Evaluating Fourier-transform infrared spectroscopy with IR Biotyper as a faster and simpler method for investigating the sources of an outbreak of legionellosis.

Fourier-transform infrared (FTIR) spectroscopy using the IR Biotyper and core genome single nucleotide polymorphism (cgSNP) analysis were performed on 12 Legionella isolates associated with an outbreak at a spa house in Kanagawa Prefecture, Japan, and 3 non-outbreak isolates. The discriminative power of FTIR spectroscopy for 48-h incubation conditions of L. pneumophila in this outbreak was lower than cgSNP-based typing but higher than serogroup typing. FTIR spectroscopy could screen outbreak isolates from a group of genetically related isolates and may be useful as an initial typing method in Legionella outbreak investigations.

New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene.

The identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB gene (∼4,107 bp) and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non-Lp isolates. The results suggest that using both the mip and rpoB genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of mip and rpoB associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of Legionella species subtypes. IMPORTANCELegionella spp. are a widely spread bacteria that cause a fatal form of pneumonia. While traditional laboratory techniques have provided valuable systems for Legionella pneumophila identification, the amplification of the mip gene has been recognized as the only useful tool for Legionella non-pneumophila species identification both in clinical and environmental samples. Several studies focused on the mip gene classification scheme showed its limitations and the need to improve the classification scheme, including other genes. Our study provides significant advantages on Legionella identification, providing a reproducible new rpoB gene classification scheme that seems to be more accurate than mip gene sequencing, bringing out greater genetic variation on Legionella species. In addition, the combined use of both the mip and rpoB genes allowed us to identify presumed new Legionella species, improving epidemiological investigations and acquiring new understanding on Legionella fields.

Characterization of the First Cultured Psychrotolerant Representative of Legionella from Antarctica Reveals Its Unique Genome Structure.

Culture-independent analysis shows that Legionella spp. inhabit a wide range of low-temperature environments, but to date, no psychrotolerant or psychrophilic strains have been reported. Here, we characterized the first cultivated psychrotolerant representative, designated strain TUM19329T, isolated from an Antarctic lake using a polyphasic approach and comparative genomic analysis. A genome-wide phylogenetic tree indicated that this strain was phylogenetically separate at the species level. Strain TUM19329T shared common physiological traits (e.g., Gram-negative, limited growth on buffered charcoal-yeast extract α-ketoglutarate [BCYEα] agar with l-cysteine requirements) with its relatives, but it also showed psychrotolerant growth properties (e.g., growth at 4°C to 25°C). Moreover, this strain altered its own cellular fatty acid composition to accumulate unsaturated fatty acid at a lower temperature, which may help maintain the cell membrane fluidity. Through comparative genomic analysis, we found that this strain possessed massive mobile genetic elements compared with other species, amounting to up to 17% of the total genes. The majority of the elements were the result of the spread of only a few insertion sequences (ISs), which were spread throughout the genome by a "copy-and-paste" mechanism. Furthermore, we found metabolic genes, such as fatty acid synthesis-related genes, acquired by horizontal gene transfer (HGT). The expansion of ISs and HGT events may play a major role in shaping the phenotype and physiology of this strain. On the basis of the features presented here, we propose a new species-Legionella antarctica sp. nov.-represented by strain TUM19329T (= GTC 22699T = NCTC 14581T). IMPORTANCE This study characterized a unique cultivated representative of the genus Legionella isolated from an Antarctic lake. This psychrotolerant strain had some common properties of known Legionella species but also displayed other characteristics, such as plasticity in fatty acid composition and an enrichment of mobile genes in the genome. These remarkable properties, as well as other factors, may contribute to cold hardiness, and this first cultivated cold-tolerant strain of the genus Legionella may serve as a model bacterium for further studies. It is worth noting that environmentally derived 16S rRNA gene phylotypes closely related to the strain characterized here have been detected from diverse environments outside Antarctica, suggesting a wide distribution of psychrotolerant Legionella bacteria. Our culture- and genome-based findings may accelerate the ongoing studies of the behavior and pathogenicity of Legionella spp., which have been monitored for many years in the context of public health.

Occurrence of the Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran; first detection of Legionella cherrii.

BACKGROUND: This study aimed to investigate the occurrence of Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran by culture and the real-time PCR of 23S-5S rRNA gene spacer region. METHODS AND RESULTS: A total of 123 clinical respiratory samples including 63 pleural aspirates, 57 bronchoalveolar lavage (BAL), and 3 sputum were collected from 65 males and 58 females with pneumonia symptoms. All samples were cultured on the Modified Wadowsky-Yee (MWY) agar. The Legionella species was identified by routine bacteriological tests. The presence of the 16S-23S rRNA spacer region gene was investigated by real-time PCR. The Legionella species were differentiated by sequencing of 16S-23S rRNA gene. A total of 2 (1.6%) BAL specimens were positive for Legionella species by culture method. No Legionella spp. were identified in pleural aspirates and sputum samples by the culture method. Using real-time PCR, 9 (7.3%) samples including 6 BAL, 1 sputum, and 2 pleural aspirates were positive for legionella species. These species were detected in 3 (5.2%) females and 6 males (9.2%). The results of sequencing showed that eight species were L. pneumophila while one was L. cherrii. Also, the 2 isolates that were identified by culture method, were confirmed as L. pneumophila by sequencing. CONCLUSIONS: The results showed that using the real-time PCR has a more efficacy for detecting of Legionella species in respiratory samples. Also, L. pneumophila was the most prevalent species circulating in the southwest region of Iran. So, periodic monitoring programs is recommended to prevent epidemics due to this bacterium.

Detection of Legionella species, the influence of precipitation on the amount of Legionella DNA, and bacterial microbiome in aerosols from outdoor sites near asphalt roads in Toyama Prefecture, Japan.

BACKGROUND: Legionellosis is caused by the inhalation of aerosolized water contaminated with Legionella bacteria. In this study, we investigated the prevalence of Legionella species in aerosols collected from outdoor sites near asphalt roads, bathrooms in public bath facilities, and other indoor sites, such as buildings and private homes, using amoebic co-culture, quantitative PCR, and 16S rRNA gene amplicon sequencing. RESULTS: Legionella species were not detected by amoebic co-culture. However, Legionella DNA was detected in 114/151 (75.5%) air samples collected near roads (geometric mean ± standard deviation: 1.80 ± 0.52 log10 copies/m3), which was comparable to the numbers collected from bathrooms [15/21 (71.4%), 1.82 ± 0.50] but higher than those collected from other indoor sites [11/30 (36.7%), 0.88 ± 0.56] (P < 0.05). The amount of Legionella DNA was correlated with the monthly total precipitation (r = 0.56, P < 0.01). It was also directly and inversely correlated with the daily total precipitation for seven days (r = 0.21, P = 0.01) and one day (r = - 0.29, P < 0.01) before the sampling day, respectively. 16S rRNA gene amplicon sequencing revealed that Legionella species were detected in 9/30 samples collected near roads (mean proportion of reads, 0.11%). At the species level, L. pneumophila was detected in 2/30 samples collected near roads (the proportion of reads, 0.09 and 0.11% of the total reads number in each positive sample). The three most abundant bacterial genera in the samples collected near roads were Sphingomonas, Streptococcus, and Methylobacterium (mean proportion of reads; 21.1%, 14.6%, and 1.6%, respectively). In addition, the bacterial diversity in outdoor environment was comparable to that in indoor environment which contains aerosol-generating features and higher than that in indoor environment without the features. CONCLUSIONS: DNA from Legionella species was widely present in aerosols collected from outdoor sites near asphalt roads, especially during the rainy season. Our findings suggest that there may be a risk of exposure to Legionella species not only in bathrooms but also in the areas surrounding asphalt roads. Therefore, the possibility of contracting legionellosis in daily life should be considered.

Improvements to the Success of Outbreak Investigations of Legionnaires' Disease: 40 Years of Testing and Investigation in New York State.

Since 1978, the New York State Department of Health's public health laboratory, Wadsworth Center (WC), in collaboration with epidemiology and environmental partners, has been committed to providing comprehensive public health testing for Legionella in New York. Statewide, clinical case counts have been increasing over time, with the highest numbers identified in 2017 and 2018 (1,022 and 1,426, respectively). Over the course of more than 40 years, the WC Legionella testing program has continuously implemented improved testing methods. The methods utilized have transitioned from solely culture-based methods for organism recovery to development of a suite of reference testing services, including identification and characterization by PCR and pulsed-field gel electrophoresis (PFGE). In the last decade, whole-genome sequencing (WGS) has further refined the ability to link outbreak strains between clinical specimens and environmental samples. Here, we review Legionnaires' disease outbreak investigations during this time period, including comprehensive testing of both clinical and environmental samples. Between 1978 and 2017, 60 outbreaks involving clinical and environmental isolates with matching PFGE patterns were detected in 49 facilities from the 157 investigations at 146 facilities. However, 97 investigations were not solved due to the lack of clinical or environmental isolates or PFGE matches. We found 69% of patient specimens from New York State (NYS) were outbreak associated, a much higher rate than observed in other published reports. The consistent application of new cutting-edge technologies and environmental regulations has resulted in successful investigations resulting in remediation efforts. IMPORTANCE Legionella, the causative agent of Legionnaires' disease (LD), can cause severe respiratory illness. In 2018, there were nearly 10,000 cases of LD reported in the United States (https://www.cdc.gov/legionella/fastfacts.html; https://wonder.cdc.gov/nndss/static/2018/annual/2018-table2h.html), with actual incidence believed to be much higher. About 10% of patients with LD will die, and as high as 90% of patients diagnosed will be hospitalized. As Legionella is spread predominantly through engineered building water systems, identifying sources of outbreaks by assessing environmental sources is key to preventing further cases LD.

LegionellaDB - A Database on Legionella Outbreaks.

LegionellaDB is the first database on Legionella outbreaks; it is based on a metadata analysis of peer-reviewed manuscripts from PubMed and SCOPUS. LegionellaDB is dynamic and extensible, allowing users to search for specific outbreaks, suggest additional information to be included after curation, visualize statistical representations on specific outbreaks, and download selected data. The database is maintained online.

Legionella spp. All Ears? The Broad Occurrence of Quorum Sensing Elements outside Legionella pneumophila.

Legionella spp. are ubiquitous bacteria principally found in water networks and ∼20 species are implicated in Legionnaire's disease. Among them, Legionella pneumophila is an intracellular pathogen of environmental protozoa, responsible for ∼90% of cases in the world. Legionella pneumophila regulates in part its virulence by a quorum sensing system named "Legionella quorum sensing," composed of a signal synthase LqsA, two histidine kinase membrane receptors LqsS and LqsT and a cytoplasmic receptor LqsR. To date, this communication system was only found in L. pneumophila. Here, we investigated 58 Legionella genomes to determine the presence of a lqs cluster or homologous receptors using TBlastN. This analysis revealed three categories of species: 19 harbored a complete lqs cluster, 20 did not possess lqsA but maintained the receptor lqsR and/or lqsS, and 19 did not have any of the lqs genes. No correlation was observed between pathogenicity and the presence of a quorum sensing system. We determined by RT-qPCR that the lqsA gene was expressed at least in four strains among different species available in our laboratory. Furthermore, we showed that the lqs genomic region was conserved even in species possessing only the receptors of the quorum sensing system, indicating an ancestral acquisition and various loss dynamics during evolution. This system could therefore function in interspecific communication as well.

Legionella septentrionalis sp. nov., isolated from aquatic environments in the northern PR China.

Four strains (km711T, km714, km542 and km524), representing a novel Legionella species, were isolated from aquatic environments in northern PR China. Cells were Gram-stain-negative, rod-shaped, microaerobic, motile and growth depended on l-cysteine. They grew at 25‒42 °C (optimum, 35‒37 °C) and could tolerate up to 1.5 % (w/v) NaCl (optimum, 0.5 %). The major fatty acids (>5 %) of the type strain km711T were C17 : 0 anteiso, C15 : 0 anteiso, iso-C16 : 0 and C16 : 1 ω7c and/or iso-C15 : 0 2OH. The pairwise comparison values were <96.1 % for 16S rRNA gene sequences, 23.3‒28.7 % interspecies variation for mip gene sequences, <93.6 % average nucleotide identity and <72.8 % average amino acid identity between these four strains and related type strains within the genus Legionella. The phylogenetic tree based on the four concatenated genes (16S rRNA, mip, rpoB and rnpB) and protein-concatamer tree based on concatenation of 21 protein markers both revealed that these four strains formed a separate phylogenetic branch cluster within the genus Legionella. The results of phenotypic and genotypic features suggest that these four strains represent a novel species of the genus Legionella, for which the name Legionella septentrionalis sp. nov. is proposed (type strain km711T=KCTC 15655T=NBRC 113219T).

Isolation and identification of Legionella spp. from hot spring water in Algeria by culture and molecular methods.

AIMS: Due to infectious risk associated with the presence of Legionella in warm water, we determined the prevalence of living Legionella spp. in hot spring water in Algeria. METHODS AND RESULTS: Detection of Legionella by culture was done by using two methods, direct culture on agar plates and co-culture with amoeba. Fifty samples were taken from different hot springs in northern Algeria, including swimming pools, showers and thermal sources. Legionella pneumophila serotypes were predominant, accounting for 60% of positive samples. Direct method allowed the isolation of 13 L. pneumophila only of 50 samples (26%), whereas co-culture using a panel of three free living amoeba allowed the isolation of 119 Legionella species from the same samples (80%) CONCLUSIONS: Amoeba co-culture allowed the isolation of several Legionella sp., while direct culture allowed the isolation of L. pneumophila only. Remarkably, Legionella longbeachae, usually isolated from soil and compost, was isolated for the first time in thermal water in three samples using Vermamoeba vermiformis co-culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Legionella in the water of hot springs in Algeria, which are mainly frequented by individuals at risk of Legionellosis, requires urgent control measures.

How Molecular Typing Can Support Legionella Environmental Surveillance in Hot Water Distribution Systems: A Hospital Experience.

In this study, we aimed to associate the molecular typing of Legionella isolates with a culture technique during routine Legionella hospital environmental surveillance in hot water distribution systems (HWDSs) to develop a risk map able to be used to prevent nosocomial infections and formulate appropriate preventive measures. Hot water samples were cultured according to ISO 11731:2017. The isolates were serotyped using an agglutination test and genotyped by sequence-based typing (SBT) for Legionella pneumophila or macrophage infectivity potentiator (mip) gene sequencing for non-pneumophila Legionella species. The isolates' relationship was phylogenetically analyzed. The Legionella distribution and level of contamination were studied in relation to temperature and disinfectant residues. The culture technique detected 62.21% of Legionella positive samples, characterized by L. pneumophila serogroup 1, Legionella non-pneumophila, or both simultaneously. The SBT assigned two sequence types (STs): ST1, the most prevalent in Italy, and ST104, which had never been isolated before. The mip gene sequencing detected L. anisa and L. rubrilucens. The phylogenetic analysis showed distinct clusters for each species. The distribution of Legionella isolates showed significant differences between buildings, with a negative correlation between the measured level of contamination, disinfectant, and temperature. The Legionella molecular approach introduced in HWDSs environmental surveillance permits (i) a risk map to be outlined that can help formulate appropriate disinfection strategies and (ii) rapid epidemiological investigations to quickly identify the source of Legionella infections.

Evaluation of Legionella spp. Colonization in Residential Buildings Having Solar Thermal System for Hot Water Production.

Despite an increase of literature data on Legionella spp. presence in private water systems, epidemiological reports assert a continuing high incidence of Legionnaires' disease infection in Italy. In this study, we report a survey on Legionella spp. colonization in 58 buildings with solar thermal systems for hot water production (TB). In all buildings, Legionella spp. presence was enumerated in hot and cold water samples. Microbiological potability standards of cold water were also evaluated. Legionella spp. was detected in 40% of the buildings. Moreover, we detected correlations between the count of Legionella spp. and the presence of the optimal temperature for the microorganism growth (less than 40 °C). Our results showed that cold water was free from microbiological hazards, but Legionella spp., was detected when the mean cold water temperature was 19.1 ± 2.2 °C. This may considered close to the suboptimal value for the Legionella growth (more then 20 °C). In conclusion, we observed the presence of a Legionnaires' disease risk and the need of some strategies aimed to reduce it, such as the application of training programs for all the workers involved in water systems maintenance.

Interpreting the Results of the Conventional Plate Culture and Gene Detection Methods for Legionella Detection in Environmental Water Samples.

The conventional plate culture method is widely used as a method for detection of Legionella in environmental water samples, but to obtain results takes more than a week. Because it is much quicker, the gene detection method has become widespread as an alternative detection method. However, the results of gene detection and plate culture methods may differ even when the same sample is examined; the gene detection method shows a higher detection ratio than the plate culture method. The reason for this difference is that the plate culture method detects Legionella cells that have the ability to form colonies on an agar plate, whereas the gene detection method detects any Legionella genes present regardless of the state of the Legionella. In this paper, we consider the factors that cause differences between the results of the plate culture and gene detection methods, and how to interpret the results of each.

Distribution of Legionella species and serogroups in patients with culture-confirmed Legionella pneumonia.

Legionella species are consistently identified as some of the most common causative agents of severe community-acquired pneumonia (CAP) or nosocomial pneumonia. Although the number of reported Legionella infection cases is gradually increasing in Japan, most cases are diagnosed by a urinary antigen test, which identifies only L. pneumophila serogroup 1. Therefore, assessment of pneumonia-causing Legionella species and serogroups would be important. The Japan Society for Chemotherapy Legionella committee has collected the isolates and clinical information on cases of sporadic community-acquired Legionella pneumonia throughout Japan. Between December 2006 and March 2019, totally 140 sporadic cases were identified, in which L. pneumophila was the most frequently isolated species (90.7%) followed by L. bozemanae (3.6%), L. dumofii (3.6%), L. micdadei (1.4%), and L. longbeachae (0.7%). Among 127 isolates of L. pneumophila, 111 isolates were of serogroup 1, two of serogroup 2, four of serogroup 3, one of serogroup 4, one of serogroup 5, seven of serogroup 6, and one was of serogroup 10. We also assessed in vitro activity of antibiotics against these isolates and showed that quinolones and macrolides have potent anti-Legionella activity. Our study showed that pneumonia-causing Legionella species and serogroup distribution was comparable to that reported in former surveillances. L. pneumophila was the most common etiologic agent in patients with community-acquired Legionella pneumonia, and L. pneumophila serogroup 1 was the predominant serogroup.

Ten-Year Retrospective Analysis of Legionella Diffusion in Hospital Water Systems and Its Serogroup Seasonal Variation.

INTRODUCTION: Legionella spp. are ubiquitous aquatic organisms found to be associated with community-acquired pneumoniae (CAP) as well as hospital-acquired pneumonia (HAP). Direct inhalation of aerosols from environmental colonisation is typically the source of infection. The aim of this study was to determine the level of colonisation in hospital water supply systems in order to assess the criticality of the water distribution network and strengthen preventive measures. METHODS: From 2009 to 2018, 769 water samples were collected and then analysed according to the standard methods indicated in ISO11731-2:2004 and ISO11731:2017 for Legionella detection. RESULTS: The samples were positive in 37.1% cases (n. 285) and negative in 62.9% cases (n. 484). The threshold of 10,000 CFU/L was exceeded in 15.1% cases and led to decolonisation as indicated by Italian and European ECDC guidelines. In the autumn-winter period SG1 showed a positivity of 41.2% (n. 40) with a decrease in the spring-summer period with 9.6% (n. 18) of positivity. In contrast, SG2-15 showed a positivity of 30.9% (n. 30) in autumn-winter, which tends to increase to 56.9% (n. 112) in spring-summer (p < 0.001). CONCLUSION: Surprisingly, besides showing a seasonal trend already described previously in the literature, the positivity of our sample was not balanced even for serogroups in the two periods. This could be due to genetic differences and ecological niches to be further investigated that could also have links with the greater pathogenicity of SG1. Environmental microbiological surveillance and risk assessment should be performed more frequently and disinfection must be carried out, especially in health facilities where people are more susceptible to infections.

A Novel Legionella Genomic Island Encodes a Copper-Responsive Regulatory System and a Single Icm/Dot Effector Protein Transcriptionally Activated by Copper.

The intracellular pathogen Legionella pneumophila utilizes the Icm/Dot type IV secretion system to translocate >300 effector proteins into host cells during infection. The regulation of some of these effector-encoding genes was previously shown to be coordinated by several global regulators, including three two-component systems (TCSs) found in all the Legionella species examined. Here, we describe the first Legionella genomic island encoding a single Icm/Dot effector and a dedicated TCS, which regulates its expression. This genomic island, which we named Lci, undergoes horizontal gene transfer in the Legionella genus, and the TCS encoded from this island (LciRS) is homologous to TCSs that control the expression of various metal resistance systems found in other bacteria. We found that the L. pneumophila sensor histidine kinase LciS is specifically activated by copper via a unique, small periplasmic sensing domain. Upon activation by LciS, the response regulator LciR directly binds to a conserved regulatory element and activates the expression of the adjacently located lciE effector-encoding gene. Thus, LciR represents the first local regulator of effectors identified in L. pneumophila Moreover, we found that the expression of the lciRS operon is repressed by the Fis1 and Fis3 regulators, leading to Fis-mediated effects on copper induction of LciE and silencing of the expression of this genomic island in the absence of copper. This island represents a novel type of effector regulation in Legionella, shedding new light on the ways by which the Legionella pathogenesis system evolves its effector repertoire and expands its activating signals.IMPORTANCELegionella pneumophila is an intracellular human pathogen that utilizes amoebae as its environmental host. The adaptation of L. pneumophila to the intracellular environment requires coordination of expression of its multicomponent pathogenesis system, which is composed of a secretion system and effector proteins. However, the regulatory factors controlling the expression of this pathogenesis system are only partially uncovered. Here, we discovered a novel regulatory system that is activated by copper and controls the expression of a single effector protein. The genes encoding both the regulatory system and the effector protein are located on a genomic island that undergoes horizontal gene transfer within the Legionella genus. This regulator-effector genomic island represents the first reported case of local regulation of effectors in Legionella The discovery of this regulatory mechanism is an important step forward in the understanding of how the regulatory network of effectors functions and evolves in the Legionella genus.

Legionnaires' Disease: State of the Art Knowledge of Pathogenesis Mechanisms of Legionella.

Legionella species are environmental gram-negative bacteria able to cause a severe form of pneumonia in humans known as Legionnaires' disease. Since the identification of Legionella pneumophila in 1977, four decades of research on Legionella biology and Legionnaires' disease have brought important insights into the biology of the bacteria and the molecular mechanisms that these intracellular pathogens use to cause disease in humans. Nowadays, Legionella species constitute a remarkable model of bacterial adaptation, with a genus genome shaped by their close coevolution with amoebae and an ability to exploit many hosts and signaling pathways through the secretion of a myriad of effector proteins, many of which have a eukaryotic origin. This review aims to discuss current knowledge of Legionella infection mechanisms and future research directions to be taken that might answer the many remaining open questions. This research will without a doubt be a terrific scientific journey worth taking.

Taking a Closer Look at the Valid Publication and Authorship of Legionella bozemanae Brenner et al. 1980, Fluoribacter bozemanae Garrity et al. 1980, Legionella pittsburghensis Pasculle et al. 1980, Legionella micdadei Hébert et al. 1980 and Tatlockia micdadei (Hébert et al. 1980) Garrity et al. 1980.

The names Legionella bozemanae Brenner et al. 1980, Fluoribacter bozemanae Garrity et al. 1980, Legionella pittsburghensis Pasculle et al. 1980, Legionella micdadei Hébert et al. 1980 and Tatlockia micdadei (Hébert et al. 1980) Garrity et al. 1980, all appeared in the same issue of the International Journal of Systematic Bacteriology. Fluoribacter bozemanae Garrity et al. 1980 appeared as the name of new taxon at the rank of species and Tatlockia micdadei (Hébert et al. 1980) Garrity et al. 1980 as a new combination, both in the same original article in the International Journal of Systematic Bacteriology. The names Legionella bozemanae Brenner et al. 1980 (originally published as Legionella bozemanii) Legionella pittsburghensis Pasculle et al. 1980 (originally published as Legionella pittsburgensis) and Legionella micdadei Hébert et al. 1980, all appeared initially in effective publications outside of the International Journal of Systematic Bacteriology and were validly published by inclusion in Validation List no 5. While it is evident from the inclusion of the names Legionella bozemanae Brenner et al. 1980, Legionella pittsburghensis Pasculle et al. 1980 and Legionella micdadei Hébert et al. 1980 on Validation List no. 5 that the authors were following the 1975 revision of the International Code of Nomenclature of Bacteria, the wording of Garrity et al. 1980 indicates that they were following the interpretation found in the 1966 revision of the International Code of Nomenclature of Bacteria. Changes to the International Code of Nomenclature of Bacteria between the 1966 and 1975 revisions introduced new criteria for the valid publication of names. In particular, there was a change from all effective publications being accepted as the publication in which valid publication of a name could occur to only one journal being accepted as the publication in which valid publication could occur (the International Journal of Systematic Bacteriology, now the International Journal of Systematic and Evolutionary Microbiology). This change has a direct effect on the order of valid publication of the names Legionella bozemanae Brenner et al. 1980, Fluoribacter bozemanae Garrity et al. 1980, Legionella pittsburghensis Pasculle et al. 1980, Legionella micdadei Hébert et al. 1980 and Tatlockia micdadei (Hébert et al. 1980) Garrity et al. 1980, their authorships, as well as determining which names should be treated as names of new taxa at the rank of species (sp. nov.) vs new combinations (comb. nov.) based on the names of existing taxa. Given the fact that Legionella pittsburghensis Pasculle et al. 1980, Legionella micdadei Hébert et al. 1980 and Tatlockia micdadei (Hébert et al. 1980) Garrity et al. 1980 share the same nomenclatural type, this also has an influence on which epithet has priority and which epithet is illegitimate.

Investigations on Contamination of Environmental Water Samples by Legionella using Real-Time Quantitative PCR Combined with Amoebic Co-Culturing.

We analyzed the contamination of environmental water samples with Legionella spp. using a conventional culture method, real-time quantitative PCR (qPCR), and real-time qPCR combined with an amoebic co-culture method. Samples (n = 110) were collected from 19 cooling towers, 31 amenity water facilities, and 60 river water sources of tap water in Japan. Legionella was detected in only three samples (3/110, 2.7%) using the culture method. The rate of Legionella detection using amoebic co-culture followed by qPCR was 74.5%, while that using qPCR without amoebic co-culture was 75.5%. A higher than 10-fold bacterial count was observed in 19 samples (19/110, 17.3%) using real-time qPCR subsequent to amoebic co-culture, compared with identical samples analyzed without co-culture. Of these 19 samples, 13 were identified as Legionella spp., including L. pneumophila and L. anisa, and the non-culturable species were identified as L. lytica and L. rowbothamii. This study showed that the detection of Legionella spp., even in those samples where they were not detected by the culture method, was possible using real-time qPCR and an amoebic co-culture method. In addition, this analytical test combination is a useful tool to detect viable and virulent Legionella spp..