Spatially and Temporally Confined Response of Gastrointestinal Antibiotic Resistance Gene Levels to Sulfadiazine and Extracellular Antibiotic Resistance Gene Exposure in Mice.

This work aims to investigate the impact of antibiotics and extracellular antibiotic resistance genes (eARGs) on the dynamics of gastrointestinal antimicrobial resistance (AMR). The antibiotic resistance gene (ARG) levels of different segments of the gastrointestinal tract of mouse models were analyzed and compared after exposure to clinical concentrations of sulfadiazine and environmental levels of eARGs carried by the conjugative plasmid pR55. Exposure to sulfadiazine and eARGs led to significant changes in ARG levels by as many as four log-folds. Further analysis showed that the response of ARG levels appeared from 12-16 days after exposure and diminished 20 days after exposure. The responses in ARG levels were also restricted to different gastrointestinal segments for sulfadiazine and eARGs. Combined exposure of sulfadiazine and eARGs was unable to further increase ARG levels. From these findings, we concluded that the short-term consumption of environmental levels of eARGs and uptake of clinical levels of antibiotics lead to a spatially and temporally confined response in gastrointestinal AMR. These findings further clarify the detrimental impacts of antibiotic and eARG uptake, and the complexity of AMR development and dissemination dynamics in the gastrointestinal tract.

Evaluation of culturable 'last-resort' antibiotic resistant pathogens in hospital wastewater and implications on the risks of nosocomial antimicrobial resistance prevalence.

Antimicrobial resistance has been recognized as an important emerging environmental pollutant. 'Last-resort' antibiotics including tigecycline, polymyxin E, daptomycin, vancomycin and linezolid are the 'last line of defence' for antibiotic resistant pathogen infections. Therefore, the presence of 'last-resort' antibiotic resistant pathogens in hospital environments and the nosocomial transmission of 'last-resort' antibiotic resistance poses a grave threat to the well-being of patients. In this work, the extent of resistance to 'last-resort' antibiotics in culturable pathogens in hospital wastewater was investigated. Resistance to 'last-resort' antibiotics were quantified for 1384 culturable Enterobacteriaceae, Enterococcus, Staphylococcus, and Pseudomonas strains. With these investigations, several significant findings were made: (1) a very high level of resistance to 'last-resort' antibiotics was found; (2) multiple resistance to antibiotics, including 'last-resort' antibiotics, was prevalent; (3) a high level of 'last-resort' antibiotic resistance phenotype-genotype inconsistency was found, suggesting knowledge gap for resistance mechanisms; 4) tet(X4)-containing tigecycline-resistant Gram-positive pathogens were found for the first time; 5) wastewater treatment processes are effective in preventing the release of 'last-resort' antibiotic resistant pathogens to the environment. This investigation reveals the severe situation on 'last-resort' resistance in the hospital environment, and implies high risk for nosocomial transmission of 'last-resort' antibiotic resistant pathogens.

In Vitro Assessment of Antimicrobial Resistance Dissemination Dynamics during Multidrug-Resistant-Bacterium Invasion Events by Using a Continuous-Culture Device.

Antimicrobial-resistant pathogens display significant public health threats by causing difficulties in clinical treatment of bacterial infection. Antimicrobial resistance (AMR) is transmissible between bacteria, significantly increasing the appearance of antimicrobial-resistant pathogens and aggravating the AMR problem. In this work, the dissemination dynamics of AMR from invading multidrug-resistant (MDR) Escherichia coli to a community of pathogenic Salmonella enterica was investigated using a continuous-culture device, and the behaviors of dissemination dynamics under different levels of antibiotic stress were investigated. Three MDR E. coli invasion events were analyzed in this work: MDR E. coli-S. enterica cocolonization, MDR E. coli invasion after antibiotic treatment of S. enterica, and MDR E. coli invasion before antibiotic treatment of S. enterica It was found that both horizontal gene transfer (HGT) and vertical gene transfer (VGT) play significant roles in AMR dissemination, although different processes contribute differently under different circumstances, that environmental levels of antibiotics promote AMR dissemination by enhancing HGT rather than leading to selective advantage for resistant bacteria, and that early invasion of MDR E. coli completely and quickly sabotages the effectiveness of antibiotic treatment. These findings contribute to understanding the drivers of AMR dissemination under different antibiotic stresses, the detrimental impact of environmental tetracycline contamination, and the danger of nosocomial presence and dissemination of MDR nonpathogens.IMPORTANCE Antimicrobial resistance poses a grave threat to public health and reduces the effectiveness of antimicrobial drugs in treating bacterial infections. Antimicrobial resistance is transmissible, either by horizontal gene transfer between bacteria or by vertical gene transfer following inheritance of genetic traits. The dissemination dynamics and behaviors of this threat, however, have not been rigorously investigated. In this work, with a continuous-culture device, we studied antimicrobial resistance dissemination processes by simulating antimicrobial-resistant Escherichia coli invasion to a pathogenic Salmonella enterica community. Using this novel tool, we provide evidence on the drivers of antimicrobial resistance dissemination, on the detrimental impact of environmental antibiotic contamination, and on the danger of antimicrobial resistance in hospitals, even if what harbors the antimicrobial resistance is not a pathogen. This work furthers our understanding of antimicrobial resistance and its dissemination between bacteria and of antibiotic therapy, our most powerful tool against bacterial infection.

The diversity of glycosylation of cellobiohydrolase I from Trichoderma reesei determined with mass spectrometry.

Cellulases are glycosylated enzymes that have wide applications in fields like biofuels. It has been widely accepted that glycosylation of cellulases impact their performance. Trichoderma reesei is the most important cellulase-producer and cellobiohydrolase I (CBHI) is the most important cellulase from T. reesei. Therefore, the glycosylation of T. reesei CBHI has been a focus of research. However, investigations have been focused on N-glycosylation of three of the four potential glycosylation sites, as well as O-glycosylation on the linker region, while a full picture of glycosylation of T. reesei CBHI is still needed. In this work, with extensive mass spectrometric investigations on CBHI from two T. reesei strains grown under three conditions, several new discoveries were made: 1) N45 and N64 are N-glycosylated with high mannose type glycans; 2) the catalytic domain of CBHI is extensively O-glycosylated with hexoses and N-acetylhexosamines; 3) experimental evidence on the mannosylation of carbohydrate binding domain (other than the linker adjacent region) was found. With structural analysis, we found several glycosylation sites (such as T383, S8, and S46) are located at the openings of the substrate-binding tunnel, and potentially involve in the binding of cellulose. These investigations provide a full and comprehensive picture on the glycosylation of CBHI from T. reesei, which benefits the engineering of CBHI by raising potential sites for modification.