Detection of carbapenemase- and ESBL-producing Klebsiella pneumoniae from bovine bulk milk and comparison with clinical human isolates in Italy.

Klebsiella pneumoniae is the most common Klebsiella species infecting animals and is one of the causing agents of mastitis in cows. The rise of antimicrobial resistance in K. pneumoniae, particularly in strains producing extended-spectrum ß-lactamases (ESBLs) and/or carbapenemases, is of concern worldwide. Recently (Regulation UE No 2022/1255), carbapenems and cephalosporins in combination with ß-lactamase inhibitors have been reserved only to human treatments in the European Union. The aim of this study was to investigate the role of cattle as carrier of human pathogenic carbapenem-resistant (CR) and ESBL-producing K. pneumoniae. On this purpose, a study involving 150 dairy farms in Parma province (Northern Italy) and 14 non replicate K. pneumoniae isolates from patients admitted at Parma University-Hospital was planned. Four multidrug resistant (MDR) K. pneumoniae strains were detected from 258 milk filters collected between 2019 and 2021. One carbapenemase KPC-3-positive K. pneumoniae ST307 (0.4 %; 95 % CI - 0.07 - 2.2) was detected in milk filters. The isolate also harboured OXA-9, CTX-M-15 and SHV-106 determinants, together with genes conferring resistance to aminoglycosides (aac(3')-IIa, aph (3″)-Ib, aph (6)-Id), fluoroquinolones (oqxA, oqxB, qnrB1), phosphonic acids (fosA6), sulphonamides (sul2), tetracyclines (tet(A)6) and trimethoprim (dfrA14). One KPC-3-producing K. pneumoniae ST307 was identified also among the human isolates, thus suggesting a possible circulation of pathogens out of the clinical settings. The remaining three bovine isolates were MDR ESBL-producing K. pneumoniae characterized by different genomic profiles: CTX-M-15, TEM-1B and SHV-187 genes (ST513); CTX-M-15 and SHV-145 (ST307); SHV-187 and DHA-1 (ST307). Occurrence of ESBL-producing K. pneumoniae in milk filters was 1.2 % (95 % CI 0.4-3.4). All the isolates showed resistance to aminoglycosides, 3rd-generation cephalosporins, and fluoroquinolones. Among the human isolates, two multidrug resistant ESBL-producing K. pneumoniae ST307 were found, thus confirming the circulation of this high-risk lineage between humans and cattle. Our findings suggest that food-producing animals can carry human pathogenic microorganisms harboring resistance genes against carbapenems and 3rd-generation cephalosporins, even if not treated with such antimicrobials. Moreover, on the MDR K. pneumoniae farms, the antimicrobial use was much higher than the Italian median value, thus highlighting the importance of a more prudent use of antibiotics in animal productions.

Pathophysiological Role of Genetic Factors Associated With Gestational Diabetes Mellitus.

Gestational Diabetes Mellitus (GDM) is a highly prevalent maternal pathology characterized by maternal glucose intolerance during pregnancy that is, associated with severe complications for both mother and offspring. Several risk factors have been related to GDM; one of the most important among them is genetic predisposition. Numerous single nucleotide polymorphisms (SNPs) in genes that act at different levels on various tissues, could cause changes in the expression levels and activity of proteins, which result in glucose and insulin metabolism dysfunction. In this review, we describe various SNPs; which according to literature, increase the risk of developing GDM. These SNPs include: (1) those associated with transcription factors that regulate insulin production and excretion, such as rs7903146 (TCF7L2) and rs5015480 (HHEX); (2) others that cause a decrease in protective hormones against insulin resistance such as rs2241766 (ADIPOQ) and rs6257 (SHBG); (3) SNPs that cause modifications in membrane proteins, generating dysfunction in insulin signaling or cell transport in the case of rs5443 (GNB3) and rs2237892 (KCNQ1); (4) those associated with enzymes such as rs225014 (DIO2) and rs9939609 (FTO) which cause an impaired metabolism, resulting in an insulin resistance state; and (5) other polymorphisms, those are associated with growth factors such as rs2146323 (VEGFA) and rs755622 (MIF) which could cause changes in the expression levels of these proteins, producing endothelial dysfunction and an increase of pro-inflammatory cytokines, characteristic on GDM. While the pathophysiological mechanism is unclear, this review describes various potential effects of these polymorphisms on the predisposition to develop GDM.

Lemon grass (Cymbopogon citratus (D.C) Stapf) polyphenols protect human umbilical vein endothelial cell (HUVECs) from oxidative damage induced by high glucose, hydrogen peroxide and oxidised low-density lipoprotein.

The aromatic herb Cymbopogon citratus Stapf is widely used in tropical and subtropical countries in cooking, as a herbal tea, and in traditional medicine for hypertension and diabetes. Some of its properties have been associated with the in vitro antioxidant effect of polyphenols isolated from their aerial parts. However, little is known about C. citratus effects on endothelial cells oxidative injury. Using chromatographic procedures, a polyphenol-rich fraction was obtained from C. citratus (CCF) and their antioxidant properties were assessed by cooper-induced LDL oxidation assay. The main constituents of the active CCF, identified by high-performance liquid chromatography with diode-array detection and mass spectrometry (HPLC-DAD-MS), were chlorogenic acid, isoorientin and swertiajaponin. CCF 10 and 100 µg/ml diminishes reactive oxidative species (ROS) production in human umbilical vein endothelial cell (HUVECs), challenged with high D-glucose (60% inhibition), hydrogen peroxide (80% inhibition) or oxidised low-density lipoprotein (55% inhibition). CCF 10 or 100 µg/ml did not change nitric oxide (NO) production. However, CCF was able to inhibit vasoconstriction induced by the thromboxane A2 receptor agonist U46619, which suggest a NO-independent vasodilatador effect on blood vessels. Our results suggest that lemon grass antioxidant properties might prevent endothelial dysfunction associated to an oxidative imbalance promoted by different oxidative stimuli.

Differential expression of functional nucleoside transporters in non-differentiated and differentiated human endothelial progenitor cells.

Extracellular adenosine removal is via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) in the endothelium, thus regulating adenosine-induced revascularization and angiogenesis. Since human endothelial progenitor cells (hEPCs) promote revascularization, we hypothesize differential expression of nucleoside transporters in hEPCs. hEPCs were cultured 3 (hEPC-3d) or 14 (hEPC-14d) days. RT-PCR for prominin 1, CD34, octamer-4, kinase insert domain receptor, oxidized low-density lipoprotein (lectin-like) receptor 1 and tyrosine endothelial kinase was used to evaluate phenotypic differentiation. Flow cytometry was used to estimate CD34(+)/KDR(-) (non-differentiated), CD34(-)/KDR(+) (differentiated) or CD34(+)/KDR(+) (mixed) cell populations. Adenosine transport was measured in absence or presence of sodium, S-(4-nitrobenzyl)-6-thio-inosine (NBTI, 1-10 µM), inosine, hypoxanthine or guanine (0.1-5 mM), hENTs protein abundance by western blot, and hENTs, hCNT1, hCNT2 and hCNT3 mRNA expression by real time RT-PCR. hEPC-3d cells were CD34(+)/KDR(-) compared with hEPC-14d cells that were CD34(-)/KDR(+). hEPC-3d cells exhibit hENT1-like adenosine transport (NBTI-sensitive, Na(+)-independent), which is absent in hEPC-14d cells. hEPC-14d cells exhibit two transport components: component 1 (NBTI insensitive, Na(+)-independent) and component 2 (NBTI insensitive, Na(+)-dependent, Hill coefficient ∼1.8), the latter resembling CNT3-like transport. hEPC-3d cells express hENT1 protein and mRNA, which is reduced (∼90%) in hEPC-14d cells, but instead only hCNT3 mRNA is expressed in this cell type. hENT2, hCNT1 and hCNT2 were undetectable in hEPCs. Thus, hEPCs exhibit a differential expression of hENT1 and hCNT3 functional nucleoside transporters, which could be related with its differentiation stage.