The development of methods to investigate the mechanisms underlying serum resistance of Ureaplasma species

The human Ureaplasma species are among the smallest and simplest self-replicating bacteria known to date. These microbes cause infection in humans, particularly in the upper genital tract during pregnancy, leading to several adverse outcomes including preterm birth, chorioamnionitis, and respiratory diseases of neonates. Little is known about the pathogenesis of Ureaplasma and mechanisms by which they avoid recognition and killing by the complement system. In this thesis, some mechanisms underlying serum resistance of Ureaplasma spp. were investigated. This goal was achieved by creating serum-resistant models of serum-sensitive laboratory Ureaplasma strains and developing and using some proteomic and molecular biology methods to study the role of potential factors, which mediate serum resistance and play a role in pathogenesis of Ureaplasma. My original contribution to the knowledge in this work was the development of transposon mutagenesis method that can now be used to study virulence genes of Ureaplasma. This method will also allow genetic manipulation of Ureaplasma for future studies. Monitoring and investigating induced serum-resistant strains using immunoblot analysis and proteomics revealed significant changes in two candidate proteins coincident with serum resistance. The first was the elongation factor Tu protein that found to be immunogenic and had altered pI isoforms. The observed change in this protein was consistent in all serum-resistant strains, which suggests a possible role in mechanism of serum resistance, possibly as a mediator for binding complement regulators, such as factor H and C4BP, at the cell surface of Ureaplasma. The second candidate protein was a novel 41 kDa protein that was uniquely expressed in all induced serum-resistant strains. Expression of this protein in all resistant strains strongly indicates its involvement in mechanism(s) of serum resistance of Ureaplasma. The possible gene that encodes for this 41 kDa protein has putatively been identified as UUR10_0137 in the genome of U. urealyticum serovar 10 (strain ATCC 33699) using the transposon mutagenesis method developed in this study. Although the gene product of UUR10_0137 gene is not known (hypothetical protein), this protein is now identified and proposed to have a role in serum resistance of Ureaplasma. The product of the UUR10_0137 gene could function as a complement regulator or inhibitor that prevents the activation of complement system, protecting Ureaplasma from the complement attack. The contribution of the multiple- banded antigen, MBA, was proven to be unimportant to serum resistance. Sole antigenic variations in this major surface antigen of Ureaplasma did not play any role in mediating serum resistance. Confirmation of a gene that mediates complement resistance would dramatically increase our understanding of Ureaplasma pathogenicity and provide a target for future human studies with preterm birth and Ureaplasma infection

The polymyxin derivative NAB739 is synergistic with several antibiotics against polymyxin-resistant strains of Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii

The antibiotic crisis has reinstated polymyxins, once abandoned because of their toxicity. Now, preclinical studies have revealed better tolerated and more effective derivatives of polymyxins such as NAB739. Simultaneously, polymyxin-resistant (PMR) strains such as the mcr-1 strains have received lots of justified publicity, even though they are still very rare. Here we show that NAB739 sensitizes the PMR strains to rifampin, a classic "anti-Gram-positive" antibiotic excluded by the intact outer membrane (OM) permeability barrier, as well as to retapamulin, the surrogate of lefamulin, an antibiotic under development against Gram-positive bacteria. Polymyxin B was used as a comparator. The combination of NAB739 and rifampin was synergistic against ten out of eleven PMR strains of Escherichia coll. (Fractional Synergy Indices, FICs, 0.14-0.19) and that of NAB739 and retapamulin against all the tested eleven strains (FICs 0.19-0.25). Against PMR Klebsiella pneumoniae (n = 7), the FICs were 0.13-0.27 for NAB739 + rifampin and 0.14-0.28 for NAB739 + retapamulin. Against Acinetobacter baumannii (n = 2), the combination of NAB739 and rifampin had the FIC of 0.09-0.19. Furthermore, NAB739 and meropenem were synergistic (FICs 0.25-0.50) against four out of five PMR strains that were simultaneously resistant to meropenem.Peer reviewe

Baseline azithromycin resistance in the gut microbiota of preterm born infants

Background Macrolides, including azithromycin, are increasingly used in preterm-born infants to treat Ureaplasma infections. The baseline carriage of macrolide resistance genes in the preterm stool microbiota is unknown. Objectives Identify carriage of azithromycin resistant bacteria and the incidence of macrolide resistant genes. Methods Azithromycin resistant bacteria were isolated from serial stool samples obtained from preterm infants (≤32 weeks’ gestation) by culturing aerobically/anaerobically, in the presence/absence of azithromycin. Using quantitative PCR, we targeted 6 common macrolide resistance genes (erm(A), erm(B), erm(C), erm(F), mef(A/E), msr(A)) in DNA extracted from selected bacteria resistant to azithromycin. Results From 89 stool samples from 37 preterm-born infants, 93.3% showed bacterial growth in aerobic or anaerobic conditions. From the 280 azithromycin resistant isolates that were identified, Staphylococcus (75%) and Enterococcus (15%) species dominated. Macrolide resistance genes were identified in 91% of resistant isolates: commonest were erm(C) (46% of isolates) and msr(A) (40%). Multiple macrolide resistance genes were identified in 18% of isolates. Conclusion Macrolide resistance is common in the gut microbiota of preterm-born infants early in life, most likely acquired from exposure to the maternal microbiota. It will be important to assess modulation of macrolide resistance, if macrolide treatment becomes routine in the management of preterm infants. Impact Statement Azithromycin resistance is present in the stool microbiota in the first month of life in preterm infants 91% of azithromycin resistant bacteria carried at least one of 6 common macrolide resistant genes Increasing use of macrolides in the preterm population makes this an important area of stud

Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-beta-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential beta-lactamase stable beta-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.Peer reviewe

Correction: Synergic Activation of Toll-Like Receptor (TLR) 2/6 and 9 in Response to Ureaplasma parvum & urealyticum in Human Amniotic Epithelial Cells.

[This corrects the article DOI: 10.1371/journal.pone.0061199.]

Synergic activation of toll-like receptor (TLR) 2/6 and 9 in response to Ureaplasma parvum & urealyticum in human amniotic epithelial cells.

Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), preterm labour (PL) pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to "sense" pathogens. One such family of PRRs are the Toll like receptor family (TLR). In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK) transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA), trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly

Isolation of separate Ureaplasma species from endotracheal secretions of twin patients

Isolation of Ureaplasma spp. from preterm neonates and the association with development of bronchopulmonary dysplasia has been previously investigated. However, few studies have contrasted the nature of infection in twins. In this article, we report that dizygotic twins (1 girl, 1 boy) born at 24 weeks gestation both yielded culturable Ureaplasma from endotracheal secretions. The samples were part of a serial blind collection cohort of ventilated premature neonates, and analysis of repeat cultures showed stable, separate infections over a period of 17 and 21 days, respectively. Immunoblot and probe-specific quantitative polymerase chain reaction analysis determined that Twin 1 was solely infected with Ureaplasma parvum (specifically, serovar 6 by gene sequencing), whereas Twin 2 was solely infected with Ureaplasma urealyticum (specifically, genotype A- serovars 2, 5, and 8 by gene sequencing). Immunoblot analysis found that the major surface antigen (multiple-banded antigen) altered relative mass for both strains during the course of infection. Quantitative polymerase chain reaction analysis of extracted endotracheal aspirates confirmed no evidence of mixed infection for either twin. Failure of sentinel ventilated preterm infants on the same ward to acquire Ureaplasma infection after the first week of birth suggests no cot-to-cot transfer of Ureaplasma infection occurred. This study demonstrated not only a contrasting clinical outcome for a set of twins infected with 2 separate species of Ureaplasma, but also the first real-time demonstration of multiple-banded antigen alteration and evolution of Ureaplasma over the course of a clinical infection

TLR and GM-1 ganglioside FRET mearurements before and after Ureaplasma stimulation.

<p>TLR and GM-1 ganglioside FRET mearurements before and after Ureaplasma stimulation of human amniotic epithelial cells (A). Energy transfer between Cy3-labelled TLR1, TLR2, TLR4 or TLR6 and GM-1 ganglioside (Cy5-cholera-toxin) before (white bar charts) and after stimulation with <i>Ureaplasma</i> (1×10⁸ bacteria/ml to 1×10⁷ cells/ml) <i>parvum SV3</i> (black barcharts), <i>U.parvum</i> SV14 (grey barcharts), <i>U. urealyticum</i> SV2 (stripped bar charts) or MBA (1 µg/ml). Energy transfer between GM1 (Cy5) and the different receptors was measured from the increase in donor (Cy3) fluorescence after acceptor (Cy5) photobleaching. The percentage of energy transfer and standard deviation was calculated from three independent experiments. Asterisks indicate statistically significant (<i>p</i><0.05) increase in energy transfer compared to corresponding unstimulated controls. (B) Inhibition of IL-6 production after lipid raft disruption. Human amniotic epithelial cells were either not treated (white barcharts) or pre-treated with nystatin and subsequently stimulated with the different Ureaplasma serovars or MBA. The supernatants were harvested and assayed for cytokine content using the Cytometric Bead Array (CBA) system (Becton Dickinson). Fluorescence was detected using a FACSCalibur (BectonDickinson). The data represents the mean ± SD of three independent experiments. Asterisks indicate statistical significance (<i>p</i><0.05).</p