Identification of Yersinia enterocolitica isolates from humans, pigs and wild boars by MALDI TOF MS

Abstract Background Yersinia enterocolitica is widespread within the humans, pigs and wild boars. The low isolation rate of Y. enterocolitica from food or environmental and clinical samples may be caused by limited sensitivity of culture methods. The main goal of present study was identification of presumptive Y. enterocolitica isolates using MALDI TOF MS. The identification of isolates may be difficult due to variability of bacterial strains in terms of biochemical characteristics. This work emphasizes the necessity of use of multiple methods for zoonotic Y. enterocolitica identification. Results Identification of Y. enterocolitica isolates was based on MALDI TOF MS, and verified by VITEK® 2 Compact and PCR. There were no discrepancies in identification of all human’ and pig’ isolates using MALDI TOF MS and VITEK® 2 Compact. However three isolates from wild boars were not decisively confirmed as Y. enterocolitica. MALDI TOF MS has identified the wild boar’ isolates designated as 3dz, 4dz, 8dz as Y. enterocolitica with a high score of matching with the reference spectra of MALDI Biotyper. In turn, VITEK® 2 Compact identified 3dz and 8dz as Y. kristensenii, and isolate 4dz as Y. enterocolitica. The PCR for Y. enterocolitica 16S rDNA for these three isolates was negative, but the 16S rDNA sequence analysis identified these isolates as Y. kristensenii (3dz, 4dz) and Y. pekkanenii (8dz). The wild boar’ isolates 3dz, 4dz and 8dz could not be classified using biotyping. The main bioserotype present within pigs and human faeces was 4/O:3. It has been shown that Y. enterocolitica 1B/O:8 can be isolated from human faeces using ITC/CIN culturing. Conclusion The results of our study indicate wild boars as a reservoir of new and atypical strains of Yersinia, for which protein and biochemical profiles are not included in the MALDI Biotyper or VITEK® 2 Compact databases. Pigs in the south-west Poland are the reservoir for pathogenic Y. enterocolitica strains. Four biochemical features included in VITEK® 2 Compact known to be common with Wauters scheme were shown to produce incompatible results, thus VITEK® 2 Compact cannot be applied in biotyping of Y. enterocolitica

Phosphine derivatives of sparfloxacin : synthesis, structures and in vitro activity

We synthesized two derivatives of sparfloxacin (HSf): aminomethyl(diphenyl)phosphine (PSf) and its oxide (OPSf). The compounds were characterized by NMR spectroscopy, MS and elemental analysis. In addition, the molecular structures of the compounds were determined using DFT and X-ray (OPSf) analysis. The antibacterial activity of HSf and both derivatives was tested against four reference and fifteen clinical Gram-positive and Gram-negative strains of bacteria (sensitive or resistant to fluoroquinolones). The results showed that the activity of PSf was similar to or higher than the activity of HSf, while OPSf was found significantly less active. The compounds were also tested in vitro toward the following cancer cell lines: mouse colon carcinoma (CT26) and human lung adenocarcinoma (A549). Regardless of the cancer cell line, derivatization of HSf resulted in the gradual increase of cytotoxicity. OPSf exhibited the highest one (4 h – incubation time: IC50(CT26) = 51.0 ± 1.2; IC50(A549) = 74.9 ± 1.4 and 24 h: IC50(CT26) = 109.2 ± 8.8; IC50(A549) = 52.7 ± 9.2)

Antibactericidal Ir(III) and Ru(II) Complexes with Phosphine-Alkaloid Conjugate and Their Interactions with Biomolecules: A Case of N-Methylphenethylamine

The phosphine ligand (Ph2PCH2N(CH3)(CH2)2Ph, PNMPEA) obtained by the reaction of the (hydroxymethyl)diphenylphosphine with naturally occurring alkaloid N-methylphenethylamine, was used to synthesize the half-sandwich iridium(III) (Ir(η5-Cp*)Cl2Ph2PCH2N(CH3)(CH2)2Ph, IrPNMPEA) and ruthenium(II) (Ru(η6-p-cymene)Cl2Ph2PCH2N(CH3)(CH2)2Ph, RuPNMPEA) complexes. They were characterized using a vast array of methods, including 1D and 2D NMR, ESI(+)MS spectrometry, elemental analysis, cyclic voltammetry (CV), electron spectroscopy in the UV-Vis range (absorption, fluorescence) and density functional theory (DFT). The initial antimicrobial activity in vitro toward Gram-positive and Gram-negative bacterial strains was examined, indicating that both complexes are selective towards Gram-positive bacteria, e. g., Staphylococcus aureus, where the IrPNMPEA has been more bactericidal compared to RuPNMPEA. Additionally, the interactions of these compounds with various biomolecules, such as DNA (ctDNA, plasmid DNA, 9-ethylguanine (9-EtG), and 9-methyladenine (9-MeA)), nicotinamide adenine dinucleotide (NADH), glutathione (GSH), and ascorbic acid (Asc) were described. The results showed that both Ir(III) and Ru(II) complexes accelerate the oxidation process of NADH, GSH and Asc that appeared to occur by an electron transfer mechanism. Interestingly, only IrPNMPEA leads to the formation of various biomolecule adducts, which can explain its higher activity. Furthermore, RuPNMPEA and IrPNMPEA have been interacting with the DNA through weak noncovalent interaction

Regulatory Protein OmpR Influences the Serum Resistance of <i>Yersinia enterocolitica</i> O:9 by Modifying the Structure of the Outer Membrane

<div><p>The EnvZ/OmpR two-component system constitutes a regulatory pathway involved in bacterial adaptive responses to environmental cues. Our previous findings indicated that the OmpR regulator in <i>Yersinia enterocolitica</i> O:9 positively regulates the expression of FlhDC, the master flagellar activator, which influences adhesion/invasion properties and biofilm formation. Here we show that a strain lacking OmpR grown at 37°C exhibits extremely high resistance to the bactericidal activity of normal human serum (NHS) compared with the wild-type strain. Analysis of OMP expression in the <i>ompR</i> mutant revealed that OmpR reciprocally regulates Ail and OmpX, two homologous OMPs of <i>Y. enterocolitica</i>, without causing significant changes in the level of YadA, the major serum resistance factor. Analysis of mutants in individual genes belonging to the OmpR regulon (<i>ail</i>, <i>ompX</i>, <i>ompC</i> and <i>flhDC</i>) and strains lacking plasmid pYV, expressing YadA, demonstrated the contribution of the respective proteins to serum resistance. We show that Ail and OmpC act in an opposite way to the OmpX protein to confer serum resistance to the wild-type strain, but are not responsible for the high resistance of the <i>ompR</i> mutant. The serum resistance phenotype of <i>ompR</i> seems to be multifactorial and mainly attributable to alterations that potentiate the function of YadA. Our results indicate that a decreased level of FlhDC in the <i>ompR</i> mutant cells is partly responsible for the serum resistance and this effect can be suppressed by overexpression of <i>flhDC</i> in <i>trans</i>. The observation that the loss of FlhDC enhances the survival of wild-type cells in NHS supports the involvement of FlhDC regulator in this phenotype. In addition, the <i>ompR</i> mutant exhibited a lower level of LPS, but this was not correlated with changes in the level of FlhDC. We propose that OmpR might alter the susceptibility of <i>Y. enterocolitica</i> O:9 to complement-mediated killing through remodeling of the outer membrane.</p></div

Phosphine derivatives of ciprofloxacin and norfloxacin, a new class of potential therapeutic agents

In this paper a new series of chalcogenides of diphenylmethylaminophosphine derived from ciprofloxacin (PPh 2 CH 2 Cp) and a new phosphine derived from norfloxacin (PPh 2 CH 2 Nr) are presented. The synthesized compounds were characterized by N MR, MS and X-ray techniques. Both phosphines exhibit antibacterial activity against: S. aureus , E. coli , K. pneumoniae and P. aeruginosa , similar to ciprofloxacin and norfloxacin. They inhibit the growth of microorganisms in relatively low concentrations. Chalcogenides are slightly less active than phosphines and unmodified a ntibiotics. All the derivatives were also tested in vitro as anticancer agents towards mouse colon carcinoma (CT26) and human lung adenocarcinoma (A549). Cytotoxicity studies revealed that phosphines and their chalcogenides are able to inhibit the proliferation of the cells at relatively low concentrations. Moreover, all the tested compounds are more active against tested cell lines than cisplatin – the main representative of antitumor drugs

The susceptibility of <i>Y. enterocolitica</i> O:9 strains lacking YadA protein to normal human serum.

<p>Strains Ye9 (wt) and AR4 (<i>ompR</i>) harboring the virulence plasmid pYV (<i>yadA</i>⁺) and their plasmid-cured derivatives (Ye9c and AR4c, respectively) were grown to exponential phase at 37°C and incubated with 50% NHS at 37°C. Aliquots removed at 0, 15, 30 and 60 min were plated for viability counts (CFU). The numbers of CFU/ml were normalized to the T₀ values as a percentage and then converted to log₁₀ to show the survival rate of the bacteria in NHS. Data are the means ±SD from at least three independent experiments. The Ye9c, pYV⁻ and the AR4c, pYV⁻ asterisks indicate statistically significant difference in comparison with the strains carrying pYV (Ye9 and AR4) (* P<0.001, ** P<0.0001, by one way ANOVA with Tukey's comparison post-test), n.s. not significant.</p

Effect of the OmpR protein on <i>ail</i> promoter activity in <i>Y. enterocolitica</i> O:9 strains.

<p>Strains Ye12 (OmpR⁺) and AR7 (OmpR⁻), representing the respective derivatives of Ye9 and AR4, harboring a transcriptional reporter <i>ail-lacZ</i> fusion, and strain AR7 with plasmid pBR3 expressing OmpR, were grown at 25 or 37°C in LB medium. The activity of β-galactosidase was assayed to evaluate <i>ail</i> expression. Values represent mean β-galactosidase activities, expressed in Miller units ± SD, from three independent experiments. Different letters (a, b) above the columns indicate statistically significant differences (P<0.005, covariance with Tukey correction).</p

The susceptibility of <i>Y. enterocolitica</i> O:9 strains with and without protein Ail to normal human serum.

<p>Strains Ye9 (wt), Ye12 (Ye9 Ail⁻), AR4 (OmpR⁻) and AR7 (AR4 Ail⁻) were grown to exponential phase at 37°C and incubated with 50% NHS at 37°C. Aliquots removed at 0, 15, 30 and 60 min were plated for viability counts (CFU). The numbers of CFU/ml were normalized to the T₀ values as a percentage and then converted to log₁₀ to show the survival rate of the bacteria in NHS. Data are the means ±SD from three independent experiments. The asterisks indicate statistically significant differences between Ye9 and AR4 and their isogenic <i>ail</i> mutant Ye12 (Ail⁻) and AR7 (Ail⁻) (* P<0.001, ** P<0.0001, by one way ANOVA with Tukey's comparison post-test).</p

The susceptibility of <i>Y. enterocolitica</i> O:9 strains with and without protein OmpC to normal human serum.

<p>Strains Ye9 (wt), OP3 (Ye9 OmpC⁻) and OP3/pBBRC4 (<i>ompC</i> mutant OP3 with plasmid pBBRC4 OmpC⁺) were grown to exponential phase at 37°C and incubated with 50% NHS at 37°C. Aliquots removed at 0, 15, 30 and 60 min were plated for viability counts (CFU). The numbers of CFU/ml were normalized to the T₀ values as a percentage and then converted to log₁₀ to show the survival rate of the bacteria in NHS. Data are the means ±SD from three independent experiments, except for strain OP3 with plasmid pBBRC4 (performed only once). The asterisks indicate statistically significant differences between Ye9 and its <i>ompC</i> mutant OP3 (* P<0.001, ** P<0.0001, by one way ANOVA with Tukey's comparison post-test).</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p