Target genes for virulence assessment of Escherichia coli isolates from water, food and the environment

The widespread species Escherichia coli includes a broad variety of different types, ranging from highly pathogenic strains causing worldwide outbreaks of severe disease to avirulent isolates which are part of the normal intestinal flora or which are well characterized and safe laboratory strains. The pathogenicity of a given E. coli strain is mainly determined by specific virulence factors which include adhesins, invasins, toxins and capsule. They are often organized in large genetic blocks either on the chromosome (‘pathogenicity islands'), on large plasmids or on phages and can be transmitted horizontally between strains. In this review we summarize the current knowledge of the virulence attributes which determine the pathogenic potential of E. coli strains and the methodology available to assess the virulence of E. coli isolates. We also focus on a recently developed procedure based on a broad-range detection system for E. coli-specific virulence genes that makes it possible to determine the potential pathogenicity and its nature in E. coli strains from various sources. This makes it possible to determine the pathotype of E. coli strains in medical diagnostics, to assess the virulence and health risks of E. coli contaminating water, food and the environment and to study potential reservoirs of virulence genes which might contribute to the emergence of new forms of pathogenic E. col

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-α-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H2O2 into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly 12−Gly13−Gly 14−Ile15−Ile16−Gly 17. Recombinant GlpO lacking these six amino acids (GlpOΔFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpOΔFAD, similarly to anti-GlpO antibodies, neutralised H2O2 production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP

Identification of a locus involved in the utilization of iron by Actinobacillus pleuropneumoniae

The cloned afu locus of Actinobacillus pleuropneumoniae restored the ability of an Escherichia coli K-12 mutant (aroB) to grow on iron-limited media. DNA sequence analysis of the fragment showed that there are three genes designated afuA, afuB and afuC (Actinobacillus ferric uptake) that encode products similar to the SfuABC proteins of Serratia marcescens, the HitABC proteins of Haemophilia influenzae, the FbpABC proteins of Neisseria gonorrhoeae and the YfuABC proteins of Yersinia enterocolitica. The three genes encode a periplasmic iron-binding protein (AfuA), a highly hydrophobic integral cytoplasmic membrane protein with two consensus permease motifs (AfuB) and one hydrophilic peripheral cytoplasmic membrane protein with Walker ATP-binding motifs (AfuC), respectively. This system has been shown to constitute a periplasmic binding protein-dependent iron transport system in these organisms. The afuABC operon is locating approximately 200 bp upstream of apxIC gene, but transcribed in opposite direction to the ApxI-toxin gene

Conservation of threatened relict trees through living ex situ collections: lessons from the global survey of the genus Zelkova (Ulmaceae)

Maintaining living ex situ collections is one of the key conservation methods in botanic gardens worldwide. Despite of the existence of many other conservation approaches used nowadays, it offers for many endangered plants an important insurance policy for the future, especially for rare and threatened relict trees. The aim of this research was to investigate the global extent of living ex situ collections, to assess and discuss their viability and inform the development of conservation approaches that respond to latest global conservation challenges. We used as a model taxon the tree genus Zelkova (Ulmaceae). The genus includes six prominent Tertiary relict trees which survived the last glaciation in disjunct and isolated refugial regions. Our comprehensive worldwide survey shows that the majority of botanic institutions with Zelkova collections are in countries with a strong horticultural tradition and not in locations of their origin. More importantly, the acutely threatened Zelkova species are not the most represented in collections, and thus safeguarded through ex situ conservation. Less than 20% of the ex situ collections surveyed contain plant material of known wild provenance while the majority (90%) of collections are generally very small (1-10 trees). Botanic gardens and arboreta particularly in regions where iconic relict trees naturally occur should play a vital role in the conservation of these species. The coordination of conservation efforts between gardens has to be enhanced to prioritise action for the most threatened relict trees. Large scale genetic studies should be undertaken, ideally at genus level, in order to verify or clarify the provenance of ex situ collections of relict trees in cultivation. For the most threatened relict tree genera, well-coordinated specialist groups should be create

Complete genome sequences of virulent mycoplasma capricolum subsp. capripneumoniae strains F38 and ILRI181

Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subsp. capripneumoniae is a severe epidemic affecting mainly domestic Caprinae species but also affects wild Caprinae species. M. capricolum subsp. capripneumoniae belongs to the “Mycoplasma mycoides cluster.” The disease features prominently in East Africa, in particular Kenya, Tanzania, and Ethiopia. CCPP also endangers wildlife and thus affects not only basic nutritional resources of large populations but also expensively built-up game resorts in affected countries. Here, we report the complete sequences of two M. capricolum subsp. capripneumoniae strains: the type strain F38 and strain ILRI181 isolated druing a recent outbreak in Kenya. Both genomes have a G+C content of 24% with sizes of 1,016,760 bp and 1,017,183 bp for strains F38 and ILRI181, respectively

Glucocorticoid sensitivity of circulating monocytes in essential hypertension

Background: Essential hypertension ranks among the strongest cardiovascular risk factors. Cytokine production by monocytes plays a key role in atherosclerosis development and acute coronary syndromes. We investigated whether stimulated monocyte cytokine release and its inhibition by glucocorticoids would differ between hypertensive and normotensive subjects. Methods: Study participants were 222 middle-aged male employees with industrial jobs. Following the criteria of the World Health Organization/International Society for Hypertension, 76 subjects were classified as being hypertensive (systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg). In vitro monocyte tumor necrosis factor (TNF)-α release after lipopolysaccharide (LPS) stimulation was assessed with and without coincubation with incremental doses of dexamethasone. Monocyte glucocorticoid sensitivity was defined as the dexamethasone concentration inhibiting TNF-α release by 50%. Results: Hypertensive subjects showed 11% higher LPS-stimulated TNF-α release than normotensive subjects (F1,181= 5.21, P = .024). In hypertensive subjects, monocyte glucocorticoid sensitivity was 21% lower than in normotensive subjects (F1,178= 4.94, P = .027), indicating that dexamethasone inhibited relatively less TNF-α release in hypertensive subjects. Results held significance when a set of classic cardiovascular risk factors was controlled for. Conclusion: The findings suggest that proinflammatory activity of circulating monocytes is higher in hypertensive than in normotensive men, providing one potential pathway to explain the increased atherosclerotic risk with essential hypertension. Am J Hypertens 2004;17:489-494 © 2004 American Journal of Hypertension, Lt

Quinolone Resistance in Gallibacterium anatis Determined by Mutations in Quinolone Resistance-Determining Region.

Control of the important pathogen, Gallibacterium anatis, which causes salpingitis and peritonitis in poultry, relies on treatment using antimicrobial compounds. Among these, quinolones and fluoroquinolones have been used extensively, leading to a rise in the prevalence of resistant strains. The molecular mechanisms leading to quinolone resistance, however, have not previously been described for G. anatis, which is the aim of this study. The present study combines phenotypic antimicrobial resistance data with genomic sequence data from a collection of G. anatis strains isolated from avian hosts between 1979 and 2020. Minimum inhibitory concentrations were determined for nalidixic acid, as well as for enrofloxacin for each included strain. In silico analyses included genome-wide queries for genes known to convey resistance towards quinolones, identification of variable positions in the primary structure of quinolone protein targets and structural prediction models. No resistance genes known to confer resistance to quinolones were identified. Yet, a total of nine positions in the quinolone target protein subunits (GyrA, GyrB, ParC and ParE) displayed substantial variation and were further analyzed. By combining variation patterns with observed resistance patterns, positions 83 and 87 in GyrA, as well as position 88 in ParC, appeared to be linked to increased resistance towards both quinolones included. As no notable differences in tertiary structure were observed between subunits of resistant and sensitive strains, the mechanism behind the observed resistance is likely due to subtle shifts in amino acid side chain properties

β-D-Glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H(2)O(2 )production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-β-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val(204), from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala(204). RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val(204), but not strains with the Bgl isoform Ala(204), do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., β-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val(204 )show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-β-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala(204). Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H(2)O(2 )production. Rather, the viability during addition of β-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val(204 )than for those with the isoform Ala(204). CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val(204 )with low hydrolysing activity are more prone to survive in environments that contain high levels of β-D-glucosides, thus contributing in some extent to mycoplasmaemia