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ABSTRACTIn this study, we focused on evaluating the occurrence of extended-spectrum β-lactamase (ESBL)-producingEscherichia coliin fecal samples of healthy ducks and environmental samples from a duck farm in South China. Duck cloacal swabs and pond water samples were cultivated on MacConkey agar plates supplemented with ceftiofur. Individual colonies were examined for ESBL production. Bacteria identified asE. coliwere screened for the presence of ESBL and plasmid-borne AmpC genes. The genetic relatedness, plasmid replicon type, and genetic background were determined. Of 245 samples analyzed, 123 hadE. coliisolates with ceftiofur MICs higher than 8 μg/ml (116 [50.4%] from 230 duck samples and 7 [46.7%] from 15 water samples).blaCTX-M,blaSHV-12,blaCMY-2, andblaDHA-1were identified in 108, 5, 9, and 1 isolates, respectively. The most commonblaCTX-Mgenes wereblaCTX-M-27(n= 34),blaCTX-M-55(n= 27),blaCTX-M-24e(n= 22), andblaCTX-M-105(n= 20), followed byblaCTX-M-14a,blaCTX-M-14b,blaCTX-M-24a, andblaCTX-M-24b. Although most of the CTX-M producers had distinct pulsotypes, clonal transmission between duck and water isolates was observed.blaCTX-Mgenes were carried by transferable IncN, IncF, and untypeable plasmids. The novel CTX-M geneblaCTX-M-105was flanked by two hypothetical protein sequences, partial ISEcp1upstream and truncated IS903D,iroN,orf1, and a Tn1721-like element downstream. It is suggested that the horizontal transfer ofblaCTX-Mgenes mediated by mobile elements and the clonal spread of CTX-M-producingE. coliisolates contributed to the dissemination ofblaCTX-Min the duck farm. Our findings highlight the importance of ducks for the dissemination of transferable antibiotic resistance genes into the environment.

This study investigated the prevalence of 16S rRNA methylase genes in 267 Enterobacteriaceae isolates collected from pets. The rmtB gene was detected in 69 isolates, most of which were clonally unrelated. The coexistence of the rmtB gene with the bla CTX-M-9 group genes and/or qepA within the same IncFII replicons was commonly detected. The two dominant types of IncF plasmids, F2:A؊:B؊, carrying rmtB-qepA, and F33:A؊: B؊, carrying the rmtB-bla CTX-M-9 group genes (and especially bla CTX-M-65 ), shared restriction patterns within each incompatibility group.Recently, the production of 16S rRNA methylases by Gramnegative bacilli has emerged as a novel mechanism for their high-level resistance to aminoglycosides (9, 31). Seven plasmid-mediated 16S rRNA methylase genes, rmtA, rmtB, rmtC, rmtD, rmtE, armA, and npmA, have been identified so far in multiple species of Enterobacteriaceae (3,6,9,13,31). These resistance determinants are globally disseminated, with rmtB and armA being the most frequently reported types worldwide (3,5,9,10,11,13,19,27,29,32). However, even though aminoglycosides are widely used in pets to treat Gram-negative bacterial infections, the prevalence of 16S rRNA methylases in bacteria from pets is not known. In addition, the 16S rRNA methylase genes, especially rmtB, are commonly associated with bla CTX-M genes (1-3, 8, 13, 19, 27, 29, 32). In our recent study on the distribution of extended-spectrum ␤-lactamase (ESBL) genes in Escherichia coli isolates obtained from pets, some CTX-M-producing isolates showed significantly reduced susceptibilities to amikacin, and this resistance to amikacin was cotransferred with CTX-M-9 subgroup genes to recipients (25). The current study investigated the prevalence of 16S rRNA methylase genes among CTX-M-producing isolates and characterized the plasmids carrying rmtB.The present study included 135 CTX-M-producing Enterobacteriaceae isolates (119 E. coli, 11 Klebsiella pneumoniae, 3 Enterobacter cloacae, and 2 Citrobacter freundii isolates) recovered from healthy or diseased pets (dogs and cats) in Guangdong, China, during 2006 and 2008. The ESBL genes in most of these strains have been characterized previously (18, 25).The gene type of bla CTX-M was confirmed by PCR and DNA sequencing (25). In addition, this study also included 132 Enterobacteriaceae isolates previously confirmed to be CTX-M negative that were collected from pets in Guangdong province of China during 2006 and 2008 (18, 25). The presence of 16S rRNA methylase genes was identified by PCR using previously designed primers (5, 6, 9). Of the 135 CTX-M producers, 60 (ϳ44%) were positive for rmtB and 5 (ϳ4%) were positive for armA (Table 1). No isolate was positive for the rmtA, rmtC, rmtD, rmtE, or npmA genes. The rmtB gene was also detected in 9 (ϳ7%) of the 132 CTX-M-negative isolates. Therefore, of the 69 rmtB-positive isolates, 60 were CTX-M producers, and most of the enzymes produced belonged to the CTX-M-9 group. Since the plasmid-mediated fluoroquinolone efflux pump gene qepA is frequently as...

MicroRNAs (miRNAs) are a class of noncoding RNAs involved in posttranscriptional regulation of gene expression and many critical roles in numerous biological processes. Porcine epidemic diarrhea virus (PEDV), the etiological agent of porcine epidemic diarrhea, causes substantial economic loss in the swine industry worldwide. Previous studies reported miRNA involvement in viral infection; however, their role in regulating PEDV infection remains unknown. In this study, we investigated the regulatory relationship between miRNA-221-5p and PEDV infection, finding that miR-221-5p overexpression inhibited PEDV replication in a dose-dependent manner, and that silencing endogenous miR-221-5p enhanced viral replication. Our results showed that miR-221-5p directly targets the 3′ untranslated region (UTR) of PEDV genomic RNA to inhibit PEDV replication, and that miR-221-5p overexpression activates nuclear factor (NF)-κB signaling via p65 nuclear translocation, thereby upregulating interferon (IFN)-β, IFN-stimulated gene 15, and MX1 expression during CH/HBTS/2017 infection. We subsequently identified NF-κB-inhibitor α and suppressor of cytokine signaling 1, negative regulators of the NF-κB pathway, as miR-221-5p targets. These results demonstrated the ability of miR-221-5p to inhibit PEDV replication by targeting the 3’ UTR of the viral genome and activating the NF-κB-signaling pathway. Our findings will aid the development of preventive and therapeutic strategies for PEDV infection.

Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs) for defense against numerous viral infections, including classical swine fever virus (CSFV). However, the mechanisms underlying the effect of ISGs on CSFV infection are rarely reported. In this study, we demonstrate that IFN-α treatment induces upregulation of ISG15 and thus attenuates CSFV replication. To determine whether ISG15 is critical for controlling CSFV replication, we established porcine alveolar macrophages (PAMs) with stable overexpression or knockdown of ISG15. Overexpression of Flag-ISG15 significantly prevented CSFV replication, whereas loss of ISG15 led to abnormal proliferation of CSFV. Furthermore, upregulated ISG15 promoted beclin-1 (BECN1) ISGylation and dysfunction and subsequently inhibited autophagy, which is indispensable for CSFV replication. In addition, HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), which functions to catalyze conjugation of ISG15 protein, was confirmed to interact with BECN1. Collectively, these results indicate that IFN-α restricts CSFV replication through ISG15-mediated BECN1 ISGylation and autophagy inhibition, providing insight into the mechanism of CSFV replication control by type I IFN. This mechanism may not be the only antiviral mechanism of ISG15; nonetheless, this study may contribute to the development of CSFV treatment and prevention strategies.