Search

In 2019, a dengue outbreak occurred with 290 confirmed cases in Wenzhou, a coastal city in southeast China. To identify the origin of the dengue virus (DENV) from this outbreak, viral RNA was extracted from four serum samples and sequenced for whole genome analysis. Then, phylogenetic analysis, gene mutation, secondary structure prediction, selection pressure analysis, and recombination analysis were performed. DENV strains Cam-03 and Cam-11 were isolated from patients traveling from Cambodia, while ZJWZ-18 and ZJWZ-62 strains were isolated from local patients without a record of traveling abroad. The whole genome sequence of all four strains was 10,735 nucleotides long. Phylogenetic tree analysis showed that the four strains belonged to genotype 1 of DENV-1, but the local Wenzhou strains and imported strains clustered in different branches. ZJWZ-18 and ZJWZ-62 were closely related to strain MF033254-Singapore-2016, and Cam-03 and Cam-11 were closely related to strain AB608788-China : Taiwan-1994. A comparison of the coding regions between the local strains and the DENV-1 standard strain (EU848545-Hawaii-1944) showed 82 amino acid mutations between the two strains. A total of 55 amino acid mutations were found between the coding regions of the local and imported strains. The overall secondary structure of the 3′ UTR of the local strains had changed: apparent changes in the head and tail position were observed when compared to DENV-1 standard strain. Furthermore, selection pressure analysis and recombination detection using the 4 isolates and 41 reference strains showed two credible positive selection sites and eight credible recombination events, which warrant further studies. This study may enhance the understanding of viral replication, infection, evolution, virulence, and pathogenicity of DENV. [ABSTRACT FROM AUTHOR]

Summary: The clinical and environmental infections caused by AmpC β‐lactamases have been increasingly reported recently. In this study, we characterize the novel chromosome‐encoded AmpC β‐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 β‐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 β‐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 β‐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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Coagulase-negative Staphylococcus warneri is an opportunistic pathogen that is capable of causing several infections, especially in patients with indwelling medical devices. Here, we determined the complete genome sequence of a clinical S. warneri strain isolated from the blood culture of a 1-year-old nursling patient with acute upper respiratory infection. Genome-wide phylogenetic analysis confirmed the phylogenetic relationships between S. warneri and other Staphylococcus species. Using comparative genomics, we identified three cell wall-anchored (CWA) proteins at the same locus (sdr), named SdrJ, SdrK, and SdrL, on the chromosome sequences of different S. warneri strains. Structural predictions showed that SdrJ/K/L have structural features characteristic of Sdr proteins but exceptionally contained an unusual N-terminal repeat region. However, the C-terminal repetitive (R) region of SdrJ contains a significantly larger proportion of alanine (142/338, 42.01%) than the previously reported SdrI (37.00%). Investigation of the genetic organization revealed that the sdrJ/K/L genes were always followed by one or two glycosyltransferase genes, gtfA and gtfB and were present in an ∼56 kb region bordered by a pair of 8 bp identical direct repeats, named Sw-Sdr. This region was further found to be located on a 160-kb region subtended by a pair of 160-bp direct repeats along with other virulence genes and resistance genes. Sw-Sdr contained a putative integrase that was probably a remnant of a functional integrase. Evidence suggests that Sw-Sdr is improbably an efficient pathogenicity island. A large-scale investigation of Staphylococcus genomes showed that sdr loci were a potential hotspot of insertion sequences (ISs), which could lead to intraspecific diversity at these loci. Our work expanded the repository of Staphylococcus Sdr proteins, and for the first time, we established the connection between sdr loci and phylogenetic relationships and compared the sdr loci in different Staphylococcus species, which provided large insights into the genetic environment of CWA genes in Staphylococcus. [ABSTRACT FROM AUTHOR]

Aminoglycosides are important options for treating life-threatening infections. However, high levels of aminoglycoside resistance (HLAR) among Klebsiella pneumoniae isolates have been observed to be increasing frequently. In this study, a total of 292 isolates of the K. pneumoniae complex from a teaching hospital in China were analyzed. Among these isolates, the percentage of HLAR strains was 13.7% (40/292), and 15 aminoglycoside resistance genes were identified among the HLAR strains, with rmtB being the most dominant resistance gene (70%, 28/40). We also described an armA -carrying Klebsiella variicola strain KP2757 that exhibited a high-level resistance to all aminoglycosides tested. Whole-genome sequencing of KP2757 demonstrated that the strain contained one chromosome and three plasmids, with all the aminoglycoside resistance genes (including two copies of armA and six AME genes) being located on a conjugative plasmid, p2757-346, belonging to type IncHI5. Comparative genomic analysis of eight IncHI5 plasmids showed that six of them carried two copies of the intact armA gene in the complete or truncated Tn 1548 transposon. To the best of our knowledge, for the first time, we observed that two copies of armA together with six AME genes coexisted on the same plasmid in a strain of K. variicola with HLAR. Comparative genomic analysis of eight armA -carrying IncHI5 plasmids isolated from humans and sediment was performed, suggesting the potential for dissemination of these plasmids among bacteria from different sources. These results demonstrated the necessity of monitoring the prevalence of IncHI5 plasmids to restrict their worldwide dissemination. [ABSTRACT FROM AUTHOR]

Purpose: An increasing frequency of antibiotic resistance has been observed in both clinical and environmental Aeromonas hydrophila isolates in recent years. However, there are still very few in-depth studies regarding the role of plasmids in the antibiotic resistance of A. hydrophila. Hence, we investigated the molecular and functional characterization of a multidrug-resistant plasmid encoding an NDM-like metallo-β-lactamase, AFM-1, in the clinical A. hydrophila isolate SS332. Methods: The minimum inhibitory concentrations (MICs) of 24 antibiotics against A. hydrophila SS332 were measured by the agar dilution method. The genome of A. hydrophila SS332 was sequenced with PacBio and Illumina platforms. Six plasmid-borne antimicrobial resistance genes were chosen for cloning, including blaAFM-1, blaOXA-1, msr(E), mph(E), aac(6ʹ)-Ib10, and aph(3ʹ)-Ia. Phylogenetic analysis, amino acid sequence alignment, and comparative genomic analysis were performed to elucidate the active site requirements and genetic context of the blaAFM-1 gene. Results: A. hydrophila SS332 showed high levels of resistance to 15 antibiotics, especially those with MIC levels at or above 1024 μg/mL, including ampicillin, cefazolin, ceftriaxone, aztreonam, spectinomycin, and roxithromycin. Six plasmid-borne resistance genes from A. hydrophila were verified to be functional in E. coli DH5α. AFM-1 shared 86% amino acid identity with NDM-1 and showed resistance to ampicillin, cefazolin, cefoxitin, and ceftazidime. In addition, the blaAFM-1 gene was associated with three different novel ISCR19-like elements, designated ISCR19-1, ISCR19-2 and ∆ISCR19-3, which may be involved in the acquisition and mobilization of the blaAFM-1 gene. Conclusion: Our investigation showed that plasmid-borne resistance genes can contribute to antibiotic resistance in A. hydrophila SS332. A novel blaNDM-like gene, blaAFM-1, was verified to be functional and associated with novel ISCR19-like elements. This fact indicated the risk of spread of blaAFM-1 genes and ISCR19-like elements. [ABSTRACT FROM AUTHOR]

Background: With the wide use of florfenicol to prevent and treat the bacterial infection of domestic animals, the emergence of the florfenicol resistance bacteria is increasingly serious. It is very important to elucidate the molecular mechanism of the bacteria's resistance to florfenicol. Methods: The minimum inhibitory concentration (MIC) levels were determined by the agar dilution method, and polymerase chain reaction was conducted to analyze the distribution of florfenicol resistance genes in 39 CoNS strains isolated from poultry and livestock animals and seafood. The whole genome sequence of one multidrug resistant strain, Staphylococcus lentus H29, was characterized, and comparative genomics analysis of the resistance gene-related sequences was also performed. Results: As a result, the isolates from the animals showed a higher resistance rate (23/28, 82.1%) and much higher MIC levels to florfenicol than those from seafood. Twenty-seven animal isolates carried 37 florfenicol resistance genes (including 26 fexA, 6 cfr and 5 fexB genes) with one carrying a cfr gene, 16 each harboring a fexA gene, 5 with both a fexA gene and a fexB gene and the other 5 with both a fexA gene and a cfr gene. On the other hand, all 11 isolates from seafood were sensitive to florfenicol, and only 3 carried a fexA gene each. The whole genome sequence of S. lentus H29 was composed of a chromosome and two plasmids (pH29-46, pH29-26) and harbored 11 resistance genes, including 6 genes [cfr, fexA, ant(6)-Ia, aacA-aphD, mecA and mph(C)] encoded on the chromosome, 4 genes [cfr, fexA, aacA-aphD and tcaA] on pH29-46 and 1 gene (fosD) on pH29-26. We found that the S. lentus H29 genome carried two identical copies of the gene arrays of radC-tnpABC-hp-fexA (5671 bp) and IS256-cfr (2690 bp), of which one copy of the two gene arrays was encoded on plasmid pH29-46, while the other was encoded on the chromosome. Conclusions: The current study revealed the wide distribution of florfenicol resistance genes (cfr, fexA and fexB) in animal bacteria, and to the best of our knowledge, this is the first report that one S. lentus strain carried two identical copies of florfenicol resistance-related gene arrays. [ABSTRACT FROM AUTHOR]

Background: This study was designed to characterize the dissemination mechanism and genetic context of Klebsiella pneumoniae carbapenemase (KPC) genes in carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates. Methods: A retrospective analysis was performed on CRKP strains isolated from a teaching hospital of Wenzhou Medical University during 2015– 2017. Polymerase chain reaction (PCR)-based amplification and whole-genome sequencing (WGS) were used to analyze the genetic context of the blaKPC-2 gene. Conjugation experiments were performed to evaluate the transferability of blaKPC-2-bearing plasmids. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to investigate the clonal relatedness of blaKPC-2-producing strains. Results: The blaKPC-2 gene was identified from 13.61% (40/294) of clinical K. pneumoniae isolates. Three different sequence types (ST11, ST15 and ST656) and 5 PFGE subtypes (A to E) were classified among them. ST11 was the dominant sequence type (92.50%, 37/40). Plasmid-oriented antibiotic resistance genes, such as extended spectrum-β-lactamases (ESBLs) and other antimicrobial resistance genes, were also found in KPC-positive K. pneumoniae (KPC-Kp) isolates. Mapping PCR and genomic sequencing revealed that the blaKPC-2-bearing sequence regions, which are related to different mobile elements, including Tn1721- and IS26-based transposons, were mainly located in but not restricted to IncFII-like plasmids and were structurally divergent. Conclusion: The blaKPC-2 genes related to divergent mobile genetic elements encoded on transferable plasmids may transfer widely, facilitating the spread of carbapenem resistance among bacteria with different genetic backgrounds. The dissemination of blaKPC-bearing plasmids that collectively carry additional multidrug resistance genes has caused widespread public concern, further limiting the antibiotics available to treat infections caused by KPC-producing pathogens. [ABSTRACT FROM AUTHOR]

To characterize the molecular structure of IncR plasmid-related sequences, comparative genomic analysis was conducted using 261 IncR plasmid backbone-related sequences. Among the sequences, 257 were IncR plasmids including the multidrug-resistance IncR plasmid pR50-74 from Klebsiella pneumoniae strain R50 of this work, and the other four were from bacterial chromosomes. The IncR plasmids were derived from different bacterial genera or species, mainly Klebsiella pneumoniae (70.82%, 182/257), Escherichia coli (11.28%, 29/257), Enterobacter cloacae (7.00%, 18/257), and Citrobacter freundii (3.50%, 9/257). The bacterial chromosomes carrying IncR plasmid backbone sequences were derived from Proteus mirabilis AOUC-001 and Klebsiella pneumoniae KPN1344, among others. The IncR backbone sequence of P. mirabilis AOUC-001 chromosome shows the highest identity with that of pR50-74. Complex class 1 integrons carrying various copies of ISCR1-sdr-qnrB6-△qacE/sul1 (ISCR1-linked qnrB6 unit) were identified in IncR plasmids. In addition to two consecutive copies of qnrB6-qacE-sul1, the other resistance genes encoded on pR50-74 are all related to mobile genetic elements, such as IS1006, IS26, and the class 1 integron. This study provides a clear understanding of the mobility and plasticity of the IncR plasmid backbone sequence and emphasizes the important role of ISCR in the recruitment of multicopy resistance genes. [ABSTRACT FROM AUTHOR]

It has been shown that circRNAs are involved in the development of heart diseases. However, few studies explored the role of circRNAs in acute myocardial infarction (AMI). The present study aims to investigate the role of circ_0060745 in the pathogenesis of AMI. We found that the expression of circ_0060745 was significantly increased in the myocardium of AMI mice and was mainly expressed in myocardial fibroblasts. The knockdown of circ_0060745 decreased myocardial infarct size and improved systolic cardiac functions after AMI. The knockdown of circ_0060745 in cardiac fibroblasts inhibited the migration of peritoneal macrophage, the apoptosis of cardiomyocytes and the expressions of IL‐6, IL‐12, IL‐1β, TNF‐α and NF‐κB under hypoxia. Overexpression of circ_0060745 caused an increase in infarct size and worsened cardiac functions after AMI. In summary, our findings showed that knockdown of circ_0060745 mitigates AMI by suppressing cardiomyocyte apoptosis and inflammation. These protective effects could be attributed to inhibition of NF‐κB activation. [ABSTRACT FROM AUTHOR]

Yokenella regensburgei , a member of the family Enterobacteriaceae, is usually isolated from environmental samples and generally resistant to early generations of cephalosporins. To characterize the resistance mechanism of Y. regensburgei strain W13 isolated from the sewage of an animal farm, whole genome sequencing, comparative genomics analysis and molecular cloning were performed. The results showed that a novel chromosomally encoded class C β-lactamase gene with the ability to confer resistance to β-lactam antibiotics, designated bla YOC –1, was identified in the genome of Y. regensburgei W13. Kinetic analysis revealed that the β-lactamase YOC-1 has a broad spectrum of substrates, including penicillins, cefazolin, cefoxitin and cefotaxime. The two functionally characterized β-lactamases with the highest amino acid identities to YOC-1 were CDA-1 (71.69%) and CMY-2 (70.65%). The genetic context of the bla YOC –1 -ampR -encoding region was unique compared with the sequences in the NCBI nucleotide database. The plasmid pRYW13-125 of Y. regensburgei W13 harbored 11 resistance genes (bla OXA –10, bla LAP –2, dfrA14 , tetA , tetR , cmlA5 , floR , sul2 , ant(3″)-IIa , arr-2 and qnrS1) within an ∼34 kb multidrug resistance region; these genes were all related to mobile genetic elements. The multidrug resistance region of pYRW13-125 shared the highest identities with those of two plasmids from clinical Klebsiella pneumoniae isolates, indicating the possibility of horizontal transfer of these resistance genes between bacteria of various origins. [ABSTRACT FROM AUTHOR]

Bacterial resistance to antibiotics has become an important concern for public health. This study was aimed to investigate the characteristics and the distribution of the florfenicol-related resistance genes in bacteria isolated from four farms. A total of 106 florfenicol-resistant Gram-negative bacilli were examined for florfenicol-related resistance genes, and the positive isolates were further characterized. The antimicrobial sensitivity results showed that most of them (100, 94.33%) belonged to multidrug resistance Enterobacteriaceae. About 91.51% of the strains carried floR gene, while 4.72% carried cfr gene. According to the pulsed-field gel electrophoresis results, 34 Escherichia coli were subdivided into 22 profiles, the genetic similarity coefficient of which ranged from 80.3 to 98.0%. The multilocus sequence typing (MLST) results revealed 17 sequence types (STs), with ST10 being the most prevalent. The genome sequencing result showed that the Proteus vulgaris G32 genome consists of a 4.06-Mb chromosome, a 177,911-bp plasmid (pG32-177), and a 51,686-bp plasmid (pG32-51). A floR located in a drug-resistant region on the chromosome of P. vulgaris G32 was with IS 91 family transposase, and the other floR gene on the plasmid pG32-177 was with an IS CR2 insertion sequence. The cfr gene was located on the pG32-51 flanked by IS 26 element and TnpA26. This study suggested that the mobile genetic elements played an important role in the replication of resistance genes and the horizontal resistance gene transfer. [ABSTRACT FROM AUTHOR]

Staphylococcus caprae, Staphylococcus capitis , and Staphylococcus epidermidis belong to the "Epidermidis Cluster Group" (ECG) and are generally opportunistic pathogens. In this work, whole genome sequencing, molecular cloning and pan-genome analysis were performed to investigate the genetic characteristics of the resistance, virulence and genome structures of 69 ECG strains, including a clinical isolate (S. caprae SY333) obtained in this work. Two resistance genes (blaZ and aadD2) encoded on the plasmids pSY333-41 and pSY333-45 of S. caprae SY333 were confirmed to be functional. The bla region in ECG exhibited three distinct structures, and these chromosome- and plasmid-encoded bla operons seemed to follow two different evolutionary paths. Pan-genome analysis revealed their pan-genomes tend to be "open." For the virulence-related factors, the genes involved in primary attachment were observed almost exclusively in S. epidermidis , while the genes associated with intercellular aggregation were observed more frequently in S. caprae and S. capitis. The type VII secretion system was present in all strains of S. caprae and some of S. epidermidis but not in S. capitis. Moreover, the isd locus (iron regulated surface determinant) was first found to be encoded on the genomes of S. caprae and S. capitis. These findings suggested that the plasmid and chromosome encoded bla operons of ECG species underwent different evolution paths, as well as they differed in the abundance of virulence genes associated with adherence, invasion, secretion system and immune evasion. Identification of isd loci in S. caprae and S. capitis indicated their ability to acquire heme as nutrient iron during infection. [ABSTRACT FROM AUTHOR]

The diversity of class D β-lactamases mediating resistance to β-lactams has been increasingly reported recently. In this study, a novel class D oxacillinase named OXA-830 was identified in a fully sequenced Aeromonas simiae strain, which was isolated from sewage discharged from a farm in southern China. OXA-830 shares the highest amino acid identity of 79.3% with an OXA-12-like variant named OXA-725. When expressed in E. coli DH5α, OXA-830 conferred resistance to penicillins and selected β-lactamase inhibitors but not to cephalosporins and carbapenems. Kinetic analysis of OXA-830 revealed a broad substrate profile including penicillins, cefazolin, cefoxitin, and ceftazidime but not carbapenems. The hydrolytic activity was significantly inhibited by sulbactam, followed by tazobactam, but was less effectively inhibited by clavulanic acid. The bla OXA–830 gene was located on the A. simiae A6 chromosome and the bla OXA–830-related region was bracketed by a pair of perfect inverted repeats. [ABSTRACT FROM AUTHOR]

The genus Citrobacter is an opportunistic pathogen causing infections in animals, and the published data for its resistance to florfenicol are scarce. In this study, we investigated the antimicrobial susceptibility and molecular characteristics of florfenicol resistance genes among Citrobacter isolates from animal and relevant environmental samples and conducted a comparative analysis of a multidrug-resistant Citrobacter freundii strain isolated from a rabbit. Among 20 Citrobacter strains isolated from animal samples, resistance was most commonly observed to ampicillin (100%), tetracycline (75%), streptomycin (65%), florfenicol (60%), chloramphenicol (60%), and aztreonam (50%), while all the strains found in environmental samples were resistant to few antibiotics. The florfenicol resistance gene floR was detected in 12 isolates (48%, 12/25) from animal samples, and all of the floR-positive isolates were resistant to florfenicol with minimum inhibitory concentration (MIC) values ≥256 μg/mL. Sequencing and comparative analysis of the plasmids from a multidrug-resistant C. freundii isolate named R47 showed that the floR-containing region in the plasmid pR47-54 was a truncated transposon-like structure and could be found on both plasmids and chromosomes of bacteria of either animal or human origin. Furthermore, a range of antimicrobial and metal resistance genes associated with mobile genetic elements could be identified in pR47-54 and the other plasmid pR47-309 of C. freundii R47. These results provide in-depth views into the phenotypic and molecular characteristics of Citrobacter isolates recovered from animal and relevant environmental samples, as well as highlight the role horizontal gene transfer plays in the dissemination of plasmid-encoded resistance genes. [ABSTRACT FROM AUTHOR]