic activity against a wide variety of microorganisms—pathogenic and nonpathogenic gram-positive and gram-negative bacteria, yeast, and fungi—was also noted with leaves ofLawsonia inermis, roots ofPlumbago zeylanica, and fruits ofTamarindus indica,Terminalia belerica, andEmblica officinalis.     

Host range and symptom differences between isolates of turnip mosaic virus obtained fromSisymbrium loeselii

Twenty-four isolates of turnip mosaic virus (TuMV) from spontaneously infectedSisymbriutn loeselii plants were collected in Bohemian localities. Single lesions on leaves ofNicotiana tabacum cv. Samsun served for inoculating petunia plants used as infection sources for twelve host species. In no case were two identical isolates obtained. 15 isolates could be transmitted toVicia faba, a new TuMV host. Almost all isolates infectedPhaseolus vulgaris cv. Prince locally andTrifolium incarnatum systemically. Seven isolates were transmissible toPisum sativum. No substantial differences between isolates were observed with infectedAn aranthus caudatus, Chenopodium quinoa, Datura innoxia andSinapis alba plants. Several isolates could not infectNicotiana glutinosa at all, other isolates caused in it latent systemic infection and some isolates only local infection contrary to normal local and systemic infections ofN. glutinosa. Attempts to transmit one isolate to cereals failed.     

Specificity among the Casuarinaceae in root nodulation byFrankia

Pure cultured isolates ofFrankia made from root nodules of plant species from among three genera of the host family Casuarinaceae were used in inoculation trials of seedlings grown in water culture. A large number of host species among the genera Allocasuarina, Casuarina and Gymnostoma from Australia, Papua New Guinea and other South Pacific Islands were tested. The most widely infectiveFrankia strains were CcI3 and AllI1; theFrankia strains with the narrowest host range within the Casuarinaceae were CcI2 and GpI1. Intrafamily cross-inoculations were uncommon. The most broadly receptive host species wasG. papuanum. For many species ofAllocasuarina tested, no infection by anyFrankia available for testing could be observed.     

A biological evaluation of five European cultures ofAnaphes flavipes [Hymenoptera: mymaridae], an egg parasite ofOulema melanopus [Coleoptera: chrysomelidae]

Comparative biological data on five European cultures ofAnaphes flavipes [Hymenoptera: Mymaridae], an egg parasite ofOulema melanopus [Coleoptera: Chrysomelidae], indicated differences in developmental rates as those individuals representing cultures from the southernmost latitudes developed the slowest at 21°C, while those from the northernmost latitudes developed the fastest at this temperature. At 15.6°C, however, the parasites from the southern latitudes developed faster than those from the northern latitudes. Variations in other biological attributes, including longevity and fecundity, were not statistically significant.     

Hydrolysis of the Leu-Gly bond of phenylazobenzyl-oxycarbonyl-l-Pro-l-Leu-Gly-l-Pro-D-Arg (a substrate of microbial collagenases) by treponemes isolated from the subgingival plaque of periodontitis patients

Cell extracts prepared from several oral treponemes isolated from the subgingival plaque of periodontitis patients showed high enzyme activity toward phenylazobenzyl-oxycarbonyl-l-prolyl-l-leucylglycyl-l-prolyl-d-arginine (a compound used as a substrate for microbial collagenases). One major enzyme hydrolyzing this substrate at the Leu-Gly bond only was partially purified from an unspeciated treponeme (strain US),Treponema denticola ATCC 35405, and 29 different clinical isolates ofT. denticola. TheTreponema US enzyme also hydrolyzed furylacryloyl-l-leucylglycyl-l-prolyl-l-alanine (another substrate of bacterial collagenases) at the Leu-Gly bond. This enzyme also hydrolyzed various collagens and collagen-derived peptides. These treponemal proteases were sensitive to metal chelators andp-chloromercury compounds. The results indicate that human oral treponemes contain enzymes that readily hydrolyze in chromogenic protease substrates the Leu-Gly bond only that is the cleavage site of these substrates also by “true” microbial collagenases.     

Distribution of Sequence-Based Types of Legionella pneumophila Serogroup 1 Strains Isolated from Cooling Towers,Hot Springs,and Potable Water Systems in China

Legionella pneumophila serogroup 1 causes Legionnaires'' disease. Water systems contaminated with Legionella are the implicated sources of Legionnaires'' disease. This study analyzed L. pneumophila serogroup 1 strains in China using sequence-based typing. Strains were isolated from cooling towers (n = 96), hot springs (n = 42), and potable water systems (n = 26). Isolates from cooling towers, hot springs, and potable water systems were divided into 25 sequence types (STs; index of discrimination [IOD], 0.711), 19 STs (IOD, 0.934), and 3 STs (IOD, 0.151), respectively. The genetic variation among the potable water isolates was lower than that among cooling tower and hot spring isolates. ST1 was the predominant type, accounting for 49.4% of analyzed strains (n = 81), followed by ST154. With the exception of two strains, all potable water isolates (92.3%) belonged to ST1. In contrast, 53.1% (51/96) and only 14.3% (6/42) of cooling tower and hot spring, respectively, isolates belonged to ST1. There were differences in the distributions of clone groups among the water sources. The comparisons among L. pneumophila strains isolated in China, Japan, and South Korea revealed that similar clones (ST1 complex and ST154 complex) exist in these countries. In conclusion, in China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environmental L. pneumophila serogroup 1 isolates, similar to its prevalence in Japan and South Korea.     

Phenotypic characteristics of thermotolerantCampylobacter from human and animal sources

Five reference strains and 314 field strains ofCampylobacter growing at 42°C, but not at 25°C, were characterized by tests of hippurate hydrolysis and sensitivity to nalidixic acid, to metronidazole, and to 2,3,5-triphenyltetrazolium chloride (TTC). All strains but seven were TTC-resistant by the disc test used. Of 168 human isolates, 87% hydrolyzed hippurate; three of these strains were nalidixic acid-resistant. Also hippurate-positive were all 23 strains from ovine abortion, 79% of 43 avian strains, two of six bovine isolates, and one of two strains of equine origin. Seventy-two porcine strains were all hippurate-negative. Metronidazole sensitivity was found to be a variable property in hippurate-positive strains, although present in all but four hippurate-negative strains. A group of eight nalidixic acid-resistant, hippurate-negative isolates of porcine and bovine origin probably represent a new group ofCampylobacter.     

Susceptibility of the tick Haemaphysalis qinghaiensis to isolates of the fungus Metarhizium anisopliae in China

Haemaphysalis qinghaiensis, a prevalent tick species in China, causes severe economic losses. In this study, we investigated the pathogenicity of six isolates of the fungus Metarhizium anisopliae to engorged female H. qinghaiensis using concentrations of 10⁶, 10⁷ and 10⁸ conidia ml^?1. The results indicated that M.aAT08 and M.aAT13 isolates were highly virulent against the ticks. Metarhizium anisopliae has potential for biocontrol of H. qinghaiensis.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> as a Probiotic Potential from Kouzeh Cheese (Traditional Iranian Cheese) and Its Antimicrobial Activity

The aim of this study is to isolate and identify Lactobacillus plantarum isolates from traditional cheese, Kouzeh, and evaluate their antimicrobial activity against some food pathogens. In total, 56 lactic acid bacteria were isolated by morphological and biochemical methods, 12 of which were identified as Lactobacillus plantarum by biochemical method and 11 were confirmed by molecular method. For analyzing the antimicrobial activity of these isolates properly, diffusion method was performed. The isolates were identified by 318 bp band dedicated for L. plantarum. The isolated L. plantarum represented an inhibitory activity against four of the pathogenic bacteria and showed different inhibition halos against each other. The larger halos were observed against Staphylococcus aureus and Staphylococcus epidermidis (15 ± 0.3 and 14.8 ± 0.7 mm, respectively). The inhibition halo of Escherichia coli was smaller than that of other pathogen and some L. plantarum did not show any inhibitory activity against E. coli, which were resistant to antimicrobial compounds produced by L. plantarum. The isolated L. plantarum isolates with the antimicrobial activity in this study had strong probiotic properties. These results indicated the nutritional value of Kouzeh cheese and usage of the isolated isolates as probiotic strains.     

Mating reaction inSaccharomyces cerevisiae VIII. Mating-type-specific substances responsible for sexual cell agglutination

The agglutination factors ofa and α mating types ofSaccharomyces cerevisiae were solubilized from isolated cell-wall fractions by treatment with snail enzyme (Glusulase) and shown to be adsorbed specifically by cells of the opposite mating type, resulting in the loss of agglutinability of these cells. The agglutination factors ofa and α types adsorbed by cells of the opposite mating type at pH 5.5 were eluted at pH 9.0. These factors were further purified on Sepharose 4B. From the elution pattern on Sepharose 4B, the molecular weights of the solubilized agglutination factors are estimated to be about one million. Thus purified agglutination factors contained carbohydrate and protein and were considerably resistant to heat treatment. Neutral protease ofBacillus subtilis inactivated botha and α type agglutination factors. Trypsin inactivated the α type agglutination factor only.     

Purification and characterization of antimicrobial substances from Bacillus licheniformis BFP011

Bacillus licheniformis BFP011 isolated from papaya (Thailand) could produce extracellular antimicrobial substances which were active against some important phytopathogens, pathogenics and spoilage microorganisms such as Colletotrichum capsici, Escherichia coli O157: H7 and Salmonella typhi ATCC 5784. The antimicrobial substances of this bacterium showed resistance to pronase enzyme and high temperature at 100 and 121°C for 15 min. They were purified by TLC on silica gel plates F254 using the different solvent mixtures. The best solvent mixture was revealed as n-butanol: ethanol: acetic acid: water (30: 60: 5: 30, v/v). The spots F4, F5 and F6 from TLC were able to inhibit growth of S. typhi ATCC 5784 assayed in vitro by the disc diffusion method. The characterization of the active fractions F4, F5 and F6 from TLC and reversedphase HPLC indicated that the antimicrobial substances of B. licheniformis BFP011 contain peptides and unsaturated fatty acids.