Design, synthesis, antibacterial/antitumor activity and in vitro stability of novel cordycepin derivatives with unsaturated fatty acid chain.

To overcome the metabolic instability of cordycepin (adenosine deaminase (ADA) metabolic deamination and plasma degradation) and obtain better bioactivity, three novel kinds of cordycepin derivatives 1a-1c containing unsaturated fatty acids including linoleic acid, arachidonic acid and α-linolenic acid, respectively, were designed and synthesized. In terms of antibacterial activity, the synthesized compounds 1a and 1c showed enhanced activity than cordycepin in the tested bacterial strains. 1a-1c also exhibited enhanced antitumor activity against four cancer cell lines (human cervical cancer cell line HeLa, human non-small cell lung cancer cell line A549, human breast cancer cell line MCF-7, and human hepatoma cell line SMMC-7721) compared with cordycepin. Notably, 1a and 1b showed better antitumor activity even compared with positive control 5-Fluorouracil (5-FU) in HeLa, MCF-7 and SMMC-7721. The cell cycle assay indicated that when compared with cordycepin, 1a and 1b could significantly inhibit the cell propagation trapped in S and G2/M phases and increase the percentage of cells trapped in G0/G1 in HeLa and A549, which might provide a synergistic antitumor mechanism evidence different from cordycepin. Last but not the least, 1a and 1b displayed improved stability both in ADA solution and mouse plasma compared with cordycepin and 1a owns a solubility of 130 µg/mL in PBS. These results offer a novel insight into the primary structure and activity relationship of how the unsaturated fatty acid chain could affect the bioactivity of cordycepin, which also represents a series of cordycepin analogs with obviously improved bioactivity and enhanced stability, therefore promoting its druggable enhancement.

Characterization of a Novel Linezolid Resistance Gene optrA and Bacitracin Resistance Locus-Carrying Multiple Antibiotic Resistant Integrative and Conjugative Element ICESsu1112S in Streptococccus Suis.

Streptococcus suis strain 1112S was isolated from a diseased pig in a feedlot from Henan, China, in 2019. The isolate harbored a linezolid resistance gene optrA. WGS data revealed that the optrA gene was associated with a single copy ETAf ISS1S, in tandem with erm(B) and tet(O), located in a novel 72,587 bp integrative and conjugative element (ICE). Notably, this novel element, designated ICESsu1112S, also carried a novel bacitracin resistance locus. ICESsu1112S could be excised from chromosome and transferred to the recipient strain S. suis P1/7 with a frequency of 5.9 × 10-6 transconjugants per donor cell. This study provided the first description of the coexistence of optrA and a novel bacitracin locus on a multiple antibiotic resistant ICE and highlighted that ICE were major vehicle and contribute to the potential transfer of clinically relevant antibiotic resistance genes. IMPORTANCE Antimicrobial resistance (AMR) caused by the imprudent use of antimicrobials has become a global problem, which poses a serious threat to treatment of S. suis infection in pigs and humans. Importantly, AMR genes can horizontally spread among commensal organisms and pathogenic microbiota, thereby accelerating the dissemination of AMR determinants. These transfers are mainly mediated by mobile genetic elements, including ICEs. In S. suis, ICEs are the major vehicles that contribute to the natural transfers of AMR genes among different bacterial pathogens. However, ICEs that carry optrA and bacitracin resistance locus are rarely investigated in S. suis isolates. Here, we investigated a S. suis isolate carrying an optrA and a novel bacitracin resistance locus, which were co-located on a novel multiple antibiotic resistant ICESsu1112S. Our study suggests that more research is needed to access the real significance of ICEs that horizontally spread clinical important resistance genes.

Coexistence of virulence and ß-lactamase genes in avian pathogenic Escherichia coli.

Emergence of multidrug resistance in E. coli and advent of newer strains is becoming serious concern which requires keen observations. This study was designed to find the ciprofloxacin resistant E. coli isolates co-existed with multi-drug resistance along with ß-lactamase production from poultry source, and finally the genome sequencing of these strains to explore genetic variations. Study constituted on isolation of n = 225 E. coli from broiler farms of central China which were further subjected to identification of resistance against ciprofloxacin followed by antibiogram of n = 26 antibiotics and identification of ß-lactamase production. Whole genome resequencing was performed using Illumina HiSeq 4000 system. PCR results revealed predominant ß-lactamase genes i.e.CTX-M, CTX-M-1, CTX-M3, TEM-1 and OXA. Furthermore, the MDR isolates were containing most of the tested virulence genes. The most prevalent virulence genes were pap-C, fim-C, fim-H, iuc-D, irp-2, tra-T, iro-N and iut-A. The single nucleotide polymorphisms (SNPs) loci mentioned in this data give valuable genetic markers to growing high-throughput techniques for fine-determination of genotyping of MDR and virulent isolates. Characterization of SNPs on functional basis shed new bits of knowledge on the evolution, disease transmission and pathogenesis of MDR E. coli isolates. In conclusion, these findings provide evidence that most of poultry E. coli are MDR, ß-lactamase producers, and virulent which could be a zoonotic threat to the humans. The whole genome resequencing data provide higher resolution of resistance and virulence characteristics in E. coli which can further be used for the development of prevention and treatment strategies.

RNA-seq-based transcriptome analysis of a cefquinome-treated, highly resistant, and virulent MRSA strain.

The emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains of animal origin that are resistant to several antibiotics is of great concern. Cefquinome is a fourth-generation cephalosporin developed specifically for veterinary use. The mechanism of MRSA resistance to cefquinome is still not established. Therefore, we designed this study to evaluate the effect of cefquinome on the transcriptome of MRSA1679a, a strain that was isolated from a chicken. The transcriptome analysis indicated that multiple efflux pumps (QacA, NorB, Bcr, and ABCb) were upregulated in MRSA1679a as a resistance mechanism to expel cefquinome. Additionally, penicillin-binding protein 1A was overexpressed, which conferred resistance to cefquinome, a ß-lactam antibiotic. Adhesion and the biofilm-forming capacity of the MRSA strain was also enhanced in addition to overexpression of many stress-related genes. Genes related to carbohydrate metabolism, secretion systems, and transport activity were also significantly upregulated in MRSA1679a. In conclusion, global transcription was triggered to overcome the stress induced by cefquinome, and the MRSA1679a showed a great genetic potential to survive in this challenging environment. This study provides a profound understanding of MRSA1679a as a potentially important pathogen and identifies key resistance characteristics of MRSA against cefquinome. Studies should be aimed to demonstrate multidrug resistance mechanisms of virulent strains by exposing to different antibiotic combinations.

Virulence and Genomic Feature of Multidrug Resistant Campylobacter jejuni Isolated from Broiler Chicken.

The aim of this study was to reveal the molecular mechanism involved in multidrug resistance and virulence of Campylobacter jejuni isolated from broiler chickens. The virulence of six multidrug resistant C. jejuni was determined by in vitro and in vivo methods. The de novo whole genome sequencing technology and molecular biology methods were used to analyze the genomic features associated with the multidrug resistance and virulence of a selected isolate (C. jejuni 1655). The comparative genomic analyses revealed a large number of single nucleotide polymorphisms, deletions, rearrangements, and inversions in C. jejuni 1655 compared to reference C. jejuni genomes. The co-emergence of Thr-86-Ile mutation in gyrA gene, A2075G mutation in 23S rRNA gene, tetO, aphA and aadE genes and pTet plasmid in C. jejuni 1655 contributed its multidrug resistance to fluoroquinolones, macrolides, tetracycline, and aminoglycosides. The combination of multiple virulence genes may work together to confer the relative higher virulence in C. jejuni 1655. The co-existence of mobile gene elements (e.g., pTet) and CRISPR-Cas system in C. jejuni 1655 may play an important role in the gene transfer and immune defense. The present study provides basic information of phenotypic and genomic features of C. jejuni 1655, a strain recently isolated from a chicken displaying multidrug resistance and relatively high level of virulence.

Serotypes and antimicrobial susceptibility of Salmonella spp. isolated from farm animals in China.

Salmonella spp. can indirectly infect humans via transfer from animals and animal-derived food products, and thereby cause potentially fatal diseases. Therefore, gaining an understanding of Salmonella infection in farm animals is increasingly important. The aim of this study was to identify the distribution of serotypes in Salmonella samples isolated from chickens (n = 837), pigs (n = 930), and dairy cows (n = 418) in central China (Henan, Hubei, and Hunan provinces) in 2010-2011, and investigate the susceptibility of strains to antimicrobial agents. Salmonella isolates were identified by PCR amplification of the invA gene, serotypes were determined by using a slide agglutination test for O and H antigens, and susceptibility to 24 antimicrobials was tested using the agar dilution method. In total, 248 Salmonella strains were identified: 105, 105, and 38 from chickens, dairy cows, and pigs, respectively. Additionally, 209 strains were identified in diseased pigs from the Huazhong Agricultural University veterinary hospital. Among these 457 strains, the dominant serotypes were Typhimurium in serogroup B, IIIb in serogroup C, and Enteritidis in serogroup D. In antimicrobial susceptibility tests, 41.14% of Salmonella spp. were susceptible to all antimicrobial agents, 48.14% were resistant to at least one, and 34.72% were resistant to more than three classes. Strains were highly resistant to sulfamethoxazole-trimethoprim (39.61%), nalidixic acid (39.17%), doxycycline (28.22%), and tetracycline (27.58%). Resistance to cephalosporins and fluoroquinolones ranged from 5.25 to 7.44% and 19.04 to 24.51%, respectively. Among penicillin-resistant and cephalosporin-resistant strains, 25 isolates produced extended-spectrum ß-lactamases (ESBLs). The multidrug-resistant and ESBL-producing Salmonella strains identified in healthy animals here will present a challenge for veterinary medicine and farm animal husbandry, and could also pose a threat to public health. The level of antibiotic resistance observed in this study further highlights the need for careful and selective use of antibiotics.

Phenotypic and molecular characterization of TEM-116 extended-spectrum beta-lactamase produced by a Shigella flexneri clinical isolate from chickens.

Extended-spectrum beta-lactamases (ESBLs) produced by a clinical isolate of Shigella flexneri from chickens were detected with confirmatory phenotypic tests of the Clinical and Laboratory Standards Institute, and minimum inhibitory concentrations of several antibacterial drugs against the isolate were determined by the twofold dilution method. The genotype and subtype of the ESBL-producing S. flexneri isolate were identified by PCR amplifying of ESBL genes and DNA sequencing analysis. The results revealed that the isolate was able to produce ESBLs. They were resistant to third-generation cephalosporins such as ceftiofur and ceftriaxone and showed characteristics of multidrug resistance. The ESBL gene from the S. flexneri isolate was of the TEM type. Sequence analysis indicated that the TEM-type gene had 99.1% and 99.2% identity to TEM-1D ESBL and TEM-1 beta-lactamase, respectively, at the nucleotide level. The amino acid sequence inferred from the TEM-type gene revealed three substitutions compared with the TEM-1 and TEM-1D enzymes: Ser51Gly, Val82Ila and Ala182Val. When it was compared with TEM-116 (99.8% identity), there were only two mutations (A(151)G and T(403)C) in the TEM-type gene, resulting in the substitution of Ser to Gly at position 51 in the amino acid sequence. The TEM type was a TEM-116 derivative.