Characterization of Resistance Patterns and Detection of Apramycin Resistance Genes in Escherichia coli Isolated from Chicken Feces and Houseflies after Apramycin Administration.

The aim of this study was to evaluate the influence of apramycin administration on the development of antibiotic resistance in Escherichia coli (E. coli) strains isolated from chicken feces and houseflies under field conditions. Chickens in the medicated group (n = 25,000) were given successive prophylactic doses (0.5 mg/l) of apramycin in their drinking water from Days 1 to 5, while no antibiotics were added to the un-medicated groups drinking water (n = 25,000). Over 40 days, a total of 1170 E. coli strains were isolated from fecal samples obtained from medicated and un-medicated chickens and houseflies from the same chicken farm. Apramycin MIC90 values for E. coli strains obtained from the medicated group increased 32-128 times from Days 2 to 6 (256-1024 µg/ml) when compared to those on Day 0 (8 µg/ml). Strains isolated from un-medicated chickens and houseflies had consistently low MIC90 values (8-16 µg/ml) during the first week, but showed a dramatic increase from Days 8 to 10 (128-1024 µg/ml). The apramycin resistance gene aac(3)-IV was detected in E. coli strains from medicated (n = 71), un-medicated (n = 32), and housefly groups (n = 42). All strains positive for aac(3)-IV were classified into 12 pulsed-field gel electrophoresis (PFGE) types. PFGE types A, E, and G were the predominant types in both the medicated and housefly groups, suggesting houseflies play an important role in spreading E. coli-resistant strains. Taken together, our study revealed that apramycin administration could facilitate the occurrence of apramycin-resistant E. coli and the apramycin resistance gene acc(3)-IV. In turn, these strains could be transmitted by houseflies, thus increasing the potential risk of spreading multi-drug-resistant E. coli to the public.

C2-ceramide enhances sorafenib-induced caspase-dependent apoptosis via PI3K/AKT/mTOR and Erk signaling pathways in HCC cells.

Sorafenib as an effective multikinase inhibitor has been approved for the clinical treatment against advanced hepatocellular carcinoma (HCC). HCC treatment requires usually combined therapy because of its complex pathogenesis. Ceramide has been confirmed to induce remarkable apoptosis in human tumor cells and has attracted increasing attention in investigations on combination therapy. In this paper, the anti-HCC effect of sorafenib combined with C2-ceramide was investigated on cell vitality, apoptosis, and migration, and the underlying mechanism was examined using flow cytometry and western blot. Bel7402 cells coincubated with sorafenib and C2-ceramide exhibited lower cell vitality and more irregular cellular morphology and cell cycle arrest. Sorafenib plus C2-ceramide stimulated significantly the production of reactive oxygen species (ROS) and mitochondrial depolarization, which promoted caspases-dependent cell apoptosis as illustrated by related protein expression including caspase 3, caspase 9, Bax, Bcl-2, and cytochrome c. Combination treatment of sorafenib and C2-ceramide inhibited obviously cell growth and proliferation via PI3K/AKT/mTOR and Erk signaling pathways. Furthermore, the combination treatment was proved to inhibit cell migration and epithelial-mesenchymal transition (EMT). These findings indicated that the combination of C2-ceramide and sorafenib provided synergistic inhibitory effects on HCC cells.

Diosgenin induces ROS-dependent autophagy and cytotoxicity via mTOR signaling pathway in chronic myeloid leukemia cells.

BACKGROUND: Diosgenin, a steroidal saponin isolated from legumes and yams, has been confirmed to possess potent anticancer effect on multifarious tumors including chronic myeloid leukemia (CML). PURPOSE: We aimed to further determine the anti-cancer activity of diosgenin and its mechanisms in CML cells. METHODS: The cell vitality was detected by MTT assay. Autophagic flux and reactive oxygen species (ROS) production were analyzed by laser scanning confocal microscope. Apoptosis was observed by flow cytometry. All proteins expression was examined by western blotting. RESULTS: Autophagy induction was demonstrated by examination of autophagic flux including autophagosomes accumulation, autophagosome-lysosome fusion and degradation of autophagosomes. Moreover, blocking autophagy with inhibitor chloroquine (CQ) and 3-methyladenine (3-MA), enhanced diosgenin-induced apoptosis, indicating the protective effect of autophagy in diosgenin-treated CML cells. Further study suggested that diosgenin-induced autophagy and cytotoxicity were accompanied by reactive oxygen species (ROS) generation and mammalian target of rapamycin (mTOR) signaling pathway inhibition. N-acetyl-L-cysteine (NAC) administration, a scavenger agent of ROS, could down-regulate diosgenin-induced autophagy via reversion of mTOR pathway inhibition. CONCLUSION: These results indicate that diosgenin obviously generates ROS and this oxidative pressure not only produces cytotoxic effect on CML cells but also induces autophagy. What's more, autophagy functions as a cytoprotective mechanism to overcome cytotoxicity of diosgenin in tumor cells and inhibition of autophagy can enhance the anti-CML activity of diosgenin.

Characteristics of Plasmids Coharboring 16S rRNA Methylases, CTX-M, and Virulence Factors in Escherichia coli and Klebsiella pneumoniae Isolates from Chickens in China.

The objective of this study was to characterize plasmids coharboring 16S rRNA methylases, blaCTX-M and virulence-associated genes in Escherichia coli and Klebsiella pneumoniae isolates from chickens in China. A total of 32 positive transconjugants exhibited coresistance to amikacin and cefotaxime in E. coli (24/281) and K. pneumoniae (8/93), and were identified by conjugation experiments and S1-pulsed-field gel electrophoresis. Polymerase chain reaction amplification assay detecting resistance genes showed that rmtB or armA gene accompanied with different blaCTX-M genes coexisted on 32 transferred plasmids. The blaCTX-M-98b gene was identified in chicken-derived E. coli and K. pneumoniae for the first time. The association between resistance genes and virulence genes was observed in the transferred plasmids; 68.8% (22/32) transferred resistance plasmids coharboring various virulence genes including traT, iutA, fyuA, msbB, and vagC genes with diverse proportions. Genetic stability tests revealed that 93.8% (30/32) transferred plasmids continued to exist in the host strain after continuous passage of 30 times in 15 days. Furthermore, 87.5% (28/32) conjugants showed no significant differences in growth rates compared with E. coli J53. Results of the growth competition assay showed that conjugants have low fitness cost, which indicated that there were no obvious negative effects on the host's growth. The combination of blaCTX-M-98b-rmtB-traT on 85-kb transferred IncF plasmids in E. coli, and blaCTX-M-14-rmtB-traT on 95-kb transferred IncF plasmids in K. pneumoniae were first identified in this study. These features of plasmids may contribute to the successful spread of resistance and virulence among pathogens of different sources and geographical origins.

PREVALENCE OF SULFONAMIDE AND FLORFENICOL RESISTANCE GENES IN ESCHERICHIA COLI ISOLATED FROM YAKS (BOS GRUNNIENS) AND HERDSMEN IN THE TIBETAN PASTURE.

To determine the antimicrobial susceptibility profiles and prevalence of resistance genes in Escherichia coli isolated from yaks (Bos grunniens) and herdsmen in nine plateau pastures in Tibet, we isolated 184 nonidentical strains of E. coli from yaks and herdsmen. Antimicrobial susceptibility testing of 15 antimicrobials was conducted and the prevalence of sulfonamide resistance genes (sul1, sul2, and sul3) and florfenicol resistance genes (floR, cfr, cmlA, fexA, pexA, and estDL136) was determined. Escherichia coli isolated from yaks had a high resistance rate to sulfamethoxazole (44%), sulphafurazole (40.4%), and florfenicol (11.4%). Escherichia coli isolated from herdsmen had a high resistance rate to sulfamethoxazole (57%) and sulphafurazole (51%). In addition, sul genes were present in 93% of sulfonamide-resistant isolates (84/90), and 17 floR genes and four cmlA genes were found in 19 florfenicol-resistant isolates. Even though florfenicol is prohibited from use in humans, three floR genes were detected in strains isolated from herdsmen. The three floR-positive isolates from herdsmen had pulsed-field gel electrophoresis patterns similar to isolates from yaks. In addition to documenting the sul and floR genes in E. coli isolated from yaks and herdsmen in the Tibetan pasture, we demonstrated the potential risk that antimicrobial-resistant E. coli could spread among herdsmen and yaks.

Two novel Salmonella genomic island 1 variants in Proteus mirabilis isolates from swine farms in China.

Four different Salmonella genomic island 1 (SGI1) variants, including two novel variants, were characterized in one Salmonella enterica serovar Rissen sequence type ST1917 isolate and three Proteus mirabilis isolates from swine farms in China. One novel variant was derived from SGI1-B with the backbone gene S021 disrupted by a 12.72-kb IS26 composite transposon containing the dfrA17-aadA5 cassettes and macrolide inactivation gene cluster mphA-mrx-mphR. The other one was an integron-free SGI1 and contained a 183-bp truncated S025 next to IS6100 and S044.

Presence and new genetic environment of pleuromutilin-lincosamide-streptogramin A resistance gene lsa(E) in Erysipelothrix rhusiopathiae of swine origin.

Erysipelothrix rhusiopathiae is a Gram-positive bacillus that causes erysipelas in swine. In recent years, erysipelas infection among swine in China has been increasing. A combined resistance phenotype to pleuromutilins, lincosamides, and streptogramin A (PLSA phenotype) was found in some E. rhusiopathiae isolates. The aim of this study was to identify the resistance genes responsible for the PLSA phenotype in E. rhusiopathiae strains and to map the genetic environment of the identified resistance gene. A total of 46 E. rhusiopathiae isolates from 31 pig farms in China were studied. Minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined by broth microdilution method. Seven were highly resistant to tiamulin (MICs 32 µg/ml) and clindamycin (MICs 64 µg/ml). Resistance genes responsible for the PLSA phenotype were screened by PCR. The lsa(E), spw, lnu(B), aadE and aphA3 genes were detected in strains had the PLSA phenotype, whereas none was detected in susceptible strains. The genetic environment of lsa(E) gene was determined by whole-genome sequencing and overlapping PCR assays. A novel multiresistance gene cluster, orf1-aadE-apt-spw-lsa(E)-lnu(B)-rec-orf2-orf1-aadE-sat4-aphA3, was found. Horizontal gene transfer experiments and whole-genome sequencing suggested that the lsa(E)-carrying multiresistance gene cluster was located in the chromosome. This is the first molecular characterization of PLSA resistance in E. rhusiopathiae. The lsa(E), spw and lnu(B) genes were found in E. rhusiopathiae for the first time. A novel lsa(E)-carrying multiresistance gene cluster was found. The location of lsa(E) in different gene cluster facilitates its persistence and dissemination.

First report of macrolide resistance gene erm(T) harbored by a novel small plasmid from Erysipelothrix rhusiopathiae.

The macrolide resistance gene erm(T) was identified for the first time in a porcine Erysipelothrix rhusiopathiae isolate from swine in China. The novel 3,749-bp small plasmid pER29, which carries erm(T), had a G+C content of 31% and four distinct open reading frames. The presence of pER29 increased by at least 128-fold the MICs of clindamycin and erythromycin for E. rhusiopathiae. The fitness cost of pER29 could be responsible for the low frequency of erm(T) in E. rhusiopathiae.

Molecular characteristics of Salmonella genomic island 1 in Proteus mirabilis isolates from poultry farms in China.

Six out of the 64 studied Proteus mirabilis isolates from 11 poultry farms in China contained Salmonella genomic island 1 (SGI1). PCR mapping showed that the complete nucleotide sequences of SGI1s ranged from 33.2 to 42.5 kb. Three novel variants, SGI1-W, SGI1-X, and SGI1-Y, have been characterized. Resistance genes lnuF, dfrA25, and qnrB2 were identified in SGI1 for the first time.

Dynamics of quinolone resistance in fecal Escherichia coli of finishing pigs after ciprofloxacin administration.

Escherichia coli resistance to quinolones has now become a serious issue in large-scale pig farms of China. It is necessary to study the dynamics of quinolone resistance in fecal Escherichia coli of pigs after antimicrobial administration. Here, we present the hypothesis that the emergence of resistance in pigs requires drug accumulation for 7 days or more. To test this hypothesis, 26 pigs (90 days old, about 30 kg) not fed any antimicrobial after weaning were selected and divided into 2 equal groups: the experimental (EP) group and control (CP) group. Pigs in the EP group were orally treated daily with 5 mg ciprofloxacin/kg of body weight for 30 days, and pigs in the CP group were fed a normal diet. Fresh feces were collected at 16 time points from day 0 to day 61. At each time point, ten E. coli clones were tested for susceptibility to quinolones and mutations of gyrA and parC. The results showed that the minimal inhibitory concentration (MIC) for ciprofloxacin increased 16-fold compared with the initial MIC (0.5 µg/ml) after ciprofloxacin administration for 3 days and decreased 256-fold compared with the initial MIC (0.5 µg/ml) after ciprofloxacin withdrawal for 26 days. GyrA (S83L, D87N/ D87Y) and parC (S80I) substitutions were observed in all quinolone-resistant E. coli (QREC) clones with an MIC ≥8 µg/ml. This study provides scientific theoretical guidance for the rational use of antimicrobials and the control of bacterial resistance.

Comparative dynamic distribution of avian infectious bronchitis virus M41, H120, and SAIBK strains by quantitative real-time RT-PCR in SPF chickens.

Avian infectious bronchitis is an acute, highly contagious disease of chickens. To study the differences of dynamic distribution between nephropathogenic infectious bronchitis virus (IBV) strains such as SAIBK and other strains (the M41 and H120 strains), relative quantitative real-time reverse transcription-polymerase chain reaction was developed by housekeeping gene selection. Glyceraldehyde-3-phosphate dehydrogenase and Ubiquitin were chosen for normalization in this experimental set. Then nine tissues, the trachea, thymus, liver, spleen, lungs, kidney, pancreas, proventriculus, and bursa of Fabricius, were analyzed and compared to determine the tropism of IBV infection. In this research, the kidney and the lung were established of the most sensitive organs in IBV infection. The pancreas and the liver are candidates for antigen detection. The trachea and the spleen can be used as references for histological diagnosis, but they are not suitable for antigen detection; proventriculus might be an important target in IBV infection; the thymus and the bursa of Fabricius were not sensitive organs in IBV infection.

Detection of clinically important ß-lactamases in commensal Escherichia coli of human and swine origin in western China.

Data correlating ß-lactamases found in commensal Escherichia coli of human and animal origin are limited. In this study, 447 commensal E. coli isolates from the faeces of humans and swine (280 human isolates from four hospitals and 167 swine isolates from seven farms) were collected between September 2006 and January 2009 in western China. For extended-spectrum ß-lactamase (ESBL)-producing and other cephalosporin-resistant isolates, the relevant ß-lactamase genes (bla(TEM), bla(SHV), bla(CTX-M-1/2/9) group, bla(CMY-2) and bla(KPC)) were detected by PCR analysis. Of the 447 isolates tested, 120 (26.8 %) were confirmed as producing ESBL. Among these, 70 and 40 human isolates carried a member of the bla(CTX-M-1 )group (13 bla(CTX-M-3), 21 bla(CTX-M-15), four bla(CTX-M-22), eight bla(CTX-M-28), four bla(CTX-M-36), 15 bla(CTX-M-55) and five bla(CTX-M-69)) or bla(SHV) (14 bla(SHV-2), seven bla(SHV-5), ten bla(SHV-12), five bla(SHV-57) and four bla(SHV-97)),respectively, whilst six and four swine isolates carried a member of the bla(CTX-M-1 )group (one bla(CTX-M-15) and five bla(CTX-M-22)) or bla(SHV) (three bla(SHV-2) and one bla(SHV-12)), respectively. Furthermore, 59 human and swine isolates and seven human isolates carried bla(CMY-2) and bla(KPC), respectively. These findings indicate that the bla(CTX-M-1) group, including the novel variant bla(CTX-M-69), and bla(SHV) are the predominant ESBL genes in both humans and swine in western China, and bla(CMY-2) is also common in both groups. The carriage rates of broad-spectrum ß-lactamases among commensal E. coli was much lower in swine than in humans, suggesting that ß-lactamase genes have not established themselves in animal ecosystems in western China.

Evaluation and target validation of indole derivatives as inhibitors of the AcrAB-TolC efflux pump.

Indole derivatives 3-amino-6-carboxyl-indole and 3-nitro-6-amino-indole were designed and synthesized based on the TolC structure. They proved to have potent synergistic antibacterial effects on chloramphenicol, tetracycline, erythromycin, and ciprofloxacin against Escherichia coli YD2 and FJ307 with decreased minimal inhibitory concentrations (MICs) at 2-64 folds. To research its functional site, Escherichia coli BL21(DE3)-3 expressing a target-site mutated TolC was constructed by red homologous recombination and the site-directed mutagenesis technique. They did not noticeably affect antimicrobial activity against BL21(DE3)-3. All the results indicate that these compounds match our design and can be developed as efflux pump inhibitors for the AcrAB-TolC efflux pump.