Lilium spp., as unnoticed environmental vector, spreading OptrA-carrying Enterococcus spp.

Flower is an essential element in the human lifestyle but its role in disseminating antimicrobial resistance (AMR) between the environment and humans is unclear. In this study, we screened fresh flowers (Lilium spp.) collected from planting bases, market and florists in Guangzhou China aiming to investigate the prevalence of AMR genes, particularly cfr, optrA and poxtA mediating resistance to linezolid, a first-line drug for the treatment of different Gram-positive bacterial infections. We found 223 Enterococcus isolates consisting of Enterococcus faecalis, Enterococcus faecium and Enterococcus mundtii, and >50% of these isolates exhibited multiple-drug resistance. Additionally, 31 optrA-positive Enterococcus including 22 E. faecalis and 9 E. mundtii strains were recovered, however cfr and poxtA were not detected. The 22 E. faecalis strains were belonged to 7 Multilocus sequence types in which ST202 and ST376 were predominant and 9 E. mundtii strains from the same plantation bases were divided into three PFGE groups. Genetically, the majority of optrA were located on the chromosome and shared similar insertion sites and transpositions mediated by Tn554 family members. Plasmid-bearing optrA were identified in 6 E. faecalis strains where IS1216 family played key roles in horizontal transfer of optrA. These findings emphasize that the prevalence of drug resistant Enterococcus in fresh flowers is a latent danger and increases the risk of AMR dissemination to humans from the environment.

Characterization of the Multi-Drug Resistance Gene cfr in Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated From Animals and Humans in China.

We investigated cfr-positive and -negative MRSA strains isolated from animals and humans in different geographical areas of China, from 2011 to 2016. Twenty cfr-positive strains (15.6%) were identified from 128 MRSA strains including 17 from food animals and three from humans. The resistance rates and prevalence of the tested antibiotic resistance genes (ARGs) in the cfr-positive MRSA isolates were higher than that in the cfr-negative MRSA isolates. All cfr-positive MRSA isolates were co-carrying fexA and ermC, and had significantly higher optrA incidence rate vs. the cfr-negative isolates (P < 0.05). In addition, multilocus sequence typing (MLST) assays showed that ST9 and spa-type t899 were the most prevalent ST and spa types in the study strains. However, all of the 20 cfr-positive and 10 randomly selected cfr-negative MRSA isolates were clonally unrelated as determined by pulsed-field gel electrophoresis (PFGE) analyses. Importantly, the cfr gene was successfully transferred to a recipient Staphylococcus aureus strain RN4220 from 13 of the 20 cfr-positive MRSA isolates by electroporation. Among these 13 cfr-positive MRSA isolates, two different genetic contexts surrounding cfr were determined and each was associated with one type of cfr-carrying plasmids. Of note, the predominant genetic context of cfr was found to be a Tn558 variant and locate on large plasmids (∼50 kb) co-harboring fexA in 11 of the 13 MRSA isolates. Furthermore, the cfr gene was also identified on small plasmids (∼ 7.1 kb) that co-carried ermC in two of the 13 MRSA isolates. Our results demonstrated a high occurrence of multi-drug resistance in cfr-positive MRSA isolates, and the spread of cfr might be attributed to horizontal dissemination of similar cfr-carrying transposons and plasmids.

Prevalence of cfr in Enterococcus faecalis strains isolated from swine farms in China: Predominated cfr-carrying pCPPF5-like plasmids conferring "non-linezolid resistance" phenotype.

The cfr gene associated with linezolid resistance has attracted wide attention. However, little is known about its prevalence and mode of transmission in Enterococcus faecalis. In this study, we investigate the prevalence and genetic environment of the cfr gene in 91 E. faecalis isolates collected from swine faecal swabs in 30 farms in Guangdong Province, China in 2012. A relatively high prevalence of cfr was identified in E. faecalis isolates (11/91, 12.1%) by PCR. All the cfr-positive E. faecalis strains had a multidrug-resistance phenotype including erythrocin, tetracycline, gentamicin, kanamicin and ciprofloxacin, except vancomycin and linezolid. Molecular typing indicated that ST475 and ST16 were the most common types in cfr-positive E. faecalis strains. In addition, we demonstrated that all the cfr genes were located on plasmids by S1-PFGE and Southern blotting. A 12 kb cfr-positive plasmid (pE30) was identified in most (9/11) E. faecalis strains, but it couldn't mediate resistance to linezolid in the transconjugant. Sequence analysis showed that the pE30 was a pCPPF5-like plasmid and the region surrounding the cfr gene was the same as a cfr-carrying ISEnfa5-composite element in Streptococcus plasmid pStrcfr with 4 bp direct repeat (GTAT) on both sides. In conclusion, the cfr gene which had no linezolid resistance phenotype was present in multidrug-resistance E. faecalis strains, and the clonal spread of ST475 and ST16 strains and the horizontal transfer of the pCPPF5-like plasmids have contributed to the dissemination of cfr.

Multilevel selection of bcrABDR-mediated bacitracin resistance in Enterococcus faecalis from chicken farms.

In this study we isolated 109 Enterococcus faecalis from chicken faecal samples in 6 provinces of China to investigate the prevalence and transmission mechanism of the bacitracin resistance locus bcrABDR in E. faecalis. Thirty-seven bcrABDR-positive E. faecalis were detected with 26 different PFGE clusters. The MLST of 14 positive strains belonged to ST16 and we also detected three new sequence types. S1-PFGE analysis indicated that the locus was located on plasmids presenting different sizes, with the most prevalent size being ~50 kb (13/37). Sequence analysis revealed that 17 out of the 37 strains harbored a 5400-bp central region, in which locus bcrABDR was bracketed by two ISEnfa1 of the same orientation. Two types of bcrABDR alleles, differing in around 10% of their sequence were found. In silico analysis showed that bcrABDR is present in a variety of bacteria including the chicken commensal Enterococcus cecorum. Our results indicate that the use of bacitracin at farms might trigger the emergence and spread of the bacitracin resistance determinant bcrABDR among human bacterial pathogens. The finding of bcrABDR in the chicken commensal E. cecorum indicates that farm animals microbiota can be an important reservoir of resistance genes with relevance for human health.