A novel incompatibility group X3 plasmid carrying blaNDM-1 encodes a small RNA that regulates host fucose metabolism and biofilm formation.

The emergence of New Delhi metallo-beta-lactamase (NDM-1) has become a major health threat to clinical managements of gram-negative bacteria infections. A novel incompatibility group X3 plasmid (IncX3) pNDM-HN380 carrying blaNDM-1 has recently been found to epidemiologically link with multiple geographical areas in China. In this paper, we studied the metabolic responses of host bacteria E. coli J53 upon introduction of pNDM-HN380. A reduction of bacterial motility was observed in J53/pNDM-HN380. We profiled the RNA repertoires of the transconjugants and found a downregulation of genes involved in flagella and chemotaxis metabolic pathways at logarithmic (log) phase. We also identified a novel intragenic region (IGR) small RNA plas2. The plasmid-transcribed sRNA IGR plas2 was further characterized as a regulator of fucRwhich controls the fucose metabolism. By knockdown of IGR plas2 using an antisense decoy, we managed to inhibit the formation of bacterial biofilm of the host. Our study demonstrated a potential way of utilizing plasmid-transcribed sRNA against infectious bacteria.

Fine-tuning carbapenem resistance by reducing porin permeability of bacteria activated in the selection process of conjugation.

Antibiotic resistance is an emerging public health issue. Plasmids are one of the popular carriers to disseminate resistance genes among pathogens. However, the response of plasmid-carrying bacteria to antibiotic treatment and how these bacteria evolve to increase their resistance remain elusive. In this study, we conjugated plasmid pNDM-HK to E. coli J53 recipient cells and selected survivors using different concentrations of the broad spectrum antibiotic meropenem. After selection, transconjugants conferred varying minimum inhibitory concentrations with respect to carbapenems. We sequenced and compared the transcriptomes of transconjugants that exhibited distinct carbapenem susceptibilities, and found that the loss of outer membrane proteins led to antibiotic resistance. Moreover, we identified a novel mutation, G63S, in transcription factor OmpR which moderates the expression of outer membrane proteins. The loss of porins was due to incapability of phosphorylation, which is essential for porin transcription and carbapenem resistance. We also characterized other genes that are regulated by ompR in this mutant, which contributed to bacterial antibiotic resistance. Overall, our studies suggest antibiotic pressure after conjugation might be an alternative pathway to promote antimicrobial resistance.

Novel RNA aptamers targeting gastrointestinal cancer biomarkers CEA, CA50 and CA72-4 with superior affinity and specificity.

Gastric cancer is the third most common cause of death from cancer in the world and it remains difficult to cure in Western countries, primarily because most patients present with advanced disease. Currently, CEA, CA50 and CA72-4 are commonly used as tumor markers for gastric cancer by immunoassays. However, the drawback and conundrum of immunoassay are the unceasing problem in standardization of quality of antibodies and time/effort for the intensive production. Therefore, there is an urgent need for the development of a standardized assay to detect gastric cancer at the early stage. Aptamers are DNA or RNA oligonucleotides with structural domain which recognize ligands such as proteins with superior affinity and specificity when compared to antibodies. In this study, SELEX (Systematic Evolution of Ligands by Exponential enrichment) technique was adopted to screen a random 30mer RNA library for aptamers targeting CEA, CA50 and CA72-4 respectively. Combined with high-throughput sequencing, we identified 6 aptamers which specifically target for these three biomarkers of gastrointestinal cancer. Intriguingly, the predicted secondary structures of RNA aptamers from each antigen showed significant structural similarity, suggesting the structural recognition between the aptamers and the antigens. Moreover, we determined the dissociation constants of all the aptamers to their corresponding antigens by fluorescence spectroscopy, which further demonstrated high affinities between the aptamers and the antigens. In addition, immunostaining of gastric adenocarcinoma cell line AGS using CEA Aptamer probe showed positive fluorescent signal which proves the potential of the aptamer as a detection tool for gastric cancer. Furthermore, substantially decreased cell viability and growth were observed when human colorectal cell line LS-174T was transfected with each individual aptamers. Taking together, these novel RNA aptamers targeting gastrointestinal cancer biomarker CEA, CA50 and CA72-4 will aid further development and standardization of clinical diagnostic method with better sensitivity and specificity, and potentially future therapeutics development of gastric cancer.

Identification of Plasmid-Encoded sRNAs in a blaNDM-1-Harboring Multidrug-Resistance Plasmid pNDM-HK in Enterobacteriaceae.

Small RNAs (sRNAs) play significant roles in regulating gene expression post-transcriptionally in response to environmental changes in bacteria. In this work, we identified and characterized six novel sRNAs from an emerging multidrug-resistance (MDR) plasmid pNDM-HK, a New Delhi metallo-ß-lactamase 1 gene (blaNDM-1)-carrying IncL/M plasmid that has caused worldwide threat in recent years. These sRNAs are located at different regions of pNDM-HK, such as replication, stability, and variable regions. Moreover, one of the plasmid-encoded sRNAs (NDM-sR3) functions in an Hfq-dependent manner and possibly plays roles in the fitness of pNDM-HK carrying bacteria. In addition, we attempted to construct the phylogenetic tree based on these novel sRNAs and surprisingly, the sRNA-phylogenetic tree provided significant information about the evolutionary pathway of pNDM-HK, including possible gene acquisition and insertion from relevant plasmids. Moreover, the sRNA-phylogenetic tree can specifically cluster the IncM2 type and distinguish it from other IncL/M subtypes. In summary, this is the first study to systematically identify and characterize sRNAs from clinically-isolated MDR plasmids. We believe that these newly found sRNAs could lead to further understanding and new directions to study the evolution and dissemination of the clinically MDR bacterial plasmids.

The CTX-M-14 plasmid pHK01 encodes novel small RNAs and influences host growth and motility.

The dissemination of extended-spectrum ß-lactamases (ESBLs) genes among bacteria is commonly achieved by plasmid conjugation. In the last decade, the CTX-M type enzyme was the most widespread and prevalent ESBLs in the world. In Hong Kong and mainland China, among the commonly found CTX-M-carrying plasmids were pHK01 and pHK01-like plasmids, which belong to incompatibility group FII (IncFII). In this work, we studied the physiological effect caused by the pHK01 plasmid in bacterial host Escherichia coli J53. The plasmid did not affect cell growth of the host but reduced their motility. The reduction of host motility was attributed to downregulation of genes that encode the flagellar system. We also identified several plasmid-encoded sRNAs, and showed that the overexpression of one of them, AS-traI, in the presence of pHK01 plasmid shortened the lag phase of host growth. In addition to the study of pHK01 in bacteria, we also developed a fast and incompatibility group-specific curing method using countertranscribed RNA, which could be of general usage for studying plasmid-host interaction in clinical aspects.

Transcriptomic Analysis of Laribacter hongkongensis Reveals Adaptive Response Coupled with Temperature.

Bacterial adaptation to different hosts requires transcriptomic alteration in response to the environmental conditions. Laribacter hongkongensis is a gram-negative, facultative anaerobic, urease-positive bacillus caused infections in liver cirrhosis patients and community-acquired gastroenteritis. It was also found in intestine from commonly consumed freshwater fishes and drinking water reservoirs. Since L. hongkongensis could survive as either fish or human pathogens, their survival mechanisms in two different habitats should be temperature-regulated and highly complex. Therefore, we performed transcriptomic analysis of L. hongkongensis at body temperatures of fish and human in order to elucidate the versatile adaptation mechanisms coupled with the temperatures. We identified numerous novel temperature-induced pathways involved in host pathogenesis, in addition to the shift of metabolic equilibriums and overexpression of stress-related proteins. Moreover, these pathways form a network that can be activated at a particular temperature, and change the physiology of the bacteria to adapt to the environments. In summary, the dynamic of transcriptomes in L. hongkongensis provides versatile strategies for the bacterial survival at different habitats and this alteration prepares the bacterium for the challenge of host immunity.