Genetic diversity and phenotypic characterization of Iodobacter limnosediminis associated with skin lesions in freshwater fish.

The relatively unknown genus Iodobacter sp. has been repeatedly isolated from skin ulcers and saprolegniosis on freshwater fish in Finland, especially farmed salmonids. Genetic characterization verified that all 23 bacterial isolates studied here belonged to the species Iodobacter limnosediminis, previously undescribed from the fish microbiota. Whole-genome pulsed-field gel electrophoresis revealed variability between the I. limnosediminis strains, suggesting that they were most likely of environmental origin. Two I. limnosediminis strains caused lesions in 27%-53% of brown trout (Salmo trutta) injected intramuscularly (p ≤ .05). The lesions represented moderate to severe tissue damage, but for most fish, the tissues had been repaired by the end of the experiment through the accumulation of fibrocytes and macrophages at the site of the lesion. I. limnosediminis was reisolated from some lesions and/or internal organs. Phenotypically and biochemically, I. limnosediminis resembles several common bacterial species found in the aquatic environment, as it grows well on several media as whitish medium-sized colonies, is Gram negative and rod-shaped. Here, we characterized I. limnosediminis strains with several methods, including MALDI-TOF. This characterization will help in further investigations into the occurrence and possible involvement of I. limnosediminis in skin lesions of freshwater fish.

Vogesella perlucida-induced bacteremia in an advanced-age patient: first case report.

BACKGROUND: Vogesella species are common aquatic, Gram-negative rod-shaped bacteria, originally described in 1997. Vogesella perlucida was first isolated from spring water in 2008. Furthermore, bacterial pathogenicity of Vogesella perlucida has never been reported. Here, we report the first case of rare Vogesella perlucida-induced bacteremia in an advanced-age patient with many basic diseases and history of dexamethasone abuse. CASE PRESENTATION: A 71-year-old female was admitted with inflamed upper and lower limbs, rubefaction, pain and fever (about 40 °C). She had been injured in a fall at a vegetable market and then touched river snails with her injury hands. A few days later, soft tissue infection of the patient developed and worsened. Non-pigmented colonies were isolated from blood cultures of the patient. Initially, Vogesella perlucida was wrongly identified as Sphingomonas paucimobilis by Vitek-2 system with GN card. Besides, we failed to obtain an acceptable identification by the MALDI-TOF analysis. Finally, the isolated strain was identified as Vogesella perlucida by 16S rRNA gene sequences. In addition, the patient recovered well after a continuous treatment of levofloxacin for 12 days. CONCLUSION: Traditional microbiological testing system may be inadequate in the diagnosis of rare pathogenic bacteria. Applications of molecular diagnostics techniques have great advantages in clinical microbiology laboratory. By using 16S rRNA gene sequence analysis, we report the the first case of rare Vogesella perlucida-induced bacteremia.

Parasutterella, in association with irritable bowel syndrome and intestinal chronic inflammation.

BACKGROUND AND AIM: Irritable bowel syndrome (IBS) is a highly prevalent chronic functional gastrointestinal disorder. Recent studies have showed increasing important role of gut microbiota in the pathophysiological changes of IBS. Our study aims to elaborate the association between intestinal flora with the genesis and the development of IBS. METHODS: Illumina high-throughput sequencing technology was applied to investigate microbial communities of IBS patients and healthy donors. Stool specimens from the IBS-D patients were equally premixed and implanted into germ free C57B/6 mice to construct IBS animal model, and the normal group was also transplanted with normal premixed feces. The post-transplant defecation and intra-epithelial lymphocyte counts were evaluated. Microbial communities were also checked by the illumina high-throughput sequencing technology. RESULTS: Fifteen genuses significantly different were found expressed in the gut flora of IBS patients, and six genuses showed significantly different abundances between the stool specimens of mice of IBS group and normal group. Among these differences, Parasutterella expression was remarkably different in both screening and validation experiments and also related to chronic intestinal inflammation; therefore, Parasutterella expression is considered in association with the development and progression of IBS. CONCLUSION: Parasutterella may be related with the genesis and development of IBS and also associated with chronic intestinal inflammation in IBS patients.

A glimpse of the endophytic bacterial diversity in roots of blackberry plants (Rubus fruticosus).

The aim of this study was to explore the diversity of culturable bacterial communities residing in blackberry plants (Rubus fruticosus). Bacterial endophytes were isolated from plant roots, and their 16S rDNA sequences were amplified and sequenced. Our results show that the roots of R. fruticosus exhibit low colony forming units of bacterial endophytes per gram of fresh tissue (6 x 102 ± 0.5 x 102). We identified 41 endophytic bacterial species in R. fruticosus by BLAST homology search and a subsequent phylogenetic analysis, belonging to the classes Actinobacteria, Bacilli, Alfaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Predominantly, genera belonging the Proteobacteria (Burkholderia, 29.4%; Herbaspirillum, 10.7%; Pseudomonas, 4.9%; and Dyella, 3.9%), Firmicutes (Bacillus, 42.1%), and Actinobacteria (two isolates showing high identity with the Streptomyces genus, 1.9%) divisions were identified. Fifty percent of the bacterial endophytes produced the phytohormone indole-acetic acid (IAA), eleven of which exhibited higher IAA production (>5.8 mg/mL) compared to the plant growth-promoting strain, Pseudomonas fluorescens UM270. Additionally, the endophytic isolates exhibited protease activity (22%), produced siderophores (26.4%), and demonstrated antagonistic action (>50% inhibition of mycelial growth) against the grey mold phytopathogen Botrytis cinerea (3.9%). These results suggested that field-grown R. fruticosus plants contain bacterial endophytes within their tissues with the potential to promote plant growth and display antagonism towards plant pathogens.

Molecular insight of putative pathogenicity markers with ESBL genes and lipopolysaccharide in Laribacter hongkongensis.

Laribacter hongkongensis is an emerging bacterial pathogen causing gastroenteritis and traveller's diarrhoea. However, pathogenicity of L. hongkongensis has not yet been properly understood. We therefore, investigated putative pathogenicity markers like elt, est, stx1, stx2, eae, eaf, EAgg, bfpA, Int I, Int II, Int III and cnf respectively in strains received from Queen Mary Hospital, Hong Kong University. Above genes were commonly found among pathogenic member genera of Enterobacteriaceae-causing diarrhoea. Received strains were confirmed microbiologically and by 16S ribosomal RNA (rRNA) sequencing. Cultivability was examined using 23 different commercially available microbial growth media followed by antibiotic susceptibility test, lipopolysaccharide (LPS) extraction and raising antisera in rabbit against heat-killed L. hongkongensis. Moreover, Escherichia coli classification genes chuA, yjaA, TspE4 and extended spectrum beta lactamase (ESBL) genes like TEM, SHV, OXA, CTXM, CTXM1 and CTXM9 were also examined by PCR assay. Results showed the possession of eae, bfpA, Int and CTXM9 respectively for putative virulence. Furthermore, purity of extracted LPS was confirmed by HPLC, and raised serum was found useful in diagnosis of LPS; bacterium thus can be employed for immunodiagnostics.

Genome implosion elicits host-confinement in Alcaligenaceae: evidence from the comparative genomics of Tetrathiobacter kashmirensis, a pathogen in the making.

This study elucidates the genomic basis of the evolution of pathogens alongside free-living organisms within the family Alcaligenaceae of Betaproteobacteria. Towards that end, the complete genome sequence of the sulfur-chemolithoautotroph Tetrathiobacter kashmirensis WT001(T) was determined and compared with the soil isolate Achromobacter xylosoxidans A8 and the two pathogens Bordetella bronchiseptica RB50 and Taylorella equigenitalis MCE9. All analyses comprehensively indicated that the RB50 and MCE9 genomes were almost the subsets of A8 and WT001(T), respectively. In the immediate evolutionary past Achromobacter and Bordetella shared a common ancestor, which was distinct from the other contemporary stock that gave rise to Tetrathiobacter and Taylorella. The Achromobacter-Bordetella precursor, after diverging from the family ancestor, evolved through extensive genome inflation, subsequent to which the two genera separated via differential gene losses and acquisitions. Tetrathiobacter, meanwhile, retained the core characteristics of the family ancestor, and Taylorella underwent massive genome degeneration to reach an evolutionary dead-end. Interestingly, the WT001(T) genome, despite its conserved architecture, had only 85% coding density, besides which 578 out of its 4452 protein-coding sequences were found to be pseudogenized. Translational impairment of several DNA repair-recombination genes in the first place seemed to have ushered the rampant and indiscriminate frame-shift mutations across the WT001(T) genome. Presumably, this strain has just come out of a recent evolutionary bottleneck, representing a unique transition state where genome self-degeneration has started comprehensively but selective host-confinement has not yet set in. In the light of this evolutionary link, host-adaptation of Taylorella clearly appears to be the aftereffect of genome implosion in another member of the same bottleneck. Remarkably again, potent virulence factors were found widespread in Alcaligenaceae, corroborating which hemolytic and mammalian cell-adhering abilities were discovered in WT001(T). So, while WT001(T) relatives/derivatives in nature could be going the Taylorella way, the lineage as such was well-prepared for imminent host-confinement.

Intercellular and intracellular signalling systems that globally control the expression of virulence genes in plant pathogenic bacteria.

Plant pathogenic bacteria utilize complex signalling systems to control the expression of virulence genes at the cellular level and within populations. Quorum sensing (QS), an important intercellular communication mechanism, is mediated by different types of small molecules, including N-acyl homoserine lactones (AHLs), fatty acids and small proteins. AHL-mediated signalling systems dependent on the LuxI and LuxR family proteins play critical roles in the virulence of a wide range of Gram-negative plant pathogenic bacteria belonging to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Xanthomonas spp. and Xylella fastidiosa, members of the Gammaproteobacteria, however, possess QS systems that are mediated by fatty acid-type diffusible signal factors (DSFs). Recent studies have demonstrated that Ax21, a 194-amino-acid protein in Xanthomonas oryzae pv. oryzae, plays dual functions in activating a rice innate immune pathway through binding to the rice XA21 pattern recognition receptor and in regulating bacterial virulence and biofilm formation as a QS signal molecule. In xanthomonads, DSF-mediated QS systems are connected with the signalling pathways mediated by cyclic diguanosine monophosphate (c-di-GMP), which functions as a second messenger for the control of virulence gene expression in these bacterial pathogens.

Evaluation of in vitro virulence characteristics of the genus Pandoraea in lung epithelial cells.

Pandoraea species are emerging opportunistic pathogens capable of causing chronic lung infections in cystic fibrosis patients. This study examined the interactions of 17 Pandoraea isolates from the five identified species (Pandoraea apista, Pandoraea norimbergensis, Pandoraea pulmonicula, Pandoraea sputorum and Pandoraea pnomenusa) plus two Pandoraea genomospecies isolates with lung epithelial cells and their ability to form biofilms in vitro. Only three isolates showed an ability to invade A549 lung epithelial cells, and only one isolate was able to form biofilms. In contrast, all isolates triggered a pronounced pro-inflammatory response, with elevation of both interleukin (IL)-6 (two- to 19-fold) and IL-8 (10- to 50-fold) above that observed for a control strain of Escherichia coli. This property is likely to be a major factor in the pathogenesis of the genus.

Epidemic spread of Pandoraea apista, a new pathogen causing severe lung disease in cystic fibrosis patients.

We show that Pandoraea apista must be added to the increasing list of pathogens capable of causing chronic lung infection in cystic fibrosis (CF) patients. It is most likely that this strain of P. apista was transmissible among patients with CF, leading to spread of infection from the index patient to 5 other patients exposed during participation in winter camps and/or hospitalization. All patients developed chronic infection with high levels of antibodies, and 4 patients had a downhill course of lung disease. P. apista must therefore be considered a new and sometimes important pathogen for CF patients. Cohort isolation prevented further spread of P. apista in our CF center.

Acidovorax valerianellae sp. nov., a novel pathogen of lamb's lettuce [Valerianella locusta (L.) Laterr].

Bacterial spot disease of lamb's lettuce [Valerianella locusta (L.) Laterr.] was first observed in fields in 1991. This new bacterial disease is localized in western France in high-technology field production of lamb's lettuce for the preparation of ready-to-use salad. Nineteen strains isolated in 1992 and 1993 from typical black leaf spots of naturally infected lamb's lettuce were characterized and compared with reference strains of Acidovorax and Delftia. The pathogenicity of the 19 strains was confirmed by artificial inoculation. Biochemical and physiological tests, fatty acid profiles, DNA-DNA hybridization and other nucleic acid-based tests were performed. A numerical taxonomic analysis of the 19 lamb's lettuce strains showed a single homogeneous phenon closely related to previously described phytopathogenic taxa of the genus Acidovorax. DNA-DNA hybridization studies showed that the lamb's lettuce strains were 91-100% related to a representative strain, strain CFBP 4730(T), and constituted a discrete DNA hybridization group, indicating that they belong to the same novel species. Results from DNA-rRNA hybridization, 16S rRNA sequence analysis and fatty acid analysis studies confirmed that this novel species belongs to the beta-subclass of the Proteobacteria and, more specifically, to the family Comamonadaceae and the genus Acidovorax. The name Acidovorax valerianellae sp. nov. is proposed for this novel taxon of phytopathogenic bacteria. The type strain is strain CFBP 4730(T) (= NCPPB 4283(T)).

Sepsis, multiple organ failure, and death due to Pandoraea pnomenusa infection after lung transplantation.

A 30-year-old man died with Pandoraea pnomenusa sepsis after lung transplantation. Pandoraea species are gram-negative rods, closely related to, and commonly misidentified as, Burkholderia cepacia complex or Ralstonia species. Heretofore considered soil bacteria and colonizers that infect patients with chronic lung diseases, Pandoraea species can produce severe infections.

Colonization of Vitis vinifera by a green fluorescence protein-labeled, gfp-marked strain of Xylophilus ampelinus, the causal agent of bacterial necrosis of grapevine.

The dynamics of Xylophilus ampelinus were studied in Vitis vinifera cv. Ugni blanc using gfp-marked bacterial strains to evaluate the relative importance of epiphytic and endophytic phases of plant colonization in disease development. Currently, bacterial necrosis of grapevine is of economic importance in vineyards in three regions in France: the Cognac, Armagnac, and Die areas. This disease is responsible for progressive destruction of vine shoots, leading to their death. We constructed gfp-marked strains of the CFBP2098 strain of X. ampelinus for histological studies. We studied the colonization of young plants of V. vinifera cv. Ugni blanc by X. ampelinus after three types of artificial contamination in a growth chamber and in a greenhouse. (i) After wounding of the stem and inoculation, the bacteria progressed down to the crown through the xylem vessels, where they organized into biofilms. (ii) When the bacteria were forced into woody cuttings, they rarely colonized the emerging plantlets. Xylem vessels could play a key role in the multiplication and conservation of the bacteria, rather than being a route for plant colonization. (iii) When bacterial suspensions were sprayed onto the plants, bacteria progressed in two directions: both in emerging organs and down to the crown, thus displaying the importance of epiphytic colonization in disease development.

Degradation and transformability of DNA from transgenic leaves.

The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the plant pathogen Ralstonia solanacearum. Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene. These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues. Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil.

Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence.

As reported previously for Ralstonia solanacearum strain GMI1000, wild-type strains AW1 and K60 were shown to produce Hrp pili. AW1 and K60 mutants lacking Hrp pili still exhibited twitching motility, which requires type 4 pili (Tfp), and electron microscopy revealed that they still made flexuous polar pili. Twitching-positive cells had an extracellular 17 kDa protein that was associated with piliation, and an internal 43-amino-acid sequence of this protein was typical of type 4 pilins. This amino acid sequence is encoded by an open reading frame, designated pilA, in the genomic sequence of GMI1000. PilA is 46% identical to a Pseudomonas aeruginosa type 4 pilin over its entire length and has all the conserved residues and motifs characteristic of type 4 group A pilins. pilA mutants did not make the 17 kDa PilA protein and did not exhibit twitching motility. When compared with its parent, an AW1 pilA mutant was reduced in virulence on tomato plants and in autoaggregation and biofilm formation in broth culture. Unlike AW1, a pilA mutant did not exhibit polar attachment to tobacco suspension culture cells or to tomato roots; it was also not naturally competent for transformation. We reported previously that twitching motility ceases in maturing AW1 colonies and that inactivation of PhcA, a global transcriptional regulator, results in colonies that continue to exhibit twitching motility. Similarly, in broth culture, expression of a pilA::lacZ fusion in AW1 decreased 10-fold at high cell density, but expression remained high in a phcA mutant. In addition, pilA::lacZ expression was positively regulated 10-fold by PehR, a response regulator that is known to be repressed by PhcA. This signal cascade is sufficient to explain why pilA expression, and thus twitching motility, decreases at high cell densities.

Genetic dissection of Ralstonia solanacearum hrp gene cluster reveals that the HrpV and HrpX proteins are required for Hrp pilus assembly.

In both plant and mammalian Gram-negative pathogenic bacteria, type III secretion systems (TTSSs) play a crucial role in interactions with the host. All these systems share conserved proteins (called Hrc in plant pathogens), but each bacterium also produces a variable number of additional type III proteins either unique or with counterparts only in a limited number of related systems. In order to investigate the role of the different proteins encoded by the hrp gene cluster of the phytopathogenic bacterium Ralstonia solanacearum, non-polar mutants in all hrp genes (except for hrcQ) were analysed for their interactions with plants, their ability to secrete the PopA protein and their production of the Hrp pilus. In addition to Hrc proteins and the HrpY major component of the Hrp pilus, four additional Hrp proteins are indispensable for type III secretion and for interactions with plants. We also provide evidence that hrpV and hrpX mutants can still target the HrpY pilin outside the bacterial cell but are impaired in the production of Hrp pili, indicating that HrpV and HrpX proteins are involved in the assembly of this appendage.

Twitching motility of Ralstonia solanacearum requires a type IV pilus system.

Twitching motility is a form of bacterial translocation over firm surfaces that requires retractile type IV pili. Microscopic colonies of Ralstonia solanacearum strains AW1, K60 and GMI1000 growing on the surface of a rich medium solidified with 1.6% agar appeared to exhibit twitching motility, because early on they divided into motile 'rafts' of cells and later developed protruding 'spearheads' at their margins. Individual motile bacteria were observed only when they were embedded within masses of other cells. Varying degrees of motility were observed for 33 of 35 strains of R. solanacearum in a selected, diverse collection. Timing was more important than culture conditions for observing motility, because by the time wild-type colonies were easily visible by eye (about 48 h) this activity ceased and the spearheads were obscured by continued bacterial multiplication. In contrast, inactivation of PhcA, a transcriptional regulator that is essential for R. solanacearum to cause plant disease, resulted in colonies that continued to expand for at least several additional days. Multiple strains with mutations in regulatory genes important for virulence were tested, but all exhibited wild-type motility. Many of the genes required for production of functional type IV pili, and hence for twitching motility, are conserved among unrelated bacteria, and pilD, pilQ and pilT orthologues were identified in R. solanacearum. Colonies of R. solanacearum pilQ and pilT mutants did not develop spearheads or rafts, confirming that the movement of cells that had been observed was due to twitching motility. Compared to the wild-type parents, both pilQ and pilT mutants caused slower and less severe wilting on susceptible tomato plants. This is the first report of twitching motility by a phytopathogenic bacterium, and the first example where type IV pili appear to contribute significantly to plant pathogenesis.

The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection.

The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.

Classification of Ralstonia pickettii biovar 3/'thomasii' strains (Pickett 1994) and of new isolates related to nosocomial recurrent meningitis as Ralstonia mannitolytica sp. nov.

Strains isolated independently from two patients could be recognized as Ralstonia pickettii biovar 3/'thomasii'. The 16S rDNA sequences of these strains and two other strains of R. pickettii biovar 3/'thomasii' clustered at less than 98% similarity versus all other described Ralstonia species and at less than 97 % versus the two other R. pickettii biovars. The separate species status of R. pickettii biovar 3/'thomasii' was confirmed by DNA-DNA hybridization, indicating less than 60% DNA homology with the R. pickettii biovars Va-1 and Va-2 and with two as-yet unclassified but biochemically similar Ralstonia strains. Phenotypically, this Ralstonia species can be distinguished from all described Ralstonia species by its acidification of D-arabitol and mannitol and by its lack of nitrate reduction and of alkalinization of tartrate and from two as-yet unclassified Ralstonia strains only by its lack of nitrate reduction. The name Ralstonia mannitolytica sp. nov. is proposed, reflecting the characteristic acidification of mannitol. Resistance to desferrioxamine is another difference from R. pickettii and Ralstonia solanacearum. Although several nosocomial outbreaks have been associated with R. mannitolytica, life-threatening infections have not yet been reported, possibly due to misidentification as Pseudomonas fluorescens or Burkholderia cepacia. In at least one of the two cases reported here, the R. mannitolytica isolate was found to be clinically relevant, causing recurrent nosocomial meningitis, with an infected implanted catheter as the source. The type strain of R. mannitolytica is NCIMB 10805T (= LMG 6866T), which was isolated during the first described outbreak as 'Pseudomonas thomasii' at St Thomas' Hospital, London, UK, in 1971.