Understanding the role of Francisella halioticida in mussel mortalities in France: an integrative approach.

Since 2014, mass mortalities of mussels Mytilus spp. have occurred in production areas on the Atlantic coast of France. The aetiology of these outbreaks remained unknown until the bacterium Francisella halioticida was detected in some mussel mortality cases. This retrospective study was conducted to assess the association between F. halioticida and these mussel mortalities. Mussel batches (n = 45) from the Atlantic coast and English Channel were selected from archived individual samples (n = 863) collected either during or outside of mortality events between 2014 and 2017. All mussels were analysed by real-time PCR assays targeting F. halioticida; in addition, 185 were analysed using histological analysis and 178 by 16S rRNA metabarcoding. F. halioticida DNA was detected by real-time PCR and 16S rRNA metabarcoding in 282 and 34 mussels, respectively. Among these individuals, 82% (real-time PCR analysis) and 76% (16S rRNA metabarcoding analysis) were sampled during a mortality event. Histological analyses showed that moribund individuals had lesions mainly characterized by necrosis, haemocyte infiltration and granulomas. Risk factor analysis showed that mussel batches with more than 20% of PCR-positive individuals were more likely to have been sampled during a mortality event, and positive 16S rRNA metabarcoding batches increased the strength of the association with mortality by 11.6 times. The role of F. halioticida in mussel mortalities was determined by reviewing the available evidence. To this end, a causation criteria grid, tailored to marine diseases and molecular pathogen detection tools, allowed more evidence to be gathered on the causal role of this bacterium in mussel mortalities.

Francisella salimarina sp. nov., isolated from coastal seawater.

Four strains (SYSU SYW-1T, SYW-2, SYW-3 and XLW-1) were isolated from seawater near the shore in Guangdong Province, China. Cells were Gram-stain-negative, aerobic, non-motile and non-spore-forming. Growth was observed at a temperature range of 16-40 °C (optimum, 32 °C), a pH range of 4-8 (optimum, pH 7) and in the presence of up to 10 % (w/v) NaCl. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and an unidentified phospholipid. The respiratory quinone was ubiquinone 8 (UQ-8), and the predominant fatty acids were C18 : 0 3-OH, C10 : 0, C14 : 0 and C18 : 1ω9c. Comparison of 16S rRNA gene and genome sequences confirmed that these strains represented a novel member of the genus Francisella, with less than 98.8 % 16S rRNA gene sequence similarity and less than 95 % genomic average nucleotide identity to recognized Francisella species. The phylogenetic tree based on 16S rRNA gene sequences and the protein-concatamer tree based on a concatenation of 28 protein marker sequences both indicated that the strains clustered with 'Francisella salina' TX07-7308 and 'Francisella marina' E95-16, but formed a distinct lineage group among the other members of the genus Francisella. The DNA G+C contents of the four strains were determined to be 32.9, 32.7, 32.9 and 32.9 %, respectively (genome). On the basis of phenotypic and genotypic features, the strains are considered to represent a novel species of the genus Francisella, for which the name Francisella salimarina sp. nov. is proposed. The type strain is SYSU SYW-1T (=CGMCC 1.17031T=NBRC 113781T).

Reclassification of Francisella noatunensis subsp. orientalis Ottem et al. 2009 as Francisella orientalis sp. nov., Francisella noatunensis subsp. chilensis subsp. nov. and emended description of Francisella noatunensis.

Francisella noatunensis is a fastidious facultative intracellular bacterial pathogen that causes 'piscine francisellosis', a serious disease affecting both marine and fresh water farmed and wild fish worldwide. Currently two F. noatunensis subspecies are recognized, i.e. F. noatunensis subsp. noatunensis and F. noatunensis subsp. orientalis. In the present study, the taxonomy of F. noatunensis was revisited using a polyphasic approach, including whole genome derived parameters such as digital DNA-DNA hybridization, whole genome average nucleotide identity (wg-ANIm), whole genome phylogenetic analysis, whole genome G+C content, metabolic fingerprinting and chemotaxonomic analyses. The results indicated that isolates belonging to F. noatunensis subsp. orientalis represent a phenotypically and genetically homogenous taxon, clearly distinguishable from F. noatunensis subsp. noatunensis that fulfils requirements for separate species status. We propose, therefore, elevation of F. noatunensis subsp. orientalis to the species rank as Francisella orientalis sp. nov. with the type strain remaining as Ehime-1T (DSM 21254T=LMG 24544T). Furthermore, we identified sufficient phenotypic and genetic differences between F. noatunensis subsp. noatunensis recovered from diseased farmed Atlantic salmon in Chile and those isolated from wild and farmed Atlantic cod in Northern Europe to warrant proposal of the Chilean as a novel F. noatunensis subspecies, i.e. Francisella noatunensis subsp. chilensis subsp. nov. with strain PQ1106T (CECT 9798T=NCTC14375T) as the type strain. Finally, we emend the description of F. noatunensis by including further metabolic information and the description of atypical strains.

Pseudofrancisella aestuarii gen. nov., sp. nov., a novel member of the family Francisellaceae isolated from estuarine seawater.

A Gram-negative, aerobic, non-motile and non-spore forming bacterium, designated strain SYSU WZ-2T, was isolated from an estuarine seawater sample. Growth of strain SYSU WZ-2T was observed at temperature range of 10-40° C (optimum, 32 °C), pH range of 6-10 (optimum, pH 7-8) and in the presence of up to 5.0% NaCl (w/v). The DNA G+C content of the novel strain was determined to be 30.1% (genome). The major polar lipids were found to be diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, an unidentified aminolipid, two unidentified aminophospholipids and two unidentified phospholipids. The major fatty acids were C18:0 3-OH (27.5%), C18:1ω9c (19.3%), C16:0 (17.0%) and C14:0 (12.9%). The respiratory quinone was found to be ubiquinone Q8. Pairwise comparison of the 16S rRNA gene sequence showed that strain SYSU WZ-2T shares high identities with members of the genera Francisella (94.8-95.9%) and Allofrancisella (93.8-94.2%). The phylogenetic dendrograms based on 16S rRNA gene sequences with the members of the family Francisellaceae showed that the strain SYSU WZ-2T formed a distinct phylogenetic lineage well separated from the members of the genera Francisella and Allofrancisella. MALDI-TOF mass spectrometric analysis also depicted a different profile for strain SYSU WZ-2T compared with those of members of the genera Francisella and Allofrancisella. Based on the above results and differences in phenotypic and chemotaxonomic features, strain SYSU WZ-2T is characterized to represent a new species of a novel genus, for which the name Pseudofrancisella aestuarii gen. nov., sp. nov. is proposed (type strain SYSU WZ-2T = KCTC 52557T = CGMCC 1.13718T).

A New Species of the γ-Proteobacterium Francisella, F. adeliensis Sp. Nov., Endocytobiont in an Antarctic Marine Ciliate and Potential Evolutionary Forerunner of Pathogenic Species.

The study of the draft genome of an Antarctic marine ciliate, Euplotes petzi, revealed foreign sequences of bacterial origin belonging to the γ-proteobacterium Francisella that includes pathogenic and environmental species. TEM and FISH analyses confirmed the presence of a Francisella endocytobiont in E. petzi. This endocytobiont was isolated and found to be a new species, named F. adeliensis sp. nov.. F. adeliensis grows well at wide ranges of temperature, salinity, and carbon dioxide concentrations implying that it may colonize new organisms living in deeply diversified habitats. The F. adeliensis genome includes the igl and pdp gene sets (pdpC and pdpE excepted) of the Francisella pathogenicity island needed for intracellular growth. Consistently with an F. adeliensis ancient symbiotic lifestyle, it also contains a single insertion-sequence element. Instead, it lacks genes for the biosynthesis of essential amino acids such as cysteine, lysine, methionine, and tyrosine. In a genome-based phylogenetic tree, F. adeliensis forms a new early branching clade, basal to the evolution of pathogenic species. The correlations of this clade with the other clades raise doubts about a genuine free-living nature of the environmental Francisella species isolated from natural and man-made environments, and suggest to look at F. adeliensis as a pioneer in the Francisella colonization of eukaryotic organisms.

Expanding Francisella models: Pairing up the soil amoeba Dictyostelium with aquatic Francisella.

The bacterial genus Francisella comprises highly pathogenic species that infect mammals, arthropods, fish and protists. Understanding virulence and host defense mechanisms of Francisella infection relies on multiple animal and cellular model systems. In this review, we want to summarize the most commonly used Francisella host model platforms and highlight novel, alternative model systems using aquatic Francisella species. Established mouse and macrophage models contributed extensively to our understanding of Francisella infection. However, murine and human cells display significant differences in their response to Francisella infection. The zebrafish and the amoeba Dictyostelium are well-established model systems for host-pathogen interactions and open up opportunities to investigate bacterial virulence and host defense. Comparisons between model systems using human and fish pathogenic Francisella species revealed shared virulence strategies and pathology between them. Hence, zebrafish and Dictyostelium might complement current model systems to find new vaccine candidates and contribute to our understanding of Francisella infection.

First identification of Francisella noatunensis subsp. orientalis causing mortality in Mexican tilapia Oreochromis spp.

Francisellosis, an emerging disease in tilapia Oreochromis spp., is caused by the facultative, intracellular bacterium Francisella noatunensis subsp. orientalis, which is present in various countries where tilapia farming is commercially important. We confirmed the presence of francisellosis in Mexican tilapia cultures in association with an outbreak during the second semester of 2012. Broodstock fish presented a mortality rate of approximately 40%, and disease was characterized by histologically classified granulomas, or whitish nodules, in different organs, mainly the spleen and kidney. Through DNA obtained from infected tissue and pure cultures in a cysteine heart medium supplemented with hemoglobin, F. noatunensis subsp. orientalis was initially confirmed through the amplification and analysis of the 16S rRNA gene and the internal transcribed spacer region. Phylogenetic analysis of these genes demonstrated close similarity with previously reported F. noatunensis subsp. orientalis sequences obtained from infected tilapia from various countries. The identification of this subspecies as the causative agent of the outbreak was confirmed using the iglC gene as a target sequence, which showed 99.5% identity to 2 F. noatunensis subsp. orientalis strains (Ethime-1 and Toba04). These findings represent the first documented occurrence of francisellosis in Mexican tilapia cultures, which highlights the importance of establishing preventative measures to minimize the spread of this disease within the Mexican aquaculture industry.

Francisella Infection in Cultured Tilapia in Thailand and the Inflammatory Cytokine Response.

Francisella infections developed in freshwater Nile Tilapia Oreochromis niloticus and red tilapia Oreochromis spp. farms in Thailand during 2012-2014. The diseased fish were lethargic and pale in color and showed numerous white nodules in their enlarged spleens. Histopathological examination and electron microscopy suggested that the white nodules were multifocal granulomas consisting of coccobacilli within vacuolated cells. Isolation of Francisella-like bacteria was achieved from 42 of 100 samples, while polymerase chain reaction confirmed Francisella infections in all samples. Analysis of the 16S rRNA gene from samples obtained from three different geographical culture areas revealed more than 99% similarity with F. noatunensis subsp. orientalis. The influence of Francisella infection on inflammatory cytokines was determined on splenic cells of fish intraperitoneally injected with the bacteria (0.8 × 10(5) colony-forming units per fish). Infected tilapia showed significantly greater expression of the pro-inflammatory genes interleukin-1ß (IL-1ß) and tumor necrotic factor-α (TNF-α) within 24 h postinjection (hpi) and for up to 96 hpi. However, down-regulation of an anti-inflammatory gene, transforming growth factor-ß (TGF-ß) was observed as early as 24 hpi. This investigation demonstrates that an imbalance between pro- and anti-inflammatory cytokines in response to the infection may account for the substantial number of granulomas in fish hematopoietic tissues that was found in the later stage of the disease. Received September 9, 2015; accepted December 13, 2015.

Characterization of Francisella species isolated from the cooling water of an air conditioning system.

Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems.

Characterization of Francisella species isolated from the cooling water of an air conditioning system

Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems.

Inflammasome priming is similar for francisella species that differentially induce inflammasome activation.

Inflammasome activation is a two-step process where step one, priming, prepares the inflammasome for its subsequent activation, by step two. Classically step one can be induced by LPS priming followed by step two, high dose ATP. Furthermore, when IL-18 processing is used as the inflammasome readout, priming occurs before new protein synthesis. In this context, how intracellular pathogens such as Francisella activate the inflammasome is incompletely understood, particularly regarding the relative importance of priming versus activation steps. To better understand these events we compared Francisella strains that differ in virulence and ability to induce inflammasome activation for their relative effects on step one vs. step two. When using the rapid priming model, i.e., 30 min priming by live or heat killed Francisella strains (step 1), followed by ATP (step 2), we found no difference in IL-18 release, p20 caspase-1 release and ASC oligomerization between Francisella strains (F. novicida, F. holarctica -LVS and F. tularensis Schu S4). This priming is fast, independent of bacteria viability, internalization and phagosome escape, but requires TLR2-mediated ERK phosphorylation. In contrast to their efficient priming capacity, Francisella strains LVS and Schu S4 were impaired in inflammasome triggering compared to F. novicida. Thus, observed differences in inflammasome activation by F. novicida, LVS and Schu S4 depend not on differences in priming but rather on their propensity to trigger the primed inflammasome.

Identification of Francisella noatunensis in novel host species French grunt (Haemulon flavolineatum) and Caesar grunt (Haemulon carbonarium).

Francisella noatunensis is an emerging pathogen of fish that has been isolated from several cultured species worldwide. Here presented is a case involving several hundred marine grunts that were caught near the Florida Keys for display in public aquaria. These fish were maintained in a recirculating system where they began to experience mortalities approximately two weeks post-stocking. Postmortem examination revealed disseminated systemic granulomatous disease most severely affecting spleen and kidney. Splenic and renal tissue homogenates inoculated in modified Thayer Martin agar media yielded colonies consistent with F. noatunensis 4 days post inoculation. Bacterial colonies and tissues were confirmed positive after real-time PCR amplification of the intracellular growth loci gene (iglC) specific for F. noatunensis subspecies orientalis. Consequently, multiple novel host species for this pathogen were identified, including the French grunt (Haemulon flavolineatum) and the Caesar grunt (Haemulon carbonarium).

Outbreaks and genetic diversity of Francisella noatunensis subsp orientalis isolated from farm-raised Nile tilapia (Oreochromis niloticus) in Brazil.

Francisella noatunensis subsp orientalis (FNO) is an emerging pathogen of warm water tilapia in a number of different countries. The disease caused by this bacterium in fish is characterized by a systemic granulomatous infection that causes high mortality rates during outbreaks. FNO has been previously described in Asia, Europe, and Central and North America. Its occurrence in South America has never been described. Since 2012, outbreaks of a granulomatous disease have been recorded in cage farms of Nile tilapia (Oreochromis niloticus L.) in Brazil. The current study aimed to identify the etiologic agent of recent francisellosis outbreaks at Brazilian tilapia farms, and to characterize the genetic diversity of the pathogen from farms with distinct geographic origins and without epidemiological connections. Bacteriological analysis of 44 diseased Nile tilapia collected from five cage farms in Brazil was performed during 2012 and 2013. The farms were in different locations and had no recent history of animal or biological material transport between each other. Sixty-two FNO isolates were identified on the basis of FNO-specific qPCR. The main predisposing factors for the occurrence of outbreaks on Brazilian farms were lower water temperature (<22°C) and life stage of fish, affecting mainly fry, fingerlings and young adults (live weight <100 g). The genetic diversity of the Brazilian FNO isolates was evaluated using repetitive extragenic palindromic-PCR. The isolates from different origins were shown to be clonally related. This is the first report of the occurrence and genetic diversity of FNO in South America.

Francisella guangzhouensis sp. nov., isolated from air-conditioning systems.

Four strains (08HL01032(T), 09HG994, 10HP82-6 and 10HL1960) were isolated from water of air-conditioning systems of various cooling towers in Guangzhou city, China. Cells were Gram-stain-negative coccobacilli without flagella, catalase-positive and oxidase-negative, showing no reduction of nitrate, no hydrolysis of urea and no production of H2S. Growth was characteristically enhanced in the presence of l-cysteine, which was consistent with the properties of members of the genus Francisella. The quinone system was composed of ubiquinone Q-8 with minor amounts of Q-9. The polar lipid profile consisted of the predominant lipids phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, two unidentified phospholipids (PL2, PL3), an unidentified aminophospholipid and an unidentified glycolipid (GL2). The polyamine pattern consisted of the major compounds spermidine, cadaverine and spermine. The major cellular fatty acids were C10 : 0, C14 : 0, C16 : 0, C18 : 1ω9c and C18 : 1 3-OH. A draft whole-genome sequence of the proposed type strain 08HL01032(T) was generated. Comparative sequence analysis of the complete 16S and 23S rRNA genes confirmed affiliation to the genus Francisella, with 95 % sequence identity to the closest relatives in the database, the type strains of Francisella philomiragia and Francisella noatunensis subsp. orientalis. Full-length deduced amino acid sequences of various housekeeping genes, recA, gyrB, groEL, dnaK, rpoA, rpoB, rpoD, rpoH, fopA and sdhA, exhibited similarities of 67-92 % to strains of other species of the genus Francisella. Strains 08HL01032(T), 09HG994, 10HP82-6 and 10HL1960 exhibited highly similar pan-genome PCR profiles. Both the phenotypic and molecular data support the conclusion that the four strains belong to the genus Francisella but exhibit considerable divergence from all recognized Francisella species. Therefore, we propose the name Francisella guangzhouensis sp. nov., with the type strain 08HL01032(T) ( = CCUG 60119(T) = NCTC 13503(T)).

Francisella noatunensis subsp. orientalis pathogenesis analyzed by experimental immersion challenge in Nile tilapia, Oreochromis niloticus (L.).

Francisella noatunensis subsp. orientalis (Fno) (syn. F. asiatica) is an emergent warmwater fish pathogen and the causative agent of francisellosis in tilapia (Oreochromis sp). To study the pathogenesis of this bacterium, tilapia fingerlings were experimentally infected by immersion challenge with wild type (WT) Fno and the distribution of bacteria to multiple organs, as well as associated lesion development, investigated after 3, 24, 48, 96, and 192h by real-time PCR and histopathological examination. Surface mucus collected 3h post-infection contained the highest number of Fno genome equivalents (GE). After 96h, marked increases of WT Fno GE were detected in spleen, anterior kidney, posterior kidney, gill, heart, liver, brain, gonad, and the gastrointestinal tract. Increases in bacterial GE also corresponded to the appearance, size and number of granulomas typical of francisellosis, predominantly in the spleen and anterior and posterior kidney segments. A simultaneous comparison was also made in tilapia challenged with an attenuated Fno strain containing a mutation in the intracellular growth locus C (iglC) gene, essential to intracellular survival. Compared to the WT, the mutant iglC strain was present in most tissues in similar numbers prior to 48h post-challenge. While the mutant did not replicate significantly or produce lesions in any tissue, it persisted for up to 192h. These findings provide insight into the pathophysiology of francisellosis in tilapia, which may also prove useful as a model for the study of mammalian tularemia, and advance our understanding of the utility of the ΔiglC mutant as a potential vaccine candidate.

Francisella asiatica as the causative agent of piscine francisellosis in cultured tilapia (Oreochromis sp.) in the United States.

Francisella asiatica is a Gram-negative, pleomorphic, facultative intracellular, bacterial pathogen that causes acute to chronic disease in a wide variety of warm-water cultured and wild fish species. Outbreaks of francisellosis in warm water fish have been documented in Taiwan, Japan, United Kingdom, Hawaii, and Latin America (including Costa Rica) but the organism has only been reported from the United States on one occasion from hybrid striped bass in California. In 2010, the bacterium was detected from diseased tilapia by culture on cystine heart agar supplemented with hemoglobin and by utilizing an F. asiatica-specific real-time polymerase chain reaction assay. The tilapia (Oreochromis niloticus) were cultured in an indoor, closed, recirculating aquaculture facility in the Midwest of the United States. The identity of isolates recovered from diseased fish was confirmed as F. asiatica by amplification and sequence comparison of the 16S ribosomal RNA and intracellular growth locus C (iglC) gene. Gross and microscopic examination of affected tissues revealed the presence of marked anterior renomegaly and splenomegaly with severe granulomatous disease.

Francisella infections in farmed and wild aquatic organisms.

Over the last 10 years or so, infections caused by bacteria belonging to a particular branch of the genus Francisella have become increasingly recognised in farmed fish and molluscs worldwide. While the increasing incidence of diagnoses may in part be due to the development and widespread availability of molecular detection techniques, the domestication of new organisms has undoubtedly instigated emergence of clinical disease in some species. Francisellosis in fish develops in a similar fashion independent of host species and is commonly characterised by the presence of multi-organ granuloma and high morbidity, with varying associated mortality levels. A number of fish species are affected including Atlantic cod, Gadus morhua; tilapia, Oreochromis sp.; Atlantic salmon, Salmo salar; hybrid striped bass, Morone chrysops × M. saxatilis and three-lined grunt, Parapristipoma trilinineatum. The disease is highly infectious and often prevalent in affected stocks. Most, if not all strains isolated from teleost fish belong to either F. noatunensis subsp. orientalis in warm water fish species or Francisella noatunensis subsp. noatunensis in coldwater fish species. The disease is quite readily diagnosed following histological examination and identification of the aetiological bacterium by culture on cysteine rich media or PCR. The available evidence may indicate a degree of host specificity for the various Francisella strains, although this area requires further study. No effective vaccine is currently available. Investigation of the virulence mechanisms and host response shows similarity to those known from Francisella tularensis infection in mammals. However, no evidence exists for zoonotic potential amongst the fish pathogenic Francisella.

Francisella infections in fish and shellfish.

A series of recent reports have implicated bacteria from the family Francisellaceae as the cause of disease in farmed and wild fish and shellfish species such as Atlantic cod, Gadus morhua L., tilapia, Oreochromis spp., Atlantic salmon, Salmo salar L., three-line grunt, Parapristipoma trilineatum (Thunberg), ornamental cichlid species, hybrid striped bass Morone chrysops x M. saxatilis and, recently, a shellfish species, the giant abalone, Haliotisgigantea Gmelin. The range of taxa affected will very probably rise as it is likely that there has been considerable under-reporting to date of these disease agents. In common with other Francisella species, their isolation and culture require specialized solid and liquid media containing cysteine and a source of iron. This likely restricted earlier efforts to identify them correctly as the cause of disease in aquatic animals. The most information to date relates to disease in cod, caused by F. noatunensis and tilapia, caused by F. noatunensis subsp. orientalis (also termed F. asiatica), both causing granulomatous inflammatory reactions. Mortalities in both species can be high and, as the disease can likely be transferred via live fish movements, they pose a significant threat to tilapia and cod aquaculture operations. Although the fish-pathogenic Francisella species are classified in the same genus as the human pathogens F. tularensis, causative agent of tularemia, and F. philomiragia, the risk to humans from the fish and shellfish pathogenic Francisella species is considered very low.

[Identification and characterization of the Francisella sp. strain 08HL01032 isolated in air condition systems].

OBJECTIVE: To identify and characterize the strain 08H101032 was isolated from air condition systems in the routine investigations of Legionella in Guangzhou, China, in 2008. METHODS: We adopted several phenotypic and genotypical methods, such as the growth status on various media, morphological, physical and biochemical characteristics, animal test, antibiotic susceptivities, PCR identification, sequence analysis of 16S RNA and RNA polymerase beta-subunit (ropB) gene etc, to determinate the phylogenetic position and outline the basic biological characteristics. RESULTS: Strain 08H101032 was Gram-negative with polymorphic short rods or coccobacillus; with no flagella; devoid of spores; well growth on buffered charcoal yeast extraction (BCYE) agar and BCYE supplemented with glycine (3 g/L), polymyxin B sulfate (80000 iu/L), vancomycin (1 mg/L) and cycloheximide (80 mg/L) (GVPC medium) within 2 days, but delayed growth on ordinary sheep blood agar untill 5 - 7 days; catalase positive; oxidase negative; no reduction of nitrate; no hydrolysis of urea; delayed fermention of glucose to produce acid; which was primarily considered as Legionella. It was lastly identified to the genus Fransicella, characterized by a variety of biochemical and molecular phylogenetic tests, which shared the highest similarities to F. Philomiragia with 95.3% to 16S rRNA gene of 1377 oligo nucleotides and 87.3% to ropB gene of 367 oligo nucleotides (GenBank accession number: FJ591095, FJ939309). Growth were observed after a treatment for 10 minutes with the KCl-HCl buffer of pH 2.2, 20 degrees C, and at 25 degrees C, 37 degrees C (optimum 25 degrees C - 28 degrees C), but not at 42 degrees C. The cells had capsule-like construction by transmission electron microscopy, however no virulence found to mice. CONCLUSIONS: Strain 08H101032 was a potential new species of the genus Fransicella with a typical characteristic of L-cysteine growth stimulating activity, distinguishingly to Legionella with L-cysteine growth dependent activity.

Direct isolation of Francisella spp. from environmental samples.

AIMS: To develop a selective medium for isolation of F. tularensis, F. novicida and F. philomiragia from environmental samples. METHODS AND RESULTS: A selective media, cysteine heart agar with 9% chocolatized sheep blood, containing polymyxin B, amphotericin B, cyclohexamide, cefepime and vancomycin (CHAB-PACCV) was developed and evaluated for growth of Francisella spp. No differences were observed in recovered colony forming units (CFUs) for F. tularensis, F. novicida and F. philomiragia on CHAB-PACCV vs nonselective CHAB. Growth of non-Francisella species was inhibited on CHAB-PACCV. When environmental samples were cultured on CHAB and CHAB-PACCV, only CHAB-PACCV allowed isolation of Francisella spp. Three new Francisella strains were isolated directly from seawater and seaweed samples by culture on CHAB-PACCV. CONCLUSIONS: CHAB-PACCV can be used for direct isolation of Francisella spp from environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Francisella spp. show a close association with environmental sources. Future utilization of CHAB-PACCV for isolation of Francisella spp. directly from environmental samples should prove valuable for investigating outbreaks and human infections attributed to environmental exposure.