A Case of Successful Treatment of Recurrent Urinary Tract Infection by Extended-Spectrum ß-Lactamase Producing Klebsiella pneumoniae Using Oral Lyophilized Fecal Microbiota Transplant.

Recurrent urinary tract infections (UTIs) are a challenging clinical entity that can be frustrating for patient and physician alike. Repeated rounds of antibiotics can select for multidrug-resistant organisms, further complicating care. We describe the successful use of fecal microbiota transplantation (FMT) for the treatment of recurrent extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae UTIs in a patient with an ileal conduit and urostomy. In the 18 months after FMT, the patient had not experienced new infections with ESBL-producing organisms. The urine and stool microbiomes of the patient were tracked before and post-FMT using 16s RNA sequencing with measurement of α-diversity. Sequencing of the recipient microbiota did not mirror the donor stool taxa at either site, but an increase in the relative proportion of the genus Bacteroides as compared with Prevotella was noted in the stool post-transplant. FMTs may be a promising treatment option for recurrent multidrug-resistant infections.

BrnQ-Type Branched-Chain Amino Acid Transporters Influence Bacillus anthracis Growth and Virulence.

Bacillus anthracis, the anthrax agent, exhibits robust proliferation in diverse niches of mammalian hosts. The metabolic attributes of B. anthracis that permit rapid growth in multiple mammalian tissues have not been established. We posit that branched-chain amino acid (BCAA) (isoleucine, leucine, and valine) metabolism is key to B. anthracis pathogenesis. Increasing evidence indicates the relationships between B. anthracis virulence and the expression of BCAA-related genes. The expression of some BCAA-related genes is altered during culture in bovine blood in vitro, and the bacterium exhibits valine auxotrophy in a blood serum mimic medium. Transcriptome analyses have revealed that the virulence regulator AtxA, which positively affects the expression of the anthrax toxin and capsule genes, negatively regulates genes predicted to be associated with BCAA biosynthesis and transport. Here, we show that B. anthracis growth in defined medium is severely restricted in the absence of exogenous BCAAs, indicating that BCAA transport is required for optimal growth in vitro. We demonstrate functional redundancy among multiple BrnQ-type BCAA transporters. Three transporters are associated with isoleucine and valine transport, and the deletion of one, BrnQ3, attenuates virulence in a murine model for anthrax. Interestingly, an ilvD-null mutant lacking dihydroxy acid dehydratase, an enzyme essential for BCAA synthesis, exhibits unperturbed growth when cultured in medium containing BCAAs but is highly attenuated in the murine model. Finally, our data show that BCAAs enhance AtxA activity in a dose-dependent manner, suggesting a model in which BCAAs serve as a signal for virulence gene expression. IMPORTANCE Infection with B. anthracis can result in systemic disease with large numbers of the bacterium in multiple tissues. We found that branched-chain amino acid (BCAA) synthesis is insufficient for the robust growth of B. anthracis; access to BCAAs is necessary for the proliferation of the pathogen during culture and during infection in a murine model for anthrax. B. anthracis produces an unusually large repertoire of BCAA-related transporters. We identified three isoleucine/valine transporters with partial functional redundancy during culture. The deletion of one of these transporters, BrnQ3, resulted in attenuated virulence. Interestingly, a BCAA biosynthesis mutant grew well in medium containing BCAAs but, like BrnQ3, was attenuated for virulence. These results suggest that BCAAs are limiting in multiple niches during infection and further our understanding of the nutritional requirements of this important pathogen.

Influence of the phosphoenolpyruvate:carbohydrate phosphotransferase system on toxin gene expression and virulence in Bacillus anthracis.

AtxA, the master virulence gene regulator of Bacillus anthracis, is a PRD-Containing Virulence Regulator (PCVR) as indicated by the crystal structure, post-translational modifications and activity of the protein. PCVRs are transcriptional regulators, named for PTS Regulatory Domains (PRDs) subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP-PTS) and for their impact on virulence gene expression. Here we present data from experiments employing physiological, genetic and biochemical approaches that support a model in which the PTS proteins HPr and Enzyme I (EI) are required for transcription of the atxA gene, rather than phosphorylation of AtxA. We show that atxA transcription is reduced 2.5-fold in a mutant lacking HPr and EI, and that this change is sufficient to affect anthrax toxin production. Mutants harboring HPr proteins altered for phosphotransfer activity were unable to restore atxA transcription to parent levels, suggesting that phosphotransfer activity of HPr and EI is important for regulation of atxA. In a mouse model for anthrax, a HPr- EI- mutant was attenuated for virulence. Virulence was restored by expressing atxA from an alternative, PTS-independent, promoter. Our data support a model in which HPr transfers a phosphate to an unidentified downstream transcriptional regulator to influence atxA gene transcription.

Pilus hijacking by a bacterial coaggregation factor critical for oral biofilm development.

The formation of dental plaque, a highly complex biofilm that causes gingivitis and periodontitis, requires specific adherence among many oral microbes, including the coaggregation of Actinomyces oris with Streptococcus oralis that helps to seed biofilm development. Here, we report the discovery of a key coaggregation factor for this process. This protein, which we named coaggregation factor A (CafA), is one of 14 cell surface proteins with the LPXTG motif predicted in A. oris MG1, whose function was hitherto unknown. By systematic mutagenesis of each of these genes and phenotypic characterization, we found that the Actinomyces/Streptococcus coaggregation is only abolished by deletion of cafA. Subsequent biochemical and cytological experiments revealed that CafA constitutes the tip of a unique form of the type 2 fimbria long known for its role in coaggregation. The direct and predominant role of CafA in adherence is evident from the fact that CafA or an antibody against CafA inhibits coaggregation, whereas the shaft protein FimA or a polyclonal antibody against FimA has no effect. Remarkably, FimA polymerization was blocked by deletion of genes for both CafA and FimB, the previously described tip protein of the type 2 fimbria. Together, these results indicate that some surface proteins not linked to a pilus gene cluster in Gram-positive bacteria may hijack the pilus. These unique tip proteins displayed on a common pilus shaft may serve distinct physiological functions. Furthermore, the pilus shaft assembly in Gram-positive bacteria may require a tip, as is true for certain Gram-negative bacterial pili.

Standard addition method for laser ablation ICPMS using a spinning platform.

A method has been developed for the fast and easy determination of Pb, Sr, Ba, Ni, Cu, and Zn, which are of geological and environmental interest, in solid samples by laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) using a spinning sample platform. The platform, containing a sample and a standard, is spun during the ablation, allowing the quasi-simultaneous ablation of both materials. The aerosols resulting from the ablation of sample and standard were mixed in the ablation cell allowing quantification of analytes by standard additions. The proportion of standard versus sample of the mixing can be increased by performing the ablation further from the axis of rotation. The ablated masses have been determined using a new strategy based on isotope dilution analysis. This spinning laser ablation method has been applied to the Allende meteorite and four powdered standard reference materials (SRMs) fused in lithium borate glasses: two sediments as well as a soil and a rock material. SRM 612 (Trace Elements in Glass) was also analyzed despite having a matrix slightly different from the glass standard obtained by lithium borate fusion. The deviation from the certified values was found to be less than 15% for most of the mass fractions for all the elements and samples studied, with an average precision of 10%. These results demonstrate the validity of the proposed method for the direct and fast analysis of solid samples of different matrixes by standard additions, using a single standard sample.

Putting a spin on LA-ICP-MS analysis combined to isotope dilution.

The determination of Zn, Sr, Ba, and Pb in solid samples has been achieved by laser ablation inductively coupled plasma isotope dilution mass spectrometry using a spinning platform. The fast rotation of a sample and an isotopically enriched spike placed close together on a sample holder allowed performing the isotope dilution directly inside the ablation cell. The proportion of spike versus sample of the aerosol mixture obtained has been determined online by isotope dilution in order to correct for differences in ablation rate although both materials were placed on the axis of rotation of the motor. Homogeneous, time-stable, and reusable samples were prepared by lithium borate fusion. A unique isotopically enriched spike glass was used to analyze four Standard Reference Materials of different matrix (after a simple polishing): two sediments Standard Reference Material (SRM) 1944 and SRM 2702 and two soils SRM 2586 and SRM 2711a. The proposed method yielded mass fractions with a deviation from the certified value usually lower than 12% and a precision of less than 9% RSD (except for Zn in SRM 2586 and 2711a). Although direct spiking of the solid before fusion could presumably provide better isotopic mixing, the presented methodology allows the reuse of the spike glass (thus, decreasing drastically the cost of the analysis) and is relatively faster because the spike does not need to be weighted, added, and evaporated each time. These results demonstrate the potential of this newly developed method for fast analysis of solid samples using isotope dilution at a low cost.