Characterization and identification of SFDC-1, a novel AmpC-type ß-lactamase in Serratia fonticola.

The clinical and environmental infections caused by AmpC ß-lactamases have been increasingly reported recently. In this study, we characterize the novel chromosome-encoded AmpC ß-lactamase SFDC-1 identified in Serratia fonticola strain R28, which was isolated from a rabbit raised on a farm in southern China. SFDC-1 shared the highest amino acid identity of 79.6% with the functionally characterized AmpC ß-lactamase gene blaYRC-1 , although it had highly homologous functionally uncharacterized relatives in the same species from different sources, including some of the clinical significance. The cloned blaSFDC-1 exhibited resistance to a broad spectrum of ß-lactam antibiotics, including most cephalosporins with the highest resistance to ampicillin, cefazolin and ceftazidime, with increased MIC levels ≥128-fold compared with the control strains. The purified SFDC-1 showed catalytic activities against ß-lactams with the highest catalytic activity to cefazolin. The genetic context of blaSFDC-1 and its relatives was conserved in the chromosome, and no mobile genetic elements were found surrounding them.

Identification and characteristics of a novel aminoglycoside phosphotransferase, APH(3')-IId, from an MDR clinical isolate of Brucella intermedia.

OBJECTIVES: To describe a novel chromosomal aminoglycoside phosphotransferase named APH(3')-IId identified in an MDR Brucella intermedia ZJ499 isolate from a cancer patient. METHODS: Species identity was determined by PCR and MALDI-TOF MS analysis. WGS was performed to determine the genetic elements conferring antimicrobial resistance. Gene cloning, transcriptional analysis and targeted gene deletion, as well as protein purification and kinetic analysis, were performed to investigate the mechanism of resistance. RESULTS: APH(3')-IId consists of 266 amino acids and shares the highest identity (48.25%) with the previously known APH(3')-IIb. Expression of aph(3')-IId in Escherichia coli decreased susceptibility to kanamycin, neomycin, paromomycin and ribostamycin. The aph(3')-IId gene in ZJ499 was transcriptionally active under laboratory conditions and the relative abundance of this transcript was unaffected by treatment with the above four antibiotics. However, deletion of aph(3')-IId in ZJ499 results in decreased MICs of these drugs. The purified APH(3')-IId showed phosphotransferase activity against kanamycin, neomycin, paromomycin and ribostamycin, with catalytic efficiencies (kcat/Km) ranging from ∼105 to 107 M-1 s-1. Genetic environment and comparative genomic analyses suggested that aph(3')-IId is probably a ubiquitous gene in Brucella, with no mobile genetic elements detected in its surrounding region. CONCLUSIONS: APH(3')-IId is a novel chromosomal aminoglycoside phosphotransferase and plays an important role in the resistance of B. intermedia ZJ499 to kanamycin, neomycin, paromomycin and ribostamycin. To the best of our knowledge, APH(3')-IId represents the fourth characterized example of an APH(3')-II enzyme.

Prevalence of Aminoglycoside Resistance Genes and Molecular Characterization of a Novel Gene, aac(3)-IIg, among Clinical Isolates of the Enterobacter cloacae Complex from a Chinese Teaching Hospital.

Members of the Enterobacter cloacae complex are important opportunistic human pathogens capable of causing a wide variety of infections. During recent decades, aminoglycoside-resistant E. cloacae complex isolates have increasingly been reported and have become a major concern. Here, we employed high-throughput sequencing in combination with specific PCR assays to investigate the prevalence of aminoglycoside resistance genes among 170 isolates of the E. cloacae complex collected from a teaching hospital in Wenzhou, China. A total of 12 known genes [aphA-1, strA, strB, aac(6')-IIc, aadA2, aac(3)-IId, aadB, aadA1, rmtB, armA, aadA5, and aac(6')-Ie-aph(2'')-Ia] and 1 novel gene [aac(3)-IIg] were identified, with aphA-1 (71.18%), strA (55.29%), and strB (52.35%) being the most prevalent, and aac(3)-IIg was detected with a positive rate of 21.76% (37/170). The aac(3)-IIg gene was 810 bp in length and encoded a protein that shared 72 to 78% identities with previously known AAC(3)-II aminoglycoside 3-N-acetyltransferases. The MICs of gentamicin and tobramycin were 512 µg/ml and 64 µg/ml, respectively, when aac(3)-IIg was cloned into Escherichia coli DH5α. All aac(3)-IIg-positive isolates exerted broad aminoglycoside resistance profiles, mediated by the coexistence of multiple resistance genes. Moreover, aminoglycoside resistance and resistance genes were found to be transferable in most strains (24/37). Nevertheless, pulsed-field gel electrophoresis (PFGE) and dendrogram analysis showed clonal diversity among these isolates. S1 nuclease PFGE, Southern hybridization, and whole-genome sequencing indicated that aac(3)-IIg was located on transferable as well as nontransferable plasmids of various sizes. The analysis of the genetic environment suggested that aac(3)-IIg is embedded within a class 1 integron, with IS26 playing an important role in its mobility.

The Structure of ampG Gene in Pseudomonas aeruginosa and Its Effect on Drug Resistance.

In order to study the relationship between the structure and function of AmpG, structure, site-specific mutation, and gene complementary experiments have been performed against the clinical isolates of Pseudomonas aeruginosa. We found that there are 51 nucleotide variations at 34 loci over the ampG genes from 24 of 35 P. aeruginosa strains detected, of which 7 nucleotide variations resulted in amino acid change. The ampG variants with the changed nucleotides (amino acids) could complement the function of ampG deleted PA01 (PA01ΔG). The ampicillin minimum inhibitory concentration (MIC) of PA01ΔG complemented with 32 ampG variants was up to 512 µg/ml, similar to the original PA01 (P. aeruginosa PA01). Furthermore, site-directed mutation of two conservative amino acids (I53 and W90) showed that when I53 was mutated to 53S or 53T (I53S or I53T), the ampicillin MIC level dropped drastically, and the activity of AmpC ß-lactamase decreased as well. By contrast, the ampicillin MIC and the activity of AmpC ß-lactamase remained unchanged for W90R and W90S mutants. Our studies demonstrated that although nucleotide variations occurred in most of the ampG genes, the structure of AmpG protein in clinical isolates is stable, and conservative amino acid is necessary to maintain normal function of AmpG.

Plasma and urine proteomics and gut microbiota analysis reveal potential factors affecting COVID-19 vaccination response.

The efficacy of COVID-19 vaccination relies on the induction of neutralizing antibodies, which can vary among vaccine recipients. In this study, we investigated the potential factors affecting the neutralizing antibody response by combining plasma and urine proteomics and gut microbiota analysis. We found that activation of the LXR/FXR pathway in plasma was associated with the production of ACE2-RBD-inhibiting antibodies, while urine proteins related to complement system, acute phase response signaling, LXR/FXR, and STAT3 pathways were correlated with neutralizing antibody production. Moreover, we observed a correlation between the gut microbiota and plasma and urine proteins, as well as the vaccination response. Based on the above data, we built a predictive model for vaccination response (AUC = 0.85). Our study provides insights into characteristic plasma and urine proteins and gut microbiota associated with the ACE2-RBD-inhibiting antibodies, which could benefit our understanding of the host response to COVID-19 vaccination.

Identification of differentially expressed proteins of Arthrospira (Spirulina) plantensis-YZ under salt-stress conditions by proteomics and qRT-PCR analysis.

Arthrospira (Spirulina) platensis as a representative species of cyanobacteria has been recognized and used worldwide as a source of protein in the food, which possesses some unusual and valuable physiological characteristics, such as alkali and salt tolerance. Based on complete genome sequencing of Arthrospira (Spirulina) plantensis-YZ, we compared the protein expression profiles of this organism under different salt-stress conditions (i.e. 0.02 M, 0.5 M and 1.0 M NaCl, respectively), using 2-D electrophoresis and peptide mass fingerprinting, and retrieved 141 proteins showing significantly differential expression in response to salt-stress. Of the 141 proteins, 114 Arthrospira (Spirulina) plantensis-YZ proteins were found with significant homology to those found in Arthrospira (76 proteins in Arthrospira platensis str. Paraca and 38 in Arthrospira maxima CS-328). The remaining 27 proteins belong to other bacteria. Subsequently, we determined the transcriptional level of 29 genes in vivo in response to NaCl treatments and verified them by qRT-PCR. We found that 12 genes keep consistency at both transcription and protein levels, and transcription of all of them but one were up-regulated. We classified the 141 differentially expressed proteins into 18 types of function categories using COG database, and linked them to their respective KEGG metabolism pathways. These proteins are involved in 31 metabolism pathways, such as photosynthesis, glucose metabolism, cysteine and methionine metabolism, lysine synthesis, fatty acid metabolism, glutathione metabolism. Additionally, the SRPs, heat shock protein and ABC transporter proteins were identified, which probably render Arthrospira (Spirulina) plantensis's resistance against high salt stress.

[Multidrug resistance of enteric bacilli and its relation to structure and molecular evolution of variable region in resistance-related class-I integron].

OBJECTIVE: To investigate the drug resistance of enteric bacilli and its relation to the drug resistance gene cassette in the variable region and molecular evolution of class-I integron. METHODS: K-B assay was applied to measure the drug resistance of E.coli, E.cloacae and A.baumannii isolated against twelve antibiotics. The class-I integron and drug resistance gene cassettes in the variable region of the integron were detected by PCR and sequencing of amplification products. The molecular evolution of drug resistance genes in the class-I integrons was analyzed using Clustal X and MEGA software. RESULTS: 54.2%-100% of A.baumannii isolates were resistant to the penicillin and cephem antibiotics, while E.coli and E.cloacae isolates had resistance rates of 41.6%-62.5% to cephem antibiotics. 62.5%(15/24) of E.coli, 67.9%(19/28) of E.cloacae and 83.3%(20/24) of A.baumannii isolates were positive for class-I integrons. 81.5% (44/54) of class-I integrons showed 4 different single band spectrums and the other class-I integrons displayed 3 different double band spectrums. In the drug resistance gene cassettes in variable regions of class-I integrons there were 7 types in 4 groups of drug resistance genes, including aac(6'), sad(3"), aad(2"), cat(4') and dfr (types 7, A13 and 15), which induced the resistance to aminoglycosides and sulfamido antibiotics and chloromycin. The class-I integrons in the isolates might be divided into 4 molecular evolution groups according to the diversity of dihydrofolate reductase encoding gene sequences. CONCLUSION: The enteric bacilli have a high drug resistance and frequently carry class-I integrons with 7 drug resistance gene cassettes which present 4 different evolutionary pathways.

Identification and characterization of a novel aminoglycoside O-nucleotidyltransferase ANT(6)-If from Paenibacillus thiaminolyticus PATH554.

Background: Paenibacillus thiaminolyticus, a species of genus Paenibacillus of the family Paenibacillaceae, exists widely in environments and habitats in various plants and worms, and occasionally causes human infections. This work aimed to characterize the function of a novel aminoglycoside O-nucleotidyltransferase resistance gene, designated ant(6)-If, from a P. thiaminolyticus strain PATH554. Methods: Molecular cloning, antimicrobial susceptibility testing, enzyme expression and purification, and kinetic analysis were used to validate the function of the novel gene. Whole-genome sequencing and comparative genomic analysis were performed to investigate the phylogenetic relationship of ANT(6)-If and other aminoglycoside O-nucleotidyltransferases, and the synteny of ant(6)-If related sequences. Results: The recombinant with the cloned ant(6)-If gene (pMD19-ant(6)-If/DH5α) demonstrated a 128-fold increase of minimum inhibitory concentration level against streptomycin, compared with the control strains (DH5α and pMD19/DH5α). The kinetic parameter kcat/Km of ANT(6)-If for streptomycin was 9.01 × 103 M-1·s-1. Among the function-characterized resistance genes, ANT(6)-If shared the highest amino acid sequence identity of 75.35% with AadK. The ant(6)-If gene was located within a relatively conserved genomic region in the chromosome. Conclusion: ant(6)-If conferred resistance to streptomycin. The study of a novel resistance gene in an unusual environmental bacterium in this work contributed to elucidating the resistance mechanisms in the microorganisms.

ANT(9)-Ic, a Novel Chromosomally Encoded Aminoglycoside Nucleotidyltransferase from Brucella intermedia.

Aminoglycoside-modifying enzymes are among the most important mechanisms of resistance to aminoglycoside antibiotics, typically conferring high-level resistance by enzymatic drug inactivation. Previously, we isolated a multidrug-resistant Brucella intermedia strain ZJ499 from a cancer patient, and whole-genome sequencing revealed several putative novel aminoglycoside-modifying enzyme genes in this strain. Here, we report the characterization of one of them that encodes an intrinsic, chromosomal aminoglycoside nucleotidyltransferase designated ANT(9)-Ic, which shares only 33.05% to 47.44% amino acid identity with the most closely related ANT(9)-I enzymes. When expressed in Escherichia coli, ANT(9)-Ic conferred resistance only to spectinomycin and not to any other aminoglycosides tested, indicating a substrate profile typical of ANT(9)-I enzymes. Consistent with this, deletion of ant(9)-Ic in ZJ499 resulted in a specific and significant decrease in MIC of spectinomycin. Furthermore, the purified ANT(9)-Ic protein showed stringent substrate specificity for spectinomycin with a Km value of 44.83 µM and a kcat/Km of 2.8 × 104 M-1 s-1, echoing the above observations of susceptibility testing. In addition, comparative genomic analysis revealed that the genetic context of ant(9)-Ic was conserved in Brucella, with no mobile genetic elements found within its 20-kb surrounding region. Overall, our results demonstrate that ANT(9)-Ic is a novel member of the ANT(9)-I lineage, contributing to the intrinsic spectinomycin resistance of ZJ499. IMPORTANCE The emergence, evolution, and worldwide spread of antibiotic resistance present a significant global public health crisis. For aminoglycoside antibiotics, enzymatic drug modification is the most common mechanism of resistance. We identify a novel chromosomal aminoglycoside nucleotidyltransferase from B. intermedia, called ANT(9)-Ic, which shares the highest identity (47.44%) with the previously known ANT(9)-Ia and plays an important role in spectinomycin resistance of the host strain. Analysis of the genetic environment and origin of ant(9)-Ic shows that the gene and its surrounding region are widely conserved in Brucella, and no mobile elements are detected, indicating that ANT(9)-Ic may be broadly important in the natural resistance to spectinomycin of Brucella species.

Class 1 integrons and multiple mobile genetic elements in clinical isolates of the Klebsiella pneumoniae complex from a tertiary hospital in eastern China.

Background: The emergence of highly drug-resistant K. pneumoniae, has become a major public health challenge. In this work, we aim to investigate the diversity of species and sequence types (STs) of clinical Klebsiella isolates and to characterize the prevalence and structure of class 1 integrons. Methods: Based on the whole genome sequencing, species identification was performed by 16S rRNA gene homology and average nucleotide identity (ANI) analysis. STs were determined in accordance with the international MLST schemes for K. pneumoniae and K. variicola. Integron characterization and comparative genomic analysis were performed using various bioinformatic tools. Results: Species identification showed that the 167 isolates belonged to four species: K. pneumoniae, K. variicola subsp. variicola, K. quasipneumoniae and K. aerogenes. Thirty-six known and 5 novel STs were identified in K. pneumoniae, and 10 novel STs were identified in K. variicola subsp. variicola. Class 1 integrons were found in 57.49% (96/167) of the isolates, and a total of 169 resistance gene cassettes encoding 19 types of resistance genes, including carbapenem resistance gene (bla IPM-4) and class D ß-lactamases gene (bla OXA-1 and bla OXA-10), were identified. Among the 17 complete genomes, 29 class 1 integrons from 12 groups were found, only 1 group was encoded on chromosomes. Interestingly, one plasmid (pKP167-261) carrying two copies of approximately 19-kb IS26-Int1 complex resistance region that contains an integron and a multidrug resistance gene fragment. Conclusion: The results of this work demonstrated that the species and STs of the clinical Klebsiella isolates were more complex by the whole genome sequence analysis than by the traditional laboratory methods. Finding of the new structure of MGEs related to the resistance genes indicates the great importance of deeply exploring the molecular mechanisms of bacterial multidrug resistance.

Identification and characterization of a novel chromosomal aminoglycoside 3'-O-phosphotransferase, APH(3')-Id, from Kluyvera intermedia DW18 isolated from the sewage of an animal farm.

Background: Aminoglycosides, as important clinical antimicrobials, are used as second-line drugs for treating multidrug-resistant tuberculosis or combined with ß-lactam drugs for treating severe infections such as sepsis. Aminoglycoside-modifying enzyme (AME) is the most important mechanism of aminoglycoside resistance and deserves more attention. Methods: The bacterium Kluyvera intermedia DW18 was isolated from the sewage of an animal farm using the conventional method. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of antimicrobials. A novel resistance gene was cloned, and the enzyme was expressed. The kinetic parameters were measured by a SpectraMax M5 multifunctional microplate reader. Bioinformatic analysis was performed to reveal the genetic context of the aph(3')-Id gene and its phylogenetic relationship with other AMEs. Results: A novel aminoglycoside 3'-O-phosphotransferase gene designated aph(3')-Id was identified in K. intermedia DW18 and shared the highest amino acid identity of 77.49% with the functionally characterized aminoglycoside 3'-O-phosphotransferase APH(3')-Ia. The recombinant plasmid carrying the novel resistance gene (pMD19-aph(3')-Id/E. coli DH5α) showed 1,024-, 512-, 128- and 16-fold increased MIC levels for kanamycin, ribostamycin, paromomycin and neomycin, respectively, compared with the reference strain DH5α. APH(3')-Id showed the highest catalytic efficiency for ribostamycin [kcat/Km of (4.96 ± 1.63) × 105 M-1/s-1], followed by paromomycin [kcat/Km of (2.18 ± 0.21) × 105 M-1/s-1], neomycin [kcat/Km of (1.73 ± 0.20) × 105 M-1/s-1], and kanamycin [kcat/Km of (1.10 ± 0.18) × 105 M-1/s-1]. Three conserved functional domains of the aminoglycoside phosphotransferase family and ten amino acid residues responsible for the phosphorylation of kanamycin were found in the amino acid sequence of APH(3')-Id. No mobile genetic element (MGE) was discovered surrounding the aph(3')-Id gene. Conclusion: In this work, a novel aminoglycoside 3'-O-phosphotransferase gene designated aph(3')-Id encoded in the chromosome of the environmental isolate Kluyvera intermedia DW18 was identified and characterized. These findings will help clinicians select effective antimicrobials to treat infections caused by pathogens with this kind of resistance gene.

BlaPSZ-1, a novel AmpC gene identified from a Pantoea isolate.

Background: Pantoea species of the family Erwiniaceae are well-known plant pathogens and animal and human conditional pathogens. Due to the widespread and continuous use of antimicrobials, multidrug-resistant strains continue to emerge, making clinical treatment difficult; therefore, there is an increasing need to clarify the mechanisms of drug resistance. Methods: A rabbit anal fecal sample was collected by a swab and the streak plate method was used to isolate single colonies. The standard agar dilution method was used to determine the minimum inhibitory concentrations (MICs) against antimicrobials. The complete genome sequence of the bacterium was obtained using Next-Generation Sequencing platforms. The potential resistance gene was annotated based on the Comprehensive Antibiotic Resistance Database (CARD) and verified by molecular cloning. The ß-lactamase PSZ-1 was expressed via the pCold I expression vector and its enzyme kinetic parameters were analyzed. The genetic environment and evolutionary process of the novel resistance gene-related sequences were analyzed by bioinformatic methods. Results: The isolate Pantoea endophytica X85 showed some degree of resistance to penicillins as well as cephalosporins. A novel AmpC resistance gene, designated blaPSZ-1 in this research, was identified to be encoded in the plasmid (pPEX85) of P. endophytica X85. BlaPSZ-1 showed resistance to penicillins and several first-, second-and third-generation cephalosporins as well as aztreonam, but it did not show resistance to the fourth-generation cephalosporins or carbapenems tested. Enzyme kinetic assays revealed that it could hydrolyze amoxicillin, penicillin G, cephalothin, and cefazolin, and its hydrolytic activity could be strongly inhibited by the inhibitor avibactam, which was generally consistent with antimicrobial susceptibility testing results. No hydrolytic activity was observed for third-generation cephalosporins or aztreonam. Conclusion: In this study, a novel AmpC ß-lactamase gene, designated blaPSZ-1, was characterized and it was encoded in the plasmid of the bacterium P. endophytica X85. It shows resistance to penicillins and several cephalosporins. The discovery of novel drug resistance mechanisms can help guide the scientific use of drugs in animal husbandry and clinical practice, effectively avoiding the abuse of antimicrobials and thus preventing the further development and spread of bacterial resistance.

Identification and characterization of a novel ß-lactamase gene, blaAMZ-1, from Achromobacter mucicolens.

Background: Achromobacter is a genus of gram-negative bacteria that can act as opportunistic pathogens. Recent studies have revealed that some species of Achromobacter show inherent resistance to ß-lactams, but the resistance mechanisms of Achromobacter mucicolens have rarely been reported. Method: The bacterium was isolated using standard laboratory procedures. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs). Genome sequencing was performed using the PacBio RS II and Illumina HiSeq 2500 platforms, and the Comprehensive Antibiotic Resistance Database (CARD) was used to annotate the drug resistance genes. The localization of the novel ß-lactamase AMZ-1 was determined, and its characteristics were determined via molecular cloning and enzyme kinetic analysis. The phylogenetic relationship and comparative genomic analysis of the resistance gene-related sequences were also analyzed. Result: Achromobacter mucicolens Y3, isolated from a goose on a farm in Wenzhou, showed resistance to multiple antibiotics, including penicillins and cephalosporins. BlaAMZ-1 showed resistance to amoxicillin, penicillin G, ampicillin, cephalothin and cefoxitin, and the resistance activity could be inhibited by ß-lactamase inhibitors. Enzyme kinetic analysis results showed that AMZ-1 has hydrolytic activity against a wide range of substrates, including cephalothin, amoxicillin, penicillin G, and cefoxitin but not ampicillin. The hydrolytic activity of AMZ-1 was greatly inhibited by avibactam but much more weakly inhibited by tazobactam. Mobile genetic elements could not be found around the blaAMZ-1-like genes, which are conserved on the chromosomes of bacteria of the genus Achromobacter. Conclusion: In this study, a novel AmpC gene, blaAMZ-1, from the animal-origin bacterium A. mucicolens Y3 was identified and characterized. It conferred resistance to some penicillins and first- and second-generation cephalosporins. The identification of this novel resistance gene will be beneficial for the selection of effective antimicrobials to treat associated infections.

The evolutionary mechanism of non-carbapenemase carbapenem-resistant phenotypes in Klebsiella spp.

Antibiotic resistance is driven by selection, but the degree to which a bacterial strain's evolutionary history shapes the mechanism and strength of resistance remains an open question. Here, we reconstruct the genetic and evolutionary mechanisms of carbapenem resistance in a clinical isolate of Klebsiella quasipneumoniae. A combination of short- and long-read sequencing, machine learning, and genetic and enzymatic analyses established that this carbapenem-resistant strain carries no carbapenemase-encoding genes. Genetic reconstruction of the resistance phenotype confirmed that two distinct genetic loci are necessary in order for the strain to acquire carbapenem resistance. Experimental evolution of the carbapenem-resistant strains in growth conditions without the antibiotic revealed that both loci confer a significant cost and are readily lost by de novo mutations resulting in the rapid evolution of a carbapenem-sensitive phenotype. To explain how carbapenem resistance evolves via multiple, low-fitness single-locus intermediates, we hypothesised that one of these loci had previously conferred adaptation to another antibiotic. Fitness assays in a range of drug concentrations show how selection in the antibiotic ceftazidime can select for one gene (blaDHA-1) potentiating the evolution of carbapenem resistance by a single mutation in a second gene (ompK36). These results show how a patient's treatment history might shape the evolution of antibiotic resistance and could explain the genetic basis of carbapenem-resistance found in many enteric-pathogens.

Identification and characterization of a novel 6'-N-aminoglycoside acetyltransferase AAC(6')-Va from a clinical isolate of Aeromonas hydrophila.

Background: Aeromonas species have been identified as agents responsible for various diseases in both humans and animals. Multidrug-resistant Aeromonas strains pose a significant public health threat due to their emergence and spread in clinical settings and the environment. The aim of this study was to determine a novel resistance mechanism against aminoglycoside antimicrobials in a clinical isolate. Methods: The function of aac(6')-Va was verified by gene cloning and antibiotic susceptibility tests. To explore the in vivo activity of the enzyme, recombinant proteins were expressed, and enzyme kinetics were tested. To determine the molecular background and mechanism of aac(6')-Va, whole-genome sequencing and bioinformatic analysis were performed. Results: The novel aminoglycoside N-acetyltransferase gene aac(6')-Va confers resistance to several aminoglycosides. Among the antimicrobials tested, ribostamycin showed the highest increase (128-fold) in the minimum inhibitory concentration (MIC) compared with the control strains. According to the MIC results of the cloned aac(6')-Va, AAC(6')-Va also showed the highest catalytic efficiency for ribostamycin [kcat/Km ratio = (3.35 ± 0.17) × 104 M-1 s-1]. Sharing the highest amino acid identity of 54.68% with AAC(6')-VaIc, the novel aminoglycoside N-acetyltransferase constituted a new branch of the AAC(6') family due to its different resistance profiles. The gene context of aac(6')-Va and its close relatives was conserved in the genomes of species of the genus Aeromonas. Conclusion: The novel resistance gene aac(6')-Va confers resistance to several aminoglycosides, especially ribostamycin. Our finding of a novel resistance gene in clinical A. hydrophila will help us develop more effective treatments for this pathogen's infections.

Codon usage patterns and adaptive evolution of marine unicellular cyanobacteria Synechococcus and Prochlorococcus.

Marine unicellular cyanobacteria, represented by Synechococcus and Prochlorococcus, dominate the total phytoplankton biomass and production in oligotrophic ocean. In this study, we employed comparative genomics approaches to extensively investigate synonymous codon usage bias and evolutionary rates in a large number of closely related species of marine unicellular cyanobacteria. Although these two groups of marine cyanobacteria have a close phylogenetic relationship, we find that they are highly divergent not only in codon usage patterns but also in the driving forces behind the diversification. It is revealed that in Prochlorococcus, mutation and genome compositional constraints are the main forces contributing to codon usage bias, whereas in Synechococcus, translational selection. In addition, nucleotide substitution rate analysis indicates that they are not evolving at a constant rate after the divergence and that the average d(N)/d(S) values of core genes in Synechococcus are significantly higher than those in Prochlorococcus. Our evolutionary genomic analysis provides the first insight into codon usage, evolutionary genetic mechanisms and environmental adaptation of Synechococcus and Prochlorococcus after divergence.

MagicViewer: integrated solution for next-generation sequencing data visualization and genetic variation detection and annotation.

New sequencing technologies, such as Roche 454, ABI SOLiD and Illumina, have been increasingly developed at an astounding pace with the advantages of high throughput, reduced time and cost. To satisfy the impending need for deciphering the large-scale data generated from next-generation sequencing, an integrated software MagicViewer is developed to easily visualize short read mapping, identify and annotate genetic variation based on the reference genome. MagicViewer provides a user-friendly environment in which large-scale short reads can be displayed in a zoomable interface under user-defined color scheme through an operating system-independent manner. Meanwhile, it also holds a versatile computational pipeline for genetic variation detection, filtration, annotation and visualization, providing details of search option, functional classification, subset selection, sequence association and primer design. In conclusion, MagicViewer is a sophisticated assembly visualization and genetic variation annotation tool for next-generation sequencing data, which can be widely used in a variety of sequencing-based researches, including genome re-sequencing and transcriptome studies. MagicViewer is freely available at http://bioinformatics.zj.cn/magicviewer/.

[Impact of CACNA1C polymorphisms on antihypertensive efficacy of calcium channel blocker].

OBJECTIVE: To explore the relationship between genetic polymorphisms of CACNA1C that encoded the a1c subunit of the L-type calcium channel and the efficacy of calcium channel blocker (CCB, Nifedipine extended release tablet/20 mg/d) in essential hypertension (EH) patients of Han Chinese in Wenzhou. METHODS: For the enrolled 103 EH patients, Multiplex Polymerase Chain Reaction (Multi-PCR) and matrix assisted laser desorption ionization time of flight MS (MLDI-TOF MS) were performed to detect their genotypes (rs216008, rs1051375, rs2299661, rs10848683, rs215976), blood pressure (BP) after CCB monotherapy was compared among patients with different genotypes. RESULTS: (1) Blood pressure was significantly reduced in all patients post CCB (P < 0.05 vs. pre-CCB). (2) Diastolic blood pressure reduction was more significant in subjects with rs2299661 C/C genotype (wild genotype) than in subjects with rs2299661C/G and rs2299661G/G genotype (mutational genotype) [(12.46 ± 7.91) mm Hg (1 mm Hg = 0.133 kPa) vs. (7.22 ± 8.01) mm Hg and (5.93 ± 9.77) mm Hg, P < 0.05]. (3) Systolic blood pressure reduction was more significant in subjects with rs216008 C/C genotype (wild genotype) than in subjects with rs216008 C/T genotype (mutational genotype) [(20.60 ± 12.35) mm Hg vs. (13.62 ± 10.21) mm Hg, P < 0.05]. (4) Blood pressure reduction was similar between subjects with genotype of rs1051375, rs10848683 and rs215976. CONCLUSION: EH patients with wild genotype of rs2299661 and rs216008 in CACNA1C are more likely to be responders of CCB monotherapy.

Early predictors of lung necrosis severity in children with community-acquired necrotizing pneumonia.

OBJECTIVE: To analyze baseline clinical and laboratory characteristics and explore the possible predictors of lung necrosis severity in children with community-acquired necrotizing pneumonia (NP). METHODOLOGY: This retrospective observational study was performed in a tertiary referral center. A total of 104 patients aged <15 years with community-acquired pneumonia and radiologically confirmed NP by computed tomography (CT) were included. Patients were classified into the mild, moderate, or massive necrosis groups. RESULTS: Among them, 29, 41, and 34 patients had mild, moderate, and massive necrosis, respectively. Moreover, 34.6% of the patients were admitted to pediatric intensive care unit. Massive necrosis was more likely to occur during winter (p < 0.05) and was associated with more severe clinical outcomes, such as longer duration of fever, longer hospitalization, increased mortality, and a higher risk of subsequent surgical intervention (p < 0.05). Multivariate analysis demonstrated that the following were independent risk factors for massive necrosis in this study: C-reactive protein (CRP) (p = 0.036), serum albumin (p = 0.009), and immunoglobulin M (IgM) (p = 0.022). Receiver operating characteristic analysis showed that when the cut-off value for CRP, serum albumin, and IgM were set at 122 mg/L, 30.8 g/L, and 95.7 mg/dl, respectively, they showed good diagnostic performance for differentiating patients with massive necrosis from all patients with NP. CONCLUSION: NP is a potentially severe complication of pediatric community-acquired pneumonia. Different severities of lung necrosis can lead to different clinical outcomes. CRP, serum albumin, and IgM levels are independent predictors of the degree of lung necrosis.

Helicobacter pylori and unignorable extragastric diseases: Mechanism and implications.

Considered as the most popular pathogen worldwide, Helicobacter pylori is intensively associated with diverse gastric diseases, including gastric ulcers, chronic progressive gastritis, and gastric cancer. Aside from its pathogenic effect on gastric diseases, growing evidences reveal that H. pylori may be related to numerous extragastric diseases. In this article, we reviewed recent studies and systematically elucidated that H. pylori may interfere with many biological processes outside the stomach and influence the occurrence of various extragastric diseases. Many epidemiological studies have indicated that H. pylori plays a pathogenic role in COVID-19, atherosclerosis, hyperemesis gravidarum and several other extragastric diseases, while the effect of H. pylori is currently under investigation in gastroesophageal reflux disease, asthma, and inflammatory bowel disease. Moreover, we also summarized the possible pathogenic mechanisms of H. pylori that may be related to chronic systemic inflammation and molecular mimicker. Taken together, this review provides a new perspective on the role of H. pylori in extragastric diseases and explores the possible mechanisms, which may help guide clinical treatment.