Mycolyltransferase is important for biofilm formation and pathogenesis of Tsukamurella keratitis.

AbstractTsukamurella, a group of multi-drug resistant, Gram-positive, aerobic and partially acid-fast bacteria, are emerging causes of bacterial conjunctivitis and keratitis. However, the pathogenesis of Tsukamurella keratitis is largely unknown. To address this, we used New Zealand White rabbits to develop the first eye infection model and conducted in vitro tests to study the pathogenesis mechanisms of Tsukamurella. There is increasing evidence that biofilms play a significant role in ocular infections, leading us to hypothesize that biofilm formation is crucial for effective Tsukamurella infection. In order to look for potential candidate genes which are important in biofilm formation and Tsukamurella keratitis. We performed genome sequencing of two ocular isolates, T. pulmonis-PW1004 and T. tyrosinosolvens-PW899, to identify potential virulence factors. Through in vitro and in vivo studies, we characterized their biological roles in mediating Tsukamurella keratitis. Our findings confirmed that Tsukamurella is an ocular pathogen by fulfilling the Koch's postulates, and using genome sequence data, we identified tmytC, encoding a mycolyltransferase, as a crucial gene in biofilm formation and causing Tsukamurella keratitis in the rabbit model. This is the first report demonstrating the novel role of mycolyltransferase in causing ocular infections. Overall, our findings contribute to a better understanding of Tsukamurella pathogenesis and provide a potential target for treatment. Specific inhibitors targeting TmytC could serve as an effective treatment option for Tsukamurella infections.

Daily oral administration of probiotics engineered to constantly secrete short-chain fatty acids effectively prevents myocardial injury from subsequent ischemic heart disease.

AIM: Given the extremely limited regeneration potential of the heart, one of the most effective strategies to reduce the prevalence and mortality of coronary artery disease is prevention. Short-chain fatty acids (SCFAs), which are by-products of beneficial probiotics, have been reported to possess cardioprotective effects. Despite their beneficial roles, delivering SCFAs and maintaining their effective concentration in plasma present major challenges. Therefore, in the present study, we aimed to devise a strategy to prevent coronary heart disease effectively by using engineered probiotics to continuously release SCFAs in vivo. METHODS AND RESULTS: We engineered a novel probiotic cocktail, EcN_TL, from the commercially available Escherichia coli Nissle 1917 strain to continuously secrete SCFAs by introducing the propionate and butyrate biosynthetic pathways. Oral administration of EcN_TL enhanced and maintained an effective concentration of SCFAs in the plasma. As a preventative strategy, we observed that daily intake of EcN_TL for 14 days prior to ischemia-reperfusion injury significantly reduced myocardial injury and improved cardiac performance compared to EcN administration. We uncovered that EcN_TL's protective mechanisms included reducing neutrophil infiltration into the infarct site and promoting the polarization of wound-healing macrophages. We further revealed that SCFAs at plasma concentration protected cardiomyocytes from inflammation by suppressing the NF-κB activation pathway. CONCLUSIONS: These data provide strong evidence to support the use of SCFA-secreting probiotics to prevent coronary heart disease. Since SCFAs also play a key role in other metabolic diseases, EcN_TL can potentially be used to treat a variety of other diseases.

Comparative Analysis of Transcriptome and Proteome Revealed the Common Metabolic Pathways Induced by Prevalent ESBL Plasmids in Escherichia coli.

Antibiotic resistance has emerged as one of the most significant threats to global public health. Plasmids, which are highly efficient self-replicating genetic vehicles, play a critical role in the dissemination of drug-resistant genes. Previous studies have mainly focused on drug-resistant genes only, often neglecting the complete functional role of multidrug-resistant (MDR) plasmids in bacteria. In this study, we conducted a comprehensive investigation of the transcriptomes and proteomes of Escherichia coli J53 transconjugants harboring six major MDR plasmids of different incompatibility (Inc) groups, which were clinically isolated from patients. The RNA-seq analysis revealed that MDR plasmids influenced the gene expression in the bacterial host, in particular, the genes related to metabolic pathways. A proteomic analysis demonstrated the plasmid-induced regulation of several metabolic pathways including anaerobic respiration and the utilization of various carbon sources such as serine, threonine, sialic acid, and galactarate. These findings suggested that MDR plasmids confer a growth advantage to bacterial hosts in the gut, leading to the expansion of plasmid-carrying bacteria over competitors without plasmids. Moreover, this study provided insights into the versatility of prevalent MDR plasmids in moderating the cellular gene network of bacteria, which could potentially be utilized in therapeutics development for bacteria carrying MDR plasmids.

Intracellular bacterial eradication using a novel peptide in vitro.

AIM: To determine the effect of a novel antimicrobial peptide (AMP; OP145) and cell-penetrating peptide (Octa-arginine/R8) conjugate on the killing of intracellular Enterococcus faecalis, compared to OP145 and an antibiotic combination recommended for regenerative endodontic procedures. METHODOLOGY: The biocompatible concentrations of OP145 and OP145-R8 were determined by assessing their cytotoxicity against human macrophages and red blood cells. Spatiotemporal internalization of the peptides into macrophages was investigated qualitatively and quantitatively by confocal laser scanning microscopy and flow cytometry respectively. Killing of extracellular and intracellular E. faecalis OG1RF by the peptides was determined by counting the colony-forming units (CFU). Intracellular antibacterial activity of the peptides was compared to a double antibiotic combination. Confocal microscopy was used to confirm the intracellular bacterial eradication. Significant differences between the different test groups were analysed using one-way analysis of variance. p < .05 was considered to be statistically significant. RESULTS: Peptides at a concentration of 7.5 µmol/L were chosen for subsequent experiments based on the results of the alamarBlue™ cell viability assay and haemolytic assay. OP145-R8 selectively internalized into lysosomal compartments and the cytosol of macrophages. Conjugation with R8 improved the internalization of OP145 into macrophages in a temporal manner (70.53% at 1 h to 77.13% at 2 h), while no temporal increase was observed for OP145 alone (60.53% at 1 h with no increase at 2 h). OP145-R8 demonstrated significantly greater extracellular and intracellular antibacterial activity compared to OP145 at all investigated time-points and concentrations (p < .05). OP145-R8 at 7.5 µmol/L eradicated intracellular E. faecalis after 2 h (3.5 log reduction compared to the control; p < .05), while the antibiotics could not reduce more than 0.5 log CFU compared to the control (p > .05). Confocal microscopy showed complete absence of E. faecalis within the OP145-R8 treated macrophages. CONCLUSIONS: The results of this study demonstrated that the conjugation of an AMP OP145 to a cell-penetrating peptide R8 eradicated extracellular and intracellular E. faecalis OG1RF without toxic effects on the host cells.

Comparative analysis of outer membrane vesicles from uropathogenic Escherichia coli reveal the role of aromatic amino acids synthesis proteins in motility.

Uropathogenic Escherichia coli (UPEC) are causative agent that causes urinary tract infections (UTIs) and the recent emergence of multidrug resistance (MDR) of UPEC increases the burden on the community. Recent studies of bacterial outer membrane vesicles (OMV) identified various factors including proteins, nucleic acids, and small molecules which provided inter-cellular communication within the bacterial population. However, the components of UPEC-specific OMVs and their functional role remain unclear. Here, we systematically determined the proteomes of UPEC-OMVs and identified the specific components that provide functions to the recipient bacteria. Based on the functional network of OMVs' proteomes, a group of signaling peptides was found in all OMVs which provide communication among bacteria. Moreover, we demonstrated that treatment with UPEC-OMVs affected the motility and biofilm formation of the recipient bacteria, and further identified aromatic amino acid (AAA) biosynthesis proteins as the key factors to provide their movement.

A Small RNA Transforms the Multidrug Resistance of Pseudomonas aeruginosa to Drug Susceptibility.

Bacteria with multiple drug resistance (MDR) have become a global issue worldwide, and hundreds of thousands of people's lives are threatened every year. The emergence of novel MDR strains and insufficient development of new antimicrobial agents are the major reasons that limit the choice of antibiotics for the treatment of bacterial infection. Thus, preserving the clinical value of current antibiotics could be one of the effective approaches to resolve this problem. Here we identified numerous novel small RNAs that were downregulated in the MDR clinical isolates of Pseudomonas aeruginosa (P. aeru), and we demonstrated that overexpression of one of these small RNAs (sRNAs), AS1974, was able to transform the MDR clinical strain to drug hypersusceptibility. AS1974 is the master regulator to moderate the expression of several drug resistance pathways, including membrane transporters and biofilm-associated antibiotic-resistant genes, and its expression is regulated by the methylation sites located at the 5' UTR of the gene. Our findings unravel the sRNA that regulates the MDR pathways in clinical isolates of P. aeru. Moreover, transforming bacterial drug resistance to hypersusceptibility using sRNA could be the potential approach for tackling MDR bacteria in the future.

Distinct mechanisms govern the phosphorylation of different SR protein splicing factors.

Serine-arginine (SR) proteins are essential splicing factors containing a canonical RNA recognition motif (RRM), sometimes followed by a pseudo-RRM, and a C-terminal arginine/serine-rich (RS) domain that undergoes multisite phosphorylation. Phosphorylation regulates the localization and activity of SR proteins, and thus may provide insight into their differential biological roles. The phosphorylation mechanism of the prototypic SRSF1 by serine-arginine protein kinase 1 (SRPK1) has been well-studied, but little is known about the phosphorylation of other SR protein members. In the present study, interaction and kinetic assays unveiled how SRSF1 and the single RRM-containing SRSF3 are phosphorylated by SRPK2, another member of the SRPK family. We showed that a conserved SRPK-specific substrate-docking groove in SRPK2 impacts the binding and phosphorylation of both SR proteins, and the localization of SRSF3. We identified a nonconserved residue within the groove that affects the kinase processivity. We demonstrated that, in contrast to SRSF1, for which SRPK-mediated phosphorylation is confined to the N-terminal region of the RS domain, SRSF3 phosphorylation sites are spread throughout its entire RS domain in vitro Despite this, SRSF3 appears to be hypophosphorylated in cells at steady state. Our results suggest that the absence of a pseudo-RRM renders the single RRM-containing SRSF3 more susceptible to dephosphorylation by phosphatase. These findings suggest that the single RRM- and two RRM-containing SR proteins represent two subclasses of phosphoproteins in which phosphorylation statuses are maintained by unique mechanisms, and pose new directions to explore the distinct roles of SR proteins in vivo.

The ribosomal maturation factor P from Mycobacterium smegmatis facilitates the ribosomal biogenesis by binding to the small ribosomal protein S12.

The ribosomal maturation factor P (RimP) is a highly conserved protein in bacteria and has been shown to be important in ribosomal assembly in Escherichia coli Because of its central importance in bacterial metabolism, RimP represents a good potential target for drug design to combat human pathogens such as Mycobacterium tuberculosis However, to date, the only RimP structure available is the NMR structure of the ortholog in another bacterial pathogen, Streptococcus pneumoniae Here, we report a 2.2 Å resolution crystal structure of MSMEG_2624, the RimP ortholog in the close M. tuberculosis relative Mycobacterium smegmatis, and using in vitro binding assays, we show that MSMEG_2624 interacts with the small ribosomal protein S12, also known as RpsL. Further analyses revealed that the conserved residues in the linker region between the N- and C-terminal domains of MSMEG_2624 are essential for binding to RpsL. However, neither of the two domains alone was sufficient to form strong interactions with RpsL. More importantly, the linker region was essential for in vivo ribosomal biogenesis. Our study provides critical mechanistic insights into the role of RimP in ribosome biogenesis. We anticipate that the MSMEG_2624 crystal structure has the potential to be used for drug design to manage M. tuberculosis infections.

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.

Matrix metalloproteinases (MMPs) mediate leukocyte recruitment during the inflammatory phase of zebrafish heart regeneration.

In zebrafish, the role of matrix metalloproteinases (MMPs) in the inflammatory phase of heart regeneration following cryoinjury remains poorly understood. Here, we demonstrated an increase in MMP enzymatic activity and elevated expression of mmp9 and mmp13 in the injured area (IA) of hearts from as early as 1 day post-cryoinjury (dpc). Treatment with the broad-spectrum MMP inhibitor, GM6001, during the first week after cryoinjury resulted in impaired heart regeneration, as indicated by the larger scar and reduced numbers of proliferating cardiomyocytes. GM6001 also significantly reduced the number of leukocytes to the IA at 0.5 dpc to 4 dpc. Specific inhibition of both MMP-9 and MMP-13 also resulted in impaired regeneration and leukocyte recruitment. However, chemokine rescue with recombinant CXCL8 and CCL2 restored the recruitment of macrophages and the cardiac regenerative capability in GM6001-treated fish. MMP-9 and MMP-13 cleaved zebrafish CXCL8 at the same site, and the truncated form was more chemotactic than the intact form. In contrast, CCL2 did not have an MMP-9 or MMP-13 cleavage site. Together, these data suggest that MMPs might play a key role in the inflammatory phase of heart regeneration in zebrafish, by mediating leukocyte recruitment via the activation of chemokines.

Comparative transcriptomics of multidrug-resistant Acinetobacter baumannii in response to antibiotic treatments.

Multidrug-resistant Acinetobacter baumannii, a major hospital-acquired pathogen, is a serious health threat and poses a great challenge to healthcare providers. Although there have been many genomic studies on the evolution and antibiotic resistance of this species, there have been very limited transcriptome studies on its responses to antibiotics. We conducted a comparative transcriptomic study on 12 strains with different growth rates and antibiotic resistance profiles, including 3 fast-growing pan-drug-resistant strains, under separate treatment with 3 antibiotics, namely amikacin, imipenem, and meropenem. We performed deep sequencing using a strand-specific RNA-sequencing protocol, and used de novo transcriptome assembly to analyze gene expression in the form of polycistronic transcripts. Our results indicated that genes associated with transposable elements generally showed higher levels of expression under antibiotic-treated conditions, and many of these transposon-associated genes have previously been linked to drug resistance. Using co-expressed transposon genes as markers, we further identified and experimentally validated two novel genes of which overexpression conferred significant increases in amikacin resistance. To the best of our knowledge, this study represents the first comparative transcriptomic analysis of multidrug-resistant A. baumannii under different antibiotic treatments, and revealed a new relationship between transposons and antibiotic resistance.

Direct detection of Mycobacterium tuberculosis and drug resistance in respiratory specimen using Abbott Realtime MTB detection and RIF/INH resistance assay.

Abbott RealTime MTB (Abbott-RT) in conjunction with Abbott RealTime MTB RIF/INH Resistance (Abbott-RIF/INH) is a new, high-throughput automated nucleic acid amplification platform (Abbott-MDR) for detection of Mycobacterium tuberculosis complex (MTBC) and the genotypic markers for rifampicin (RIF) and isoniazid (INH) resistance directly from respiratory specimens. This prospective study evaluated the diagnostic performance of this new platform for MTBC and multidrug-resistant tuberculosis (MDR-TB) using 610 sputum specimens in a tuberculosis high-burden setting. Using conventional culture results and clinical background as reference standards, Abbott-RT exhibited an overall sensitivity and specificity of 95.2% and 99.8%, respectively. Genotypic RIF/INH resistance of 178 "MTB detected" specimens was subsequently analyzed by Abbott-RIF/INH. Compared to phenotypic drug susceptibility test results, Abbott-RIF/INH detected resistance genotypic markers in 84.6% MDR-TB, 80% mono-RIF-resistant and 66.7% mono-INH-resistant specimens. Two of the RIF-resistant specimens carried a novel single, nonsense mutation at rpoB Q513 and in silico simulation demonstrated that the truncated RpoB protein failed to bind with other subunits for transcription. Overall, Abbott-MDR platform provided high throughput and reliable diagnosis of MDR-TB within a TB high-burden region.

Gut-homing Δ42PD1+Vδ2 T cells promote innate mucosal damage via TLR4 during acute HIV type 1 infection.

The innate immune cells underlying mucosal inflammatory responses and damage during acute HIV-1 infection remain incompletely understood. Here, we report a Vδ2 subset of gut-homing γδ T cells with significantly upregulated Δ42PD1 (a PD1 isoform) in acute (~20%) HIV-1 patients compared to chronic HIV-1 patients (~11%) and healthy controls (~2%). The frequency of Δ42PD1+Vδ2 cells correlates positively with plasma levels of pro-inflammatory cytokines and fatty-acid-binding protein before detectable lipopolysaccharide in acute patients. The expression of Δ42PD1 can be induced by in vitro HIV-1 infection and is accompanied by high co-expression of gut-homing receptors CCR9/CD103. To investigate the role of Δ42PD1+Vδ2 cells in vivo, they were adoptively transferred into autologous humanized mice, resulting in small intestinal inflammatory damage, probably due to the interaction of Δ42PD1 with its cognate receptor Toll-like receptor 4 (TLR4). In addition, blockade of Δ42PD1 or TLR4 successfully reduced the cytokine effect induced by Δ42PD1+Vδ2 cells in vitro, as well as the mucosal pathological effect in humanized mice. Our findings have therefore uncovered a Δ42PD1-TLR4 pathway exhibited by virus-induced gut-homing Vδ2 cells that may contribute to innate immune activation and intestinal pathogenesis during acute HIV-1 infection. Δ42PD1+Vδ2 cells may serve as a target for the investigation of diseases with mucosal inflammation.

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.

Discovering Protein-DNA Binding Cores by Aligned Pattern Clustering.

Understanding binding cores is of fundamental importance in deciphering Protein-DNA (TF-TFBS) binding and gene regulation. Limited by expensive experiments, it is promising to discover them with variations directly from sequence data. Although existing computational methods have produced satisfactory results, they are one-to-one mappings with no site-specific information on residue/nucleotide variations, where these variations in binding cores may impact binding specificity. This study presents a new representation for modeling binding cores by incorporating variations and an algorithm to discover them from only sequence data. Our algorithm takes protein and DNA sequences from TRANSFAC (a Protein-DNA Binding Database) as input; discovers from both sets of sequences conserved regions in Aligned Pattern Clusters (APCs); associates them as Protein-DNA Co-Occurring APCs; ranks the Protein-DNA Co-Occurring APCs according to their co-occurrence, and among the top ones, finds three-dimensional structures to support each binding core candidate. If successful, candidates are verified as binding cores. Otherwise, homology modeling is applied to their close matches in PDB to attain new chemically feasible binding cores. Our algorithm obtains binding cores with higher precision and much faster runtime ( ≥ 1,600x) than that of its contemporaries, discovering candidates that do not co-occur as one-to-one associated patterns in the raw data. AVAILABILITY: http://www.pami.uwaterloo.ca/~ealee/files/tcbbPnDna2015/Release.zip.

A highly versatile platform based on geometrically well-defined 3D DNA nanostructures for selective recognition and positioning of multiplex targets.

We develop a versatile recognition system based on 3D triangular-shaped DNA nanotubes by integrating three different aptamer sequences along the three edges. This would allow multiple binding activities to be combined into a single system. The versatility of this nanotube platform can also provide a framework for spatial orientation and positioning of different aptamer-binding ligands in a 'pea-pod' architecture.

Novel Mutations L228I and Y232H Cause Nonnucleoside Reverse Transcriptase Inhibitor Resistance in Combinational Pattern.

The emergence of drug resistance mutations is increasing after the implementation of highly active antiretroviral therapy. To characterize two novel mutations L228I and Y232H in the primer grip of reverse transcriptase (RT) of HIV-1 circulating recombination form 08_BC (CRF08_BC) subtype, both mutant clones were constructed to determine their impacts on viral phenotypic susceptibility and replication capacity (RC). Results showed that the novel mutation, L228I, conferred a low-level resistance to etravirine by itself. L228I in combination with Y188C displayed a high level of cross-resistance to both nevirapine (NVP) and efavirenz (EFV). The copresence of A139V and Y232H induced a moderate level of resistance to NVP and EFV. Mutations Y188C/L228I, A139V, Y232H, and A139V/Y232H reduced more than 55% of viral RC compared with that of the wild-type (WT) reference virus. Modeling study suggested that the copresence of Y188C/L228I or A139V/Y232H might induce conformational changes to RT, which might result in reduced drug susceptibility and viral RC due to abolished hydrogen bonding or complex interaction with vicinal residues. Our results demonstrated that L228I and Y232H were novel accessory nonnucleoside reverse transcriptase inhibitor resistance-related mutations and provided valuable information for clinicians to design more effective treatment to patients infected with HIV-1 subtype CRF08_BC.

Prevalence and characterization of hybrid blaCTX-M among Escherichia coli isolates from livestock and other animals.

This study investigated 248 extended-spectrum ß-lactamase-producing Escherichia coli isolates from 2012 to 2013 for hybrid blaCTX-M genes. blaCTX-M genes were detected in 228 isolates of which 14 isolates were hybrid blaCTX-M positive (6 blaCTX-M-123, 6 blaCTX-M-64, and 2 blaCTX-M-132). The 14 hybrid blaCTX-M-carrying isolates (8 from chickens, 2 each from pigs and cattle, 1 each from dog and rodent) were genetically diverse. All but 2 hybrid blaCTX-M were carried on IncI1 (5 blaCTX-M-123) and IncI2 (6 blaCTX-M-64 and one blaCTX-M-132) plasmids. Our IncI1 and IncI2 plasmids had pHNAH4-1-like and pHN1122-1-like restriction fragment length polymorphism patterns, respectively. Genetic relatedness of the plasmids to pHNAH4-1 and pHN1122-1 were confirmed by complete sequencing of 3 plasmids, pCTXM123_C0996, pCTXM64_C0967, and pCTXM132_P0421. Plasmids closely related to pHNAH4-1 and pHN1122-1 and carrying different blaCTX-M alleles have been reported from multiple geographic areas in China previously. The findings highlighted the wide dissemination of hybrid blaCTX-M variants in different parts of China.

A novel mutation, D404N, in the connection subdomain of reverse transcriptase of HIV-1 CRF08_BC subtype confers cross-resistance to NNRTIs.

OBJECTIVES: Growing evidence suggests that mutations in the connection domain of the HIV-1 reverse transcriptase (RT) can contribute to viral resistance to RT inhibitors. This work was designed to determine the effects of a novel mutation, D404N, in the connection subdomain of RT of HIV-1 CRF08_BC subtype on drug resistance, viral replication capacity (RC) and RT activity. METHODS: Mutation D404N, alone or together with the other reported mutations, was introduced into an HIV-1 CRF08_BC subtype infectious clone by site-directed mutagenesis. Viral susceptibility to nine RT inhibitors, viral RC and the DNA polymerase activity of viral RT of the constructed virus mutants were investigated. A modelling study using the server SWISS-MODEL was conducted to explore the possible structure-related drug resistance mechanism of the mutation D404N. RESULTS: Single mutations D404N and H221Y conferred low-level resistance to nevirapine, efavirenz, rilpivirine and zidovudine. Double mutations Y181C/D404N and Y181C/H221Y significantly reduced susceptibility to NNRTIs. The most pronounced resistance to NNRTIs was observed with the triple mutation Y181C/D404N/H221Y. Virus containing D404N as the only mutation displayed ∼50% RC compared with the WT virus. The modelling study suggested that the D404N mutation might abolish the hydrogen bonds between residues 404 and K30 in p51 or K431 in p66, leading to impaired RT subunit structure and enhanced drug resistance. CONCLUSIONS: These results indicate that D404N is a novel NNRTI-associated mutation in the HIV-1 subtype CRF08_BC and provides information valuable for the monitoring of clinical RTI resistance.