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1. Piglets harbor carbapenem resistant E. coli and have great public health significance. 2. Apart from carbapenemase, efflux pump is also important for carbapenem resistance. 3. This is the first report of blaNDM in the piglets from India.

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Prostaglandins (PG) are involved in several female reproductive processes, and their action is regulated at the levels of biosynthesis, catabolism, and signal transduction. Facilitated transport across cell membranes emerges as an additional checkpoint regulating PG action. We have already reported on the influx transporter solute carrier organic anion transporting polypeptide (SLCO2A1) [PG transporter (PGT)] in relation to PG action in the bovine endometrium. In the present study, we report on the functional expression and regulation of multidrug resistance-associated protein 4 (MRP4)/ATP-binding cassette carrier 4, an alternate PG transporter belonging to the ATP-binding cassette carrier (ABC) family. We have found that MRP4 protein was present throughout the estrous cycle and exhibited a pattern of expression similar to that of PGT with maximal expression during early-mid luteal phase in the bovine endometrium. Functional expression and regulation of MRP4 was studied in vitro using the newly developed bovine endometrial epithelial bEEL and stromal CSC cell lines. Oxytocin (OT) stimulated PGF2α production and MRP4 mRNA and protein in a time- and dose-dependent manner but had no effect on PGT. OT induced preferred accumulation of PG outside the cells and secretion toward the basolateral side of polarized bEEL cells grown on membrane inserts. MK-571 and indomethacin, two documented inhibitors of MRP4 activity, blocked preferred accumulation of PG, but interferon-τ and NS-398 had no effect on MRP4 expression or the direction of PG transport. Our results suggest that MRP4 is a functional PG carrier under the regulation of OT in the bovine endometrium.

Interferon-tau (IFNtau) is the embryonic signal responsible for pregnancy recognition in ruminants. The primary action of IFNtau is believed to be mediated through inhibition of prostaglandin F(2alpha) (PGF(2alpha)) released from the endometrial epithelial cells in response to oxytocin (OT). Our working hypothesis was that the antiluteolytic effect of IFNtau also involved modulation of PG production downstream of OT receptor (OTR) and/or cyclooxygenase 2 (COX2). There is currently no OT-sensitive endometrial cell line to study the molecular mechanisms underlying our hypotheses. Therefore, we established an immortalized bovine endometrial epithelial cell line (bEEL) exhibiting OT response. These cells were cytokeratin positive, expressed steroid receptors, and exhibited preferential accumulation of PGF(2alpha) over PGE(2). The bEEL cells were highly sensitive to OT, showing time- and concentration-dependent increase in COX2 transcript and protein and PGF(2alpha) accumulation. Interestingly, IFNtau (20 ng/ml) significantly reduced OT-induced PGF(2alpha) accumulation, but surprisingly, the effect was not mediated through down-regulation of either OTR or COX2. Rather, IFNtau up-regulated COX2 in a time- and concentration-dependent manner while decreasing OT-induced PG accumulation. This suggests that COX2 is not a primary target for the antiluteolytic effect of IFNtau. Because IFNtau reduced OT-stimulated PGF(2alpha) accumulation within 3 h, the mechanism likely involves a direct interference at the level of the OT signaling or transcription in addition to the down-regulation of OTR observed in vivo. In summary, bEEL cells offer a unique in vitro model for investigating the cellular and molecular mechanisms underlying OT and IFNtau response in relation with luteolysis and recognition of pregnancy in the bovine.

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