Background: FslE and FupA are Francisella-specific paralogous proteins involved in iron acquisition. Results: fslE mutation disrupts siderophore-mediated ferric iron uptake, fupA mutation impairs high affinity ferrous iron uptake, and both mutations impact virulence. Conclusion: Optimal iron acquisition and virulence require both paralogs. Significance: Iron acquisition mechanisms are potential targets for preventive or therapeutic intervention in F. tularensis infections.

BackgroundPan-bacterial 16S rRNA microbiome surveys performed with massively parallel DNA sequencing technologies have transformed community microbiological studies. Current 16S profiling methods, however, fail to provide sufficient taxonomic resolution and accuracy to adequately perform species-level associative studies for specific conditions. This is due to the amplification and sequencing of only short 16S rRNA gene regions, typically providing for only family- or genus-level taxonomy. Moreover, sequencing errors often inflate the number of taxa present. Pacific Biosciences’ (PacBio’s) long-read technology in particular suffers from high error rates per base. Herein, we present a microbiome analysis pipeline that takes advantage of PacBio circular consensus sequencing (CCS) technology to sequence and error correct full-length bacterial 16S rRNA genes, which provides high-fidelity species-level microbiome data.ResultsAnalysis of a mock community with 20 bacterial species demonstrated 100% specificity and sensitivity with regard to taxonomic classification. Examination of a 250-plus species mock community demonstrated correct species-level classification of > 90% of taxa, and relative abundances were accurately captured. The majority of the remaining taxa were demonstrated to be multiply, incorrectly, or incompletely classified. Using this methodology, we examined the microgeographic variation present among the microbiomes of six sinonasal sites, by both swab and biopsy, from the anterior nasal cavity to the sphenoid sinus from 12 subjects undergoing trans-sphenoidal hypophysectomy. We found greater variation among subjects than among sites within a subject, although significant within-individual differences were also observed. Propiniobacterium acnes (recently renamed Cutibacterium acnes) was the predominant species throughout, but was found at distinct relative abundances by site.ConclusionsOur microbial composition analysis pipeline for single-molecule real-time 16S rRNA gene sequencing (MCSMRT, https://github.com/jpearl01/mcsmrt) overcomes deficits of standard marker gene-based microbiome analyses by using CCS of entire 16S rRNA genes to provide increased taxonomic and phylogenetic resolution. Extensions of this approach to other marker genes could help refine taxonomic assignments of microbial species and improve reference databases, as well as strengthen the specificity of associations between microbial communities and dysbiotic states.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0569-2) contains supplementary material, which is available to authorized users.

Shigella species represent one of the growing numbers of antimicrobial-resistant bacteria in developing countries. Fluoroquinolone-resistant strains of Shigella dysenteriae type1 and Shigella flexneri type 2a emerged in India during 2002 and 2003, respectively. Sixty strains of Shigella from different parts of India were analysed for antimicrobial susceptibility, the presence of the qnr plasmid, mutations in the quinolone resistance determining regions (QRDRs), fluoroquinolone accumulation, and the presence of other genes encoding resistance to various antimicrobials. Fluoroquinolone-resistant strains had mutations in gyrA and parC genes and had an active efflux system. They were also resistant to several other antimicrobials but were susceptible to azithromycin and ceftriaxone. The majority of the strains harboured genes encoding resistance to ampicillin (97 %), tetracycline (95 %), streptomycin (95 %) and chloramphenicol (94 %). PFGE analysis revealed clonality among strains of S. dysenteriae types 1 and 5, S. flexneri type 2a and Shigella boydii type 12. INTRODUCTIONShigellosis remains an important public health problem in developing countries with Shigella sonnei in Europe and the US and Shigella flexneri in Asian and African countries being of epidemiological importance. Antimicrobial therapy is advocated for shigellosis to shorten the duration of illness (Salam & Bennish, 1991). However, in Asia and Africa, antimicrobial resistance is an emerging problem among Shigella species (von Seidlein et al., 2006) and treatment options are becoming limited globally (Salam & Bennish, 1991;Kariuki & Hart, 2001). The World Health Organization has recommended that ciprofloxacin should be considered a first-line antibiotic for the treatment of shigellosis, and the use of nalidixic acid is not encouraged, even in areas where it is still effective against Shigella (WHO, 2004). Similar to the prevalence of different serotypes, antimicrobial-resistance patterns of strains also differ from country to country and even within the same country (Pazhani et al., 2005;von Seidlein et al., 2006), which may be due to the spread of resistant clones as found for multidrug-resistant strains of Shigella dysenteriae type 1 in eastern parts of India (Pazhani et al., 2004). In this study, we have investigated the mechanisms of antibiotic resistance and clonal relatedness of Shigella strains isolated from epidemic and endemic cases of shigellosis in different parts of India. METHODSBacterial strains. We examined 60 strains of Shigella species (20 S. dysenteriae, 16 S. flexneri, 7 Shigella boydii and 17 S. sonnei) isolated from dysentery outbreaks from different parts of India and sporadic hospitalized cases of shigellosis in Kolkata and Goa between 2001 and 2004. Strains were confirmed as Shigella spp. by standard biochemical tests (WHO, 1987) and serotyped using commercially available antisera (Denka Seiken).Antimicrobial susceptibility testing. Antimicrobial susceptibility tests were performed by a disc diffusion method in accordance with...

The Gram-negative pathogen Francisella tularensis secretes a siderophore to obtain essential iron by a TonB-independent mechanism. The fslABCDE locus, encoding siderophore-related functions, is conserved among different Francisella strains. In the virulent strain Schu S4, fslE is essential for siderophore utilization and for growth under conditions of iron limitation. In contrast, we found that deletion of fslE did not affect siderophore utilization by the attenuated live vaccine strain (LVS). We found that one of the fslE paralogs encoded in the LVS genome, FTL_0439 (fupA/B), was able to partially complement a Schu S4 ⌬fslE mutant for siderophore utilization. We generated a deletion of fupA/B in LVS and in the LVS ⌬fslE background. The ⌬fupA/B mutant showed reduced growth under conditions of iron limitation. It was able to secrete but was unable to utilize siderophore. Mutation of both fupA/B and fslE resulted in a growth defect of greater severity. The ⌬fupA/B mutants showed a replication defect in J774.1A cells and decreased virulence following intraperitoneal infection in mice. Complementation of the ⌬fupA/B mutation in cis restored the ability to utilize siderophore and concomitantly restored virulence. Our results indicate that fupA/B plays a significant role in the siderophore-mediated iron uptake mechanism of LVS whereas fslE appears to play a secondary role. Variation in iron acquisition mechanisms may contribute to virulence differences between the strains.

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We surveyed urine microbiota of females diagnosed with interstitial cystitis/bladder pain syndrome (IC/BPS) and matched control participants enrolled in the National Institutes of Health (NIH) Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network using the culture-independent methodology. Midstream urine specimens were analyzed with the Plex-ID molecular diagnostic platform that utilizes polymerase chain reaction–electrospray ionization–time-of-flight–mass spectrometry (PCR-ESI-TOF MS) to provide a comprehensive identification of bacterial and select fungal species. IC/BPS and control participants were evaluated for differences (presence, diversity, and abundance) in species and genus. Urine specimens obtained from 181 female IC/BPS and 182 female control participants detected a total of 92 species (41 genera). Mean (SD) species count was 2.49 (1.48) and 2.30 (1.28) among IC/BPS and control participants, respectively. Overall species composition did not significantly differ between IC/BPS and control participants at any level (p = 0.726 species level, p = 0.222 genus level). IC/BPS participants urine trended to an overabundance of Lactobacillus gasseri (p = 0.09) detected but had a lower prevalence of Corynebacterium compared with control participants (p = 0.002). The relative abundance data analysis mirrored the prevalence data differences with no significant differences in most species or genus abundance other than Lactobacillus gasseri and Corynebacterium (p = 0.08 and p = 0.001, respectively). No cause and/or effect conclusion can be drawn from this observation, but it suggests that a more comprehensive evaluation (vaginal, bowel, catheterized bladder and/or tissue-based specimens) of the lower urinary tract microbiota in IC/BPS patients is warranted.

Francisella tularensis is a highly infectious Gram-negative pathogen that replicates intracellularly within the mammalian host. One of the factors associated with virulence of F. tularensis is the protein FupA that mediates high-affinity transport of ferrous iron across the outer membrane. Together with its paralogue FslE, a siderophore-ferric iron transporter, FupA supports survival of the pathogen in the host by providing access to the essential nutrient iron. The FupA orthologue in the attenuated live vaccine strain (LVS) is encoded by the hybrid gene fupA/B, the product of an intergenic recombination event that significantly contributes to attenuation of the strain. We used 55 Fe transport assays with mutant strains complemented with the different paralogues to show that the FupA/B protein of LVS retains the capacity for high-affinity transport of ferrous iron, albeit less efficiently than FupA of virulent strain Schu S4. 55 Fe transport assays using purified siderophore and siderophore-dependent growth assays on iron-limiting agar confirmed previous findings that FupA/B also contributes to siderophore-mediated ferric iron uptake. These assays further demonstrated that the LVS FslE protein is a weaker siderophore-ferric iron transporter than the orthologue from Schu S4, and may be a result of the sequence variation between the two proteins. Our results indicate that iron-uptake mechanisms in LVS differ from those in Schu S4 and that functional differences in the outer membrane iron transporters have distinct effects on growth under iron limitation.

Iron acquisition mechanisms in Francisella tularensis, the causative agent of tularemia, include the Francisella siderophore locus (fsl) siderophore operon and a ferrous iron–transport system comprising outer‐membrane protein FupA and inner‐membrane transporter FeoB. To characterize these mechanisms and to identify any additional iron uptake systems in the virulent subspecies tularensis, single and double deletions were generated in the fsl and feo iron acquisition systems of the strain Schu S4. Deletion of the entire fsl operon caused loss of siderophore production that could be restored by complementation with the biosynthetic genes fslA and fslC and Major Facilitator Superfamily (MFS) transporter gene fslB. 55Fe‐transport assays demonstrated that siderophore‐iron uptake required the receptor FslE and MFS transporter FslD. A ΔfeoB′ mutation resulted in loss of ability to transport ferrous iron (55Fe2+). A ΔfeoB′ ΔfslA mutant that required added exogenous siderophore for growth in vitro was unable to grow within tissue culture cells and was avirulent in mice, indicating that no compensatory cryptic iron uptake systems were induced in vivo. These studies demonstrate that the fsl and feo pathways function independently and operate in parallel to effectively support virulence of F. tularensis.